AmScope MU USB3.0 Operation & User’s Manual

User’s Manual Table of Contents Before Use ......................................................................................................... 5 Introduction ........................................................................................................................5 Safety Precautions ..............................................................................................................6 Parts .................................................................................................................. 7 MU USB2.0 Camera ............................................................................................................7 MU USB3.0 Camera ............................................................................................................8 Definition of Parts ...............................................................................................................9 Definition of Parts (Continued) .........................................................................................10 Getting Started ................................................................................................ 11 Quick Start ........................................................................................................................11 Installing the Software (CD) ..............................................................................................12 Installing the Software (Download) ..................................................................................17 Using the Software .......................................................................................... 30 Activating the Camera ......................................................................................................30 Capturing & Saving a Still Image ......................................................................................31 Opening a Saved Still Image .............................................................................................31 Recording & Saving a Video ..............................................................................................32 Left Hand Navigation Bar (Camera Bar) ............................................................ 38 Understanding the Software Options ...............................................................................38 Camera List .......................................................................................................................39 Capture & Resolution ........................................................................................................39 Exposure & Gain ...............................................................................................................40 White Balance...................................................................................................................41 Color Adjustment ..............................................................................................................42 Power Frequency (Anti-Flicker) .........................................................................................43 Frame Rate .......................................................................................................................43 Color/Grey ........................................................................................................................44 Flip ....................................................................................................................................44 Sampling ...........................................................................................................................44 Bit Depth ...........................................................................................................................45 ROI (Region of Interest) ....................................................................................................45 Histogram .........................................................................................................................45 Dark Field Correction ........................................................................................................46 Cooling ..............................................................................................................................46 Misc ..................................................................................................................................47 Parameters .......................................................................................................................47 Top Toolbar ...................................................................................................... 48 File Shortcuts ....................................................................................................................48 Measurement Swap Shortcuts ..........................................................................................48 Selection & Movement Tools ............................................................................................49 Drawing and Annotation Tools .........................................................................................49 Calibration Shortcuts ........................................................................................................50 Table of Contents

User’s Manual Image Enhancement Tools ................................................................................................50 Top Navigation Bar ........................................................................................... 51 File Menu ..........................................................................................................................51 Edit Menu .........................................................................................................................53 View Menu ........................................................................................................................54 Browse Menu ....................................................................................................................56 Setup Menu ......................................................................................................................57 Capture Menu ...................................................................................................................58 Image Menu .....................................................................................................................59 Process Menu ...................................................................................................................60 Layer Menu .......................................................................................................................63 Measurement Menu .........................................................................................................64 Options Menu ...................................................................................................................65 Window Menu ..................................................................................................................66 Help Menu ........................................................................................................................67 Process Menu Tools ......................................................................................... 68 Stitch .................................................................................................................................68 High Dynamic Range ........................................................................................................72 Extended Depth of Focus (EDF) .........................................................................................74 Deinterlace ........................................................................................................................80 Color Composite ................................................................................................................81 Segmentation & Count .....................................................................................................84 Denoise .............................................................................................................................98 Sharpen ...........................................................................................................................100 Color Toning ....................................................................................................................102 Filter ................................................................................................................................105 Image Stacking ...............................................................................................................113 Line Profile ......................................................................................................................114 Line Profile ......................................................................................................................115 Surface Plot .....................................................................................................................117 Pseudo Color ...................................................................................................................120 Range ..............................................................................................................................122 Binary .............................................................................................................................123 Layer Menu Functions .................................................................................... 124 Layer Menu .....................................................................................................................124 Measurement Menu Functions ...................................................................... 125 Measurement Menu .......................................................................................................125 Options Menu Functions ................................................................................ 126 Options Menu .................................................................................................................126 Preferences Menu ...........................................................................................................127 Measurement Menu .......................................................................................................133 Magnification Menu .......................................................................................................139 Calibration Menu ............................................................................................................140 Edit Dye List Menu ..........................................................................................................142

User’s Manual Auto Correction Menu ....................................................................................................143 Window Menu Functions ............................................................................... 144 Window Menu ................................................................................................................144 Troubleshooting ............................................................................................. 145 Common Issues ...............................................................................................................145

User’s Manual Before Use Introduction Congratulations on the purchase of your new AmScope microscope camera! This manual is designed for the MU camera series. Please be sure to take a few minutes to familiarize yourself with the features and functions of your new AmScope microscope camera. If you’d like more information on microscopes, parts, or accessories, please visit our website at: www.amscope.com We highly recommend reading this manual thoroughly before operating the microscope cam- era, and that it be kept on hand for future reference. If lost or forgotten, a new copy can be downloaded from our website using the included passcode from your packing list. Please keep all packing materials, as they are required for any warranty service to guarantee safe shipment to and from our location, and the packing list includes your password to access this manual at any time. If you have additional questions or need assistance, please do not hesitate to email us at: [email protected] Or give us a call during business hours (6AM-5PM Pacific Standard Time, GMT-8) at: 888-950-2000 Copyright © 2017 United Scope Inc. DBA AmScope. All rights reserved. Copyright Notice This document, which is in draft form and subject to change without notice, is an unpublished work containing confidential, trade secret information that is proprietary to AmScope, and is protected by laws pertaining to such materials. This document, the information in this document and all rights thereto are the sole and exclusive prop- erty of AmScope and are not to be copied, used or disclosed to anyone, in whole or in part, without the express written permission of AmScope. AmScope assumes no liability for any damages incurred, directly or indirectly, from any errors, omissions or discrepancies between the use of this microscope and the information contained in this document.

User’s Manual Before Use Safety Precautions 1. As the microscope camera is a precision instrument, always handle it with care, avoiding impact or abrupt movement during transporation. Do not shake the package. 2. Do not place the microscope camera in direct sunlight or in high heat. Keap it indoors in a dry and clean place with temperateures between 32-100 degrees F (0-40 degrees C), and in maximum relative humidity of 85%. 3. Avoid touching the lenses on the cmaera body and the reduction lenses so that oil and dirt from your fingerprints do not obstruct your view. Additional details regarding maintenance and safety can be found on page 10.

User’s Manual Parts MU USB2.0 Camera Adapter Sleeves Reduction Lens (SKU: RU050) Camera Body USB Cable

User’s Manual Parts MU USB3.0 Camera Adapter Sleeves Reduction Lens (SKU: RU050) Camera Body USB Port (3.0 is Blue, 2.0 is White) USB Cable

User’s Manual Parts Definition of Parts Adapters/Spacers Metal cylindrical sleeves that adapt a larger size ocular tube down to 23.2mm for the camera to fit. Camera Body Square metallic housing that holds the camera sensor and circuit board, as well as has a port to con- nect a USB cable.

User’s Manual Parts Definition of Parts (Continued) Reduction Lens/Adapter A lens that both reduces the incoming magnification from the microscope to better fit the image to the sensor’s size in the camera, and adapts the C-mount connection down to a 23.2mm port size, common on AmScope microscope models. Software CD CD that includes a copy of the software and driver for the camera, which allows the Windows PC to both inter- face with the camera, and allows the user to interact with the camera via the on screen controls. USB Port A standardized port (universal serial bus) that accepts a USB cable, allowing a connection between the computer’s USB port and the camera’s USB port to facilitate communication between devices.

User’s Manual Getting Started Quick Start 1. Check the packing list on www.amscope.com or included in your package to ensure that you’re received all items that are to be included: - 1 Camera Body (SKU on sticker) (USB2.0 or USB3.0) - 1 Reduction Lens/Adapter - 1 Camera Adapter (30mm to 23.2mm) - 1 Camera Adapter (30.5mm to 23.2mm) - 1 Software CD - 1 USB Cable 2. The camera can be mounted and assembled in two different primary ways: C-Mount: If you intend to attach the camera via C-mount to your microscope, you can take the camera body and thread it onto your male C-mount port (25.4mm outer diameter, threaded). Reduction Lens/Adapter: If you intend to attach via the reduction lens/adapter (23.2mm port size compatibil- ity), please thread the reduction lens onto the camera body. 3. Insert the software CD into the CD tray of your computer. If your device does not have a CD drive, you can download the software from our website at www.amscope.com/software-download. For instructions on down- loading the software and installing it, please see the following sections. 4. Install both the driver and the software by following the on screen instructions. For detailed instructions on installing the software and driver, please see the following sections depending on if installing from the CD or from a downloaded file from our website (pages 10 - 12). 5. Once the software and driver are installed, connect the included USB cable to the camera body’s USB port, and then the USB port on your computer. 6. Activate the software by double clicking the AmScope/ToupView shortcut icon on your desktop or Start Menu. 7. Once the software has loaded, on the left hand side, you should see the “Device List,” showing your camera. It may be either named the file name of the driver, or the SKU of your camera (MU300, for example). If you see the camera listed there, click the name to open up the live feed from the camera 8. Install the camera into your microscope, whether through a 23.2mm, 30mm, or 30.5mm eyepiece, or trin- ocular port, or via C-mount. Open any shutter ports on your microscope if applicable, and turn the light on the microscope. You should see an image (although it may require refocusing) from the camera.

User’s Manual Getting Started Installing the Software (CD) 1. Begin by placing the software CD in your computer’s CD tray, and insert the CD. 2. Windows should either bring up the AutoPlay function. When asked, select the first option “Run autorun. exe” in order to access the AmScope/ToupView installation menu. 3. Next, click “Install Application” to start the Installation Wizard.

User’s Manual Getting Started Installing the Software (CD) (Continued) 4. Once the Installation Wizard has opened, please click “Next” to continue.

User’s Manual Getting Started Installing the Software (CD) (Continued) 5. The next window will let you choose where you wish to install the program to. If unsure, leave as default, and press “Install” to continue. Pressing “Browse” will allow you to navigate to the folder you wish to install the software to using the File Browser, or, if known, you may type in the address to the Destination Folder directly. When done, press “Install” to begin the installation process.

User’s Manual Getting Started Installing the Software (CD) (Continued) 6. The Installation Wizard will then begin installing the program onto your computer’s hard drive in the folder designated by you on the previous page. Please wait until the bar has been completely filled, upon which the Installation Wizard will automatically prog- ress to the final Installation Wizard window.

User’s Manual Getting Started Installing the Software (CD) (Continued) 7. The final page of the Installation Wizard will advise whether the software and driver package was success- fully installed or not, and if so, will allow you to immediately open the software. By keeping the “Run AmScope 3.7” box ticked, upon hitting “Finish,” the software will open. If unticked, pressing “Finish” will simply close the Installation Wizard.

User’s Manual Getting Started 1. Begin by identifying which specific MU series camera model you have. This can be viewed on the SKU on the sticker of the camera body, and starts with “MU” followed by a series of numbers. 2. Open your web browser (Google Chrome, Internet Explorer/Edge, Mozilla, Safari, Opera, etc), and in the ad- dress bar, type “www.amscope.com/software-download”, and press Enter. Note: Alternatively, you may input “www.amscope.com” and navigate to the bottom of the page to find a section titled “Customer Support,” and click “Camera Software Downloads” to open the page. 3. Once on the Camera Software Downloads page, select the first drop down menu under “Select Camera” by clicking the arrow in the box, and from the menu that drops down, select your camera model (MU Series) by clicking on it. Installing the Software (Download)

User’s Manual Getting Started 4. To the right of the “Select Camera” drop down menu, there is a second drop down menu titled “Select Oper- ating System.” Click the arrow to drop the menu down, and select “Windows” by clicking on it. Note: If you’d rather manually search for the software/driver, you can also simply scroll down the page until you find “MU Series” and select the version of the driver and software you’d like to download. 5. Once you’ve selected both camera series and operating system, a new section will appear just below the drop down menus, listing the various versions of the software we have available. Please select the most recent release (listed at the top of the list), and to the right, under “Download Link,” click “download,” and do the same for the DirectShow driver (or Twain, if your application requires it--DirectShow is best for most users). Note: Both driver AND software must be installed for the camera to function. Installing the Software (Download) (Continued)

User’s Manual Getting Started 6. By clicking “download,” your web browser will automatically begin downloading the installer package to the location that is set as your Downloads folder. Some web browsers will show the download on the bottom of the bar once completed, or will appear in a “Downloads List” funtion in your web browser’s menu. Once it is marked as complete, proceed to the next step. Below is an example using Google Chrome. Note: Different web browsers have different default locations for your “Downloads” folder. We cannot be sure where your file goes if you have changed it, so, please be aware of where on your computer’s hard drive you have your Downloads folder set to, as that will be where the installer package is located once it finishes. 7. Locate the installer package where the web browser placed it. A quick way to locate it is to right click on the download in your browser’s download list or bottom bar, and click “Show in Folder” which will open a window of that folder’s contents. Below’s example is from Google Chrome, but, all web browsers have a variant of this function. Installing the Software (Download) (Continued)

User’s Manual Getting Started 9. The installer file is packaged in a .zip file, which is a compressed file format that allows you to send multiple files over the internet as a single file, and compresses them so that the file size is smaller, and thus, faster/easi- er to transfer. Because of this, you cannot immediately open it like you would a program (a .exe file) or a folder. Think of a .zip file as a cardboard box or envelope with multiple things in it. You must first open the cardboard box with a knife, or the letter with a letter opener, in order to access the contents. Luckily, Windows 7 and higher editions have .zip extraction functionality built into them. Simply right click the .zip file, and select “Extract All.” Note: If you are using an older version of Windows, such as XP, you may have to download and install Win- RAR, which is a freely available software. The below image is shown using WinRAR’s context menu options, which work with all Windows versions, however, the native Windows options are similarly named. 10. Follow the on screen instructions, hitting “Next” until it shows “Finish,” which after clicking, will show you your newly extracted files. Installing the Software (Download) (Continued)

User’s Manual Getting Started 11. Look for the installer file, which has a file extension (ending) of .exe, and a type of Application. Double click this in order to run the Installation Wizard. 12. When the Installation Wizard window opens up, please click “Next” to proceed. Installing the Software (Download) (Continued)

User’s Manual Getting Started 13. The second page of the Installation Wizard lists the software license terms and conditions of use. Please read through them and click “I Agree” when complete to proceed. If you do not agree with the license terms, the software cannot be installed and used. Installing the Software (Download) (Continued)

User’s Manual Getting Started 14. Now, you will be given an option to select a location to install the software to. You can leave this at the default location if unsure or you do not have a preference, or, press “Browse” to select a folder to install the program to. Alternatively, you can type in the folder’s address in the Destination Folder bar. When ready, click “Install” to proceed with installing the software. Installing the Software (Download) (Continued)

User’s Manual Getting Started 15. Once complete, the Installation Wizard will display a final confirmation window, and the “Next” button will change to a “Finish” button. The driver still must be installed before the camera will function, so untick the “Run AmScope” box, and click “Finish.” 16. Navigate back to the folder to which the driver download is located. We offer two drivers (DirectShow and Twain), however, DirectShow works with most applications for most users. The installation procedure is the same for both. Double click your desired driver installer to start the Installation Wizard. Installing the Software (Download) (Continued)

User’s Manual Getting Started 17. When the Installation Wizard window opens up, please click “Next” to proceed. Installing the Software (Download) (Continued)

User’s Manual Getting Started 18. The second page of the Installation Wizard lists the software license terms and conditions of use. Please read through them and click “I Agree” when complete to proceed. If you do not agree with the license terms, the driver cannot be installed and used. Installing the Software (Download) (Continued)

User’s Manual Getting Started 19. Now, you will be given an option to select a location to install the driver to. You can leave this at the default location if unsure or you do not have a preference, or, press “Browse” to select a folder to install the program to. Alternatively, you can type in the folder’s address in the Destination Folder bar. When ready, click “Install” to proceed with installing the DirectShow or Twain driver. Installing the Software (Download) (Continued)

User’s Manual Getting Started 20. The Installation Wizard will proceed to install the DirectShow or Twain driver. When it has completed, it will automatically move to the final page. Installing the Software (Download) (Continued)

User’s Manual Getting Started 21. Once complete, the Installation Wizard will display a final confirmation window, and the “Next” button will change to a “Finish” button. 22. You are now ready to open the software and use the camera. Please plug in the camera, and navigate to your desktop. You will now see either a yellow “A” or a yellow “T” (AmScope / ToupView) depending on what version of the software you chose to install. Double click either to open the software. Installing the Software (Download) (Continued)

User’s Manual Using the Software Activating the Camera 1. Begin by opening the software. Double click your AmScope/ToupView shortcut icon (the yellow “A” or “T”) from the desktop, or single click the AmScope/ToupView shortcut icon from the start menu. 2. Once loaded, locate the “Device List” on the left hand navigation bar, and click the camera name to activate the live view from the camera. 3. What the camera is currently seeing will appear in the gray area to the right of the navigation bar. While view- ing the camera’s feed, adjust the focusing knobs on the microscope until the feed is in clear view of your desired sample. 4. From here, you can adjust the software settings as desired to fine tune the image manipulation options, cap- ture a photo, or record video as desired. Instructions for taking a photo, video, calibration for measurement, and fine tuning image settings are covered in the following sections.

User’s Manual Using the Software Capturing & Saving a Still Image Opening a Saved Still Image 1. To capture an image, once you are focused on the sample and it is in clear view in the software view, simply press the “Snap” button with the photo of the camera. Be sure to set the “Snap” resolution to the resolution desired of the still image recording prior to pressing “Snap.” 2. A new tab will open over the camera view area, noted by the different colored tabs at the top. The “Video [Camera SKU Here]” tab will always be the live view of what the camera is feeding the software. The following tab will be the number of photo taken--in this case, “0001*” meaning the first photo of this session. 3. To save the photo, click on “File” at the top navigation bar of the software, and “Save” (or “Save As”). “Save” will immediately save it in the desired directory in the default format, while “Save As” will allow you to select not only a directory and a name, but also the file type (such as .jpeg, .png, etc.). Select either method, name your photo, and select where you’d like it saved to. 1. To recall a previously saved photo, simply click “File” from the top navigation bar, and click “Open Image.” This will open a navigation window, allowing you to locate and select which photo you would like to open. 2. Once you have located and selected the file you wish to open, click it, and click “Open.” The image should ap- pear as a tab in the view area with the file name as the name of the tab.

User’s Manual Using the Software Recording & Saving a Video 1. To record a video, once you are focused on the sample and it is in clear view in the software view, simply press the “Record” button with the photo of the “Play” button (the triangle pointing to the right). Be sure to set the “Snap” resolution to the resolution desired of the video recording prior to pressing “Record.” 2. The “Video File” window will pop up, asking you to name your video file, and select a location to save the re- cording to. Select your desired location, and type in the desired name for your video, then press “Next.”

User’s Manual Using the Software Recording & Saving a Video 3. The “Video Format and Encoder” menu will appear next, asking which file format you would like to save and encode your video in. Most users will use .wmv or .avi. If unsure, use .wmv, and press “Next.”

User’s Manual Using the Software Recording & Saving a Video (Continued) 4. The “Encoder” menu will appear next, asking what parameters you would like to use to encode your video, such as what bitrate you would like to use, the quality, and the key frame spacing. If unsure, leave these at de- fault. “Bitrate (Mbps)” is the numbers of megabits per second that can be transmitted along a digital network. In this case, it is how many megabits per second the video will record to the file. A larger number will have more bits, and thus, a higher playback quality, however, will also have a much larger file size, and require greater processing power for both recording and playback. “Quality” is a percentage in this case, and can be set anywhere between 0 and 100 to improve file size, ease of playback/transmittal. It is set to 100 by default, however, can be adjusted as needed. “Key Frames Spacing” refers to the time between frames in which the encoder will re-evaluate the video image and record a full frame, rather than recording just the changes. Unlike traditional film, digital recordings often only record what has changed in an image in between frames, rather than capturing an entire frame each time, in an effort to speed up frame rates and minimize file sizes. Adjusting this higher will cause your encoder to speed up recording, however, will drop image quality if you have rapid motion in your sample. Lowering it will increase your quality at the expense of frame rate. When done, please press “Next” to continue.

User’s Manual Using the Software Recording & Saving a Video (Continued) 5. The “Display Information” menu will appear next, allowing you to set the ID3 tags for your video. These are file properties that will show up in certain playback softwares, and is useful for cataloging results. While not neces- sary, you may fill in this section. When completed, please press “Next” to continue.

User’s Manual Using the Software Recording & Saving a Video (Continued) 6. The “Start to Record” menu will appear next, allowing you to set how long you wish the video to record for (if you wish to set a limit), and the time-lapse (time between captured frames). These can be adjusted if desired, or left alone to have a normal, smooth video that will record until you hit the “Record” button again to stop. Press “Finish” when ready to begin recording your video. Note: At any time from when the “Video File” window (step 2) is open, you can press “Finish” instead of “Next” to immediately begin recording, and leave all other settings as default. 7. When you have finished recording, you can press the “Record” button again to stop the video, or simply wait for the time that was set in step 6 to elapse. The icon next to “Record” will change to a red circle with a gray square in it, much like the “Stop” button on most recording devices.

User’s Manual Using the Software Recording & Saving a Video (Continued) 8. The video recording is automatically saved to the folder designated on the “Video File” menu (step 2), so no further saving procedure is necessary. 9. If you wish to load a video for playback, you can navigate to the file folder containing the video and play it using your desired media playback software. We recommend Video Lan Controller (VLC), which is a free to use software available on the internet, and has a wide compatibility with different codecs and encoding methods. Note: We at AmScope are not affiliated with VLC in any way, shape, or form.

User’s Manual Using the Software Understanding the Software Options Left Hand Navigation Bar (Camera Bar) This section covers the various options and adjustments that the software is capable of. Understanding these software options is essential to successful operation of the software, and in getting the desired results. Every camera sensor is a little different, even from the same assembly line, and every environment and sample has differences, thus, the options here are designed to allow you the flexibility to compensate for those factors.

User’s Manual Left Hand Navigation Bar (Camera Bar) Camera List Capture & Resolution This section displays the connected device(s) to your system. Note that only one camera will be recognized at a time by the software, usually whichever is the most recently attached device. Clicking a camera in this section will activate that camera in the viewing area to the right of the navigation bar. This section houses the “Snap” and “Record” buttons, which allow you to snap a still image, or record a video. Additionally, this section contains the resolution adjustments available for the camera. “Live” resolution refers to the resolution that the camera’s live view is fed in. “Snap” refers to the resolution that both still images and recorded videos are captured at. Note: The lower the resolution selected, the faster the camera can respond, as there is less data being pro- cessed per frame. This results in a faster frames per second (FPS) rate. You can lower the “Live” resolution while keeping the “Snap” resolution up to focus/adjust the microscope more easily, while not sacrificing reso- lution for your still images. Unlike still images, video recorded frame rates will be affected by the “Snap” resolu- tion as well, causing an FPS drop the higher the resolution is.

User’s Manual Left Hand Navigation Bar (Camera Bar) Exposure & Gain This section contains various settings regarding exposure, which is how much light the camera sensor accepts per frame, and gain, which is a brightness setting controlling how dark “black” colors appear. By default, the “Auto Exposure” box is ticked, meaning the camera itself will automatically adjust these set- tings to compensate for different amounts of light coming from the microscope’s light source. The drawback of the Auto Exposure feature is that it can cost a few frames per second as the camera is constantly adjusting the exposure target and time, requiring additional processing time. Turning this off and manually setting the sliders is recommended if you need faster frame rates from the camera, or the image does not appear at the desired brightness level even after adjusting the “Exposure Target.”

User’s Manual Left Hand Navigation Bar (Camera Bar) White Balance This section allows for adjustments in “Color Temperature” and “Tint” in order to ensure that the camera sen- sor is capturing the correct colors. Often times, cameras can come out of the box seeing different colors differ- ently, and thus displaying them as such. Pressing the “White Balance” button at the bottom of this section will automatically make adjustments to Color Temperature and Tint to balance out at a point where items that are known to be white appear as white, and all other colors are adjusted based on that known point in the color spectrum. When clicking the button, an adjustable red box will appear in the viewing area. If your sample has an area that is supposed to be white (blank space between the sample’s elements, or a sheet of thin white paper if calibrat- ing without a sample), drag and resize the box so that everything inside the box is supposed to be white. Press the “White Balance” button again, and your sample will be adjusted properly for correct white balance. The sliders can be used to further adjust as necessary. “Defaults” will return it to factory settings so you may readjust and start the process over. “Color Temperature” adjusts colors between cyan on the low end (cooler color temperatures) and yellow on the high end (higher color temperatures). “Tint” adjusts colors between magenta on the low end and green on the high end. Usually, the automatic “White Balance” button, when used as above, sets this very accurately without use of the sliders.

User’s Manual Left Hand Navigation Bar (Camera Bar) Color Adjustment Much like the section before it, this section allows you greater control over the colors as they appear in your image through various sliders. The “Hue” adjustment allows you to shift the colors displayed between the entire visible color spectrum. Ad- just as needed if your colors are entirely shifted the wrong way. The “Saturation” adjustment slider allows you to adjust how powerful your colors appear in your image, affect- ing their vibrancy. A higher value on the slider will have more vibrant and powerful colors, while a lower value will be toned down or grayed out. The “Brightness” adjustment slider defines how intense the “white” colors in your image are. A higher value will appear more brightly lit (or washed out if too bright), while a lower value will dim your image’s white col- ors down for a darker overall image. The “Contrast” slider adjusts how strong the difference is between light and dark pixels that neighbor each other. A higher value will add more contrast between them, better defining but also darkening your image, while a lower value will brighten up the image, but also create less definition between elements in your sam- ple. The “Gamma” slider serves as an adjustment in how the sensor processes light captured from the sensor. A higher value will darken the image, while a lower value will brighten the image.

User’s Manual Left Hand Navigation Bar (Camera Bar) Power Frequency (Anti-Flicker) This section is only available by turning “Auto Exposure” off in the “Exposure & Gain” section. It allows you to set the CMOS sensor to capture frames in alignment with the frequency of local power supply powering the microscope. CMOS sensors use a rolling shutter (captures each row of pixels from top to bottom sequentially), so if the light is not flickering at the same rate as capture, a flickering effect or rolling lines will be seen. If you are detecting a series of rolling lines or flickering when viewing your sample, the “Anti-Flicker” system may help resolve the issue. Turn “Auto Exposure” off and select the power frequency of nearby devices to the camera (such as the microscope, computer, monitor, etc). Frame Rate This section covers manual adjustment of the camera’s frame rate (FPS). By default, it is set to the max speed possible, as users are looking for the closest to live experience possible from cameras. If your application deems it necessary, you can lower the frame rate as desired here. Note: Although this section exists, the greatest effect on frame rate will come from the resolution seelcted, whether the device in use is a USB2.0/3.0 device (in a compliant USB2.0/3.0 USB port), and the Exposure & Gain settings (as well as the Sampling section, covered below).

User’s Manual Left Hand Navigation Bar (Camera Bar) Color/Grey This section allows you to switch the camera sensor between “Color” mode and “Gray” mode. Color captures images in color, while Gray captures images in grayscale (black and white). Flip Sampling This section allows you to flip the image received from the camera along either the “Horizontal” or “Vertical” axes. Horizontal will flip the image from left to right, while vertical will flip the image from top to bottom. The “Sampling” section contains two options: “Bin” and “Skip.” “Bin” option refers to the method of averaging pixels in blocks of neighboring same color pixels to resize the video to a lower resolution. It provides a small speed increase while maintaining image integrity. “Skip” (also “Decimation”) skips a pre-determined amount of pixels (either horizontally, vertically, or in both axes) to reduce the resolution of the resulting video, increasing speed. This method introduces subsampling artifacts, however. Skip is on by default.

User’s Manual Left Hand Navigation Bar (Camera Bar) Bit Depth ROI (Region of Interest) Histogram Bit depth refers to the color information stored in an image. The higher the bit depth, the more colors it can store. 1 bit stores 2 values, so a 16 bit image can store 32 different color values. This section allows you to select a specific area in your sample’s view area as the region that one is interested in viewing. It is simply a visual aid for the user. This section is a graph that illustrates how pixels in an image are distributed by graphing the number of pixels at each color intensity level, including shadows (low values), mid-tones (middle of the range), and highlights (top of the range). This is mainly used to ensure that the image captured has enough detail to use other correc- tions effectively. The drop down menu (by default set to “RGB”) allows you to view all or a single color channel. “Left” and “Right” allow you to set boundaries to limit the range for best image quality. Clicking “Auto” will automatically set these boundaries for the software’s detected ideal range.

User’s Manual Left Hand Navigation Bar (Camera Bar) Dark Field Correction Cooling Darkfield images are a specialized type of microscopy used to create additional contrast in a sample by using a dark background instead of a light background, as well as lighting the sample from oblique angles (lighting the edges of the sample by angled lighting). Because of the nature of darkfield, normal image capturing methods with cameras are not ideal. This section allows one to correct the image digitally for a superior final result. First, cover the objective lens of your microscope to ensure the field is totally black. You may also simply close the shutter on your microscope’s trinocular port if it has one. Next, hit the “Capture” button. This will capture a black image, enabling the dark field correction function. Simply hit “Enable” and focus on your darkfield sample for best results, and use the software as normal. Hitting “Enable” again will turn correction off. The MU series cameras do not have a cooling mechanism in their design, however, this section is used to con- trol the cooling functionality of other cameras that use this software.

User’s Manual Left Hand Navigation Bar (Camera Bar) Misc Parameters This section only contains a single function, “Negative,” which inverts the colors in the image. This section allows you to save all of the above options as a configuration file, which can be later recalled to avoid having to set them each time. The “Save” button allows you to save a series of settings, which can then be recalled using the “Load” button. Clicking “Save” allows you to name the settings, and adds it to the drop down menu. Select it from the drop down menu, and hit “Load” to import them. “Overwrite” will save your current settings onto an existing saved parameter group. “Management” will let you sort, delete, or copy your saved parameters.

User’s Manual Top Toolbar File Shortcuts Measurement Swap Shortcuts This section is located above the left navigation bar and viewing area, and contains shortcuts to many tools the software has to further enhance your image, create annotations, or simple quality of life improvements. Each one will be covered in the following sections. The first four icons on the toolbar (from left to right): Open Image - Open a single image file. Save - Save the currently open image file. Quick Save - Saves the currently open image file with a default name. Browse - Changes the left navigation bar from the “Camera Bar” to the “Folder Bar,” providing quick access to open files on your hard drive in the viewing area. Note: To change back to the “Camera Bar,” click the tab below the left hand navigation bar titled “Camera,” and click the first tab in your viewing area to return to the camera’s live view. The following two drop down menus are useful when you have calibrated your software to capture measure- ments in real distance while looking at a sample through your MU series camera. If you have not yet per- formed the calibration procedures using a micrometer slide or other unit of known measurement, these func- tions will not actually change anything. From left to right, the options are: Measurement - Allows you to quickly swap between measurement sizes, such as milimeters (mm) and nano- meters (nm). Magnification - Allows you to quickly change between calibrations performed at different magnifications. These are important because the camera itself only sees an image in pixels. By telling it how many pixels are in a given unit of measurement at a specific magnification level, the camera software can perform measurements in real distance by extrapolating how many pixels are in the requested line or arc, and converting it to real measurement. Without calibrating the software, it will always read and feed back in pixels, as that is the only thing the camera sensor and software can actually quantify. For more details on calibration, please see “Cali- brating the Software” on page 140.

User’s Manual Top Toolbar Selection & Movement Tools Drawing and Annotation Tools The following two tools are used to select or highlight an area in an image or video, and if zoomed in, to move the zoomed in area around to scan. From left to right: Select - Allows you to highlight an area of interest in the viewing area. Similar to the ROI function. Track - Allows you to grab and move the image, if the image is zoomed in more than the screen display area. The following tool set is used for drawing measurements, lines, arcs, and annotating the captured images. From left to right: Object Selection - Enables the mouse cursor to select a previously drawn object. Angle - Creates either a three or four point angle and provides the measurement data of the angle drawn. Point - Creates a point and provides X and Y coordinates in the selected measurement size. Line - Creates one line and provides X and Y coordinates of each end, as well as the distance between points. Parallel - Creates two lines, locking the angle of the second to the angle of the first, and providing measure- ment information of length, angle, start, end, and distance between lines. Two Parallel - Creates two pairs of parallel lines with a midpoint line, and provides above measurements. Vertical - Creates a line and a second line perpendicular to it, and provides above measurements. Rectangle - Draws a rectangle and provides perimeter, width, height, and area. Ellipse - Draws a circle with dissimilar axes, providing length of each, perimeter, area, and center point. Circle - Draws a circle with similar axes, with either a diameter, radius, or triple point methods. Annulus - Draws multiple concentric circles with a common center point. Two Circles - Draws two circles sequentially, and provides distance between center points. Arc - Creates an arc after setting the first point, providing center point, diameter, perimeter, and angle. Text - Creates a text box for putting notes on the image for items of interest. Polygon - Draws a polygon of the user’s choice number of points, and provides area and perimeter. Right click to close the polygon, left click to add a point.

User’s Manual Top Toolbar Calibration Shortcuts Image Enhancement Tools The following two tools are used as shortcuts to access calibration menus. From left to right: Calibration - Opens the Calibration menu, allowing you to use a unit of known measurement and a given mag- nification to provide the software with the means to extrapolate real distances from pixels counted on screen. Grey Calibration - Provides the average gray value in the image per frame displayed. The following three tools are special tools used to improve the image as captured on screen in various man- ners using software manipulation. From left to right: Stitch - The Stitch tool allows users to take images taken at different points along the X and Y axes, and put them together into a single image. It is particularly useful when the field of view is not large enough to capture the entire region of interest in a single photo, allowing the images to be stitched together in a linear or grid format. Extended Depth of Focus (EDF) - The EDF tool allows users to take a sequential series of photos from different focal points, and have the software digitally mesh them together, creating a more deeply focused image. This is useful when you need high magnification values, but, cannot get a large enough depth of field to have the whole area in focus at one time due to contrasting sample topographies. High Dynamic Range (HDR) - The HDR tool allows users to capture images with a larger range between how dark and how bright an image can be in terms of contrast.

User’s Manual Top Navigation Bar File Menu Above the tool bar is the top navigation bar, which begins with the “File” drop down menu, and ends with the “Help” drop down menu. These menus contain the full range of tools and options that the software is capable of, although since there are many shortcuts on the tool bar and the left hand navigation bar for commonly used procedures, using the menus is generally only as a last resort if a feature you need is not available via a tool or shortcut. The following functions are available in the “File” drop down menu: From top to bottom: Open Image - Selecting “Open Image” will bring up the file navigator, allowing you to select a previously saved photograph to open in a new tab in the viewing area. Control + O is the keyboard shortcut for this function. Open Video - Seleting “Open Video” will bring up the file navigator, allowing you to select a previously saved video to open in a new tab in the viewing area.

User’s Manual Top Navigation Bar File Menu (Continued) Open Broadcast - Seleting “Open Broadcast” will bring up the Open Broadcast menu, allowing users to input an IP address and port to receive video from. Performance is bandwidth dependent. Save - Selecting “Save” will immediately save a copy of the image tab currently open to the default folder, , while still leaving the image tab active. If the file has not been saved before, it will default to the “Save As...” function, and require a name to be input, and a file type selected. Control + S is the keyboard shortcut for this option. Save As... - “Save As” will, regardless whether the image has been saved before or not, open a dialog menu requiring file name and file type selection, and allows you to save the file. Batch Save - This menu option allows the user to save multiple captured images at once, using a designated file prefix, name, and file type, and automatically appending a numerical value to the image. Quick Save - “Quick Save” saves the currently opened image tab without requiring name or format input. It will default to the values set in the “Options -> Preferences” menu. Control + Q is the keyboard shortcut for this option. Paste as New File - If an image file is copied onto the clipboard in Windows, this function will allow the user to paste that image into a new image tab. Twain: Select Device - AmScope/ToupView software supports other Twain compatible image capture devices. Unfortunately, we at AmScope do not support other devices on our software, nor do we support third party softwares for our camera devices. This menu option is used to select a Twain compatible source to use within the software. All AmScope/ToupTek cameras are part of the AmCam/Toupcam Twain source. Twain: Acquire - Allows the user to acquire an image from a Twain compatible device. Please see above. Print Setup - Activating “Print Setup” brings up a menu to allow the user to select which printer, and what printing options, they would like to use to print the currently active image prior to printing. Print Preview - “Print Preview” shows the user a preview of what to expect to come from their selected printer if the image in the active tab is printed (size and orientation particularly). Control + Shift + P is the keyboard shortcut for this function. Print - Allows the user to print the currently active tab with their default printer and printing options. Control + P is the keyboard shortcut for this option. Recent Files - Contains a list of recently saved and opened files for ease of access. Exit - Closes the AmScope/ToupView software.

User’s Manual Top Navigation Bar Edit Menu The “Edit” menu contains options for manipulating items on the clipboard, and the current active window. Cut - This menu option allows the user to remove a selection from the image attached and place it on the clip- board to be placed somewhere else. Control + X is the keyboard shortcut for this function. Copy - “Copy” performs the same action as “Cut,” however, instead of removing the selected area from an im- age, it creates a copy, leaving the original selection as a part of the image. Control + X is the keyboard shortcut for this function. Paste - If an image file is copied onto the clipboard in Windows, this function will allow the user to paste that image or selection onto the currently active image. Control + V is the keyboard shortcut for this function. Paste Shortcut - If an image file is copied onto the clipboard in Windows, this function will allow the user to paste that image or selection onto the currently active image. Delete - The “Delete” function allows a user to delete the current selection from the image. The “Delete” key is the shortcut for this function (not the “Backspace” key). Image Select - This tool will allow you to select an area of interest in the currently active image tab in the view- ing area, and use that for the above functions. Select All - This tool selects the entire image in the currently active tab in the viewing area. Control + A is the keyboard shortcut for this function. Select None - Removes any selections in the viewing area in the currently active tab. Control + D is the key- board shortcut for this. Also known as “Deselect.”

User’s Manual Top Navigation Bar View Menu The “View” menu contains options for manipulating the current active view, navigation bars, and opened tabs. Browse - This menu option opens the file explorer box, allowing you to navigate and select an image or video to open from your hard drive. Control + B is the keyboard shortcut for this function. Measurement Sheet - This option brings up the “Measurement Sheet” from the bottom of the software win- dow, which allows you to see a table of measurements that have been performed. This can also be accessed by clicking “Measurement Sheet” at the bottom of the screen. Sidebar - This menu folds out into another menu, which allows the user to turn different tabs of the left hand navigation bar on/off as desired. The potential side bars are Camera, Folders (Quick Browse), Undo/Redo, Lay- ers, and Measurements. One can change between these tabs at the bottom of the left hand navigation bar as well. Grids - This function allows the user to create custom sized grids superimposed over the currently active im- age. It folds out into a new menu, allowing the software to draw the grid automatically with “Auto,” the user to draw their own with “Manual,” or the “Settings” window for more detailed customization. Best Fit - This tool will zoom the displayed image to fit the viewing area best in either the vertical or horizontal dimention. Note: This is NOT increasing your magnification, just enlarging the currently displayed image. Actual Size - This function adjusts the size of the image or camera feed in the viewing area to fit the actual size it is capturing and displaying at (the “Live” resolution or “Snap” resolution if looking at a previously captured image). Full Screen - This tool enlarges the image in the viewing area to fit the entire screen. The “Escape” key can be used to return to the software view.

User’s Manual Top Navigation Bar View Menu (Continued) Track - When zoomed in to the image to the point where the image is larger than the display area (or using a high resolution setting to view from the camera), the “Track” or “Hand” tool is used to slide the image to scan different areas. It is only usable when zoomed in. If the image is entirely fitted to the viewing area, it will not move the image.

User’s Manual Top Navigation Bar Browse Menu The “Browse” menu is disabled unless a file browsing tab is open in the viewing area. This can be done by click- ing the “Folders” tab in the bottom section of the left navigation bar, then clicking the “Browse” button. Sort - The “Sort” function allows the user to sort how the files or folders are displayed in terms of order, by various categories (file type, name, size, etc). This allows you to more easily find the files you are looking for. Icons - The “Icons” function allows the user to change the properties of the displayed icons for the folders and files in the “Browse” tab to either be large or small icons. Refresh - “Refresh” is a command that tells the “Browse” tab to recheck the folder to see if any changes have been made, and display the latest version of that folder. This is useful if a file was saved or modified after open- ing the “Browse” tab, but is not displayed. Properties - This tool brings up the associated properties with the file or folder currently selected in the “Browse” tab.

User’s Manual Top Navigation Bar Setup Menu The “Setup” menu is used primarily to adjust settings related to producing video, such as watermarks, file properties, and overlays. Start/Pause - The “Start/Pause” command allows the user to freeze the image provided from the camera or the video being viewed in place. Using the command again will unpause playback. View Properties - This function brings up the “View Properties” menu, which will display detailed information about the device being used, such as device name, pixel size, and resolution. Video Overlay - The “Video Overlay” function brings up a menu that allows the user to customize a digital overlay or marker on their recording. Adjustments range from position and size, to color and type, such as scale, date/time, clarity factor, etc for overlays, and circles or crosses (or a combination) for markers. Video Watermark - The “Video Watermark” function brings up the Watermark menu, allowing the user to select an image to place on their video (with opacity controls to allow it to be seen in front of the video) to protect against unauthorized reproduction or redistribution of the video, if captured video/images are propri- etary intellectual property. Watermarks are hard encoded onto the video, preventing easy removal. Move Watermark- The “Move Watermark” function allows the user to reposition the watermark. Common places are spanning the entire image but more translucent than most, or in any of the four corners of the im- age, with more opacity. Rotate Watermark - “Rotate Watermark” will allow the user to pivot the watermark among different angles to best fit their preferences. Gray Calibration - This tool brings up the “Gray Calibration” menu, feeding the user a value of gray in the im- age used to calibrate their device for gray reproduction accuracy.

User’s Manual Top Navigation Bar Capture Menu The “Capture” menu encompasses tools that are used to take a photo, record a video, or start a broadcast. Capture Image - This function captures a still image, much like the “Snap” button. F8 is the keyboard shortcut for this function. Start Time-lapse (Auto Capture)... - This function opens up the “Time-lapse” menu, which allows you to desig- nate the software to capture a still image at given intervals of time, up to a given length of time. Start Record... - The “Start Record...” command is much like the “Record” button, which opens the “Record Video” menus, and allows you to begin or stop recording (depending on whether you are currently recording a video or not). Start Broadcast - The “Start Broadcast” function opens the Broadcast menu, which allows you to designate an a port and the encoding settings for a live video to be broadcast over an intranet or the internet.

User’s Manual Top Navigation Bar Image Menu The “Image” menu allows you to process the user’s current active image much like the selection modification in the “Edit” menu, with tools such as rotation, adjustment levels, cropping, scaling, and more. Mode - This menu option encompasses three camera mode options: “Color Quantize,” “Grey Scale,” and “Con- trast Preserving Decoloratization.” While sounding complex, they are rather simple. “CQ” allows you to select image bit values (most commonly used is 8 bit or 24 bit), “Grey Scale” allows you to convert the image from color to grey, and “CPD” does the same as grey scale, while preserving contrast for additional detail. Adjust - Much like Photoshop, the “Adjust” section encompasses a number of potential adjustments to your image, ranging from brightness/contrast to enhancing details, refining edges, and color replacement. Rotate - The “Rotate” section allows the user to turn the image angularly in preset degrees, or arbitrarily with an input value. Crop - The “Crop” tool is used to remove excess sections from the image outside of the region of interest. Shift + C is the keyboard shortcut for the “Crop” tool. Scale - The “Scale” function is used with a captured image to enlarge or reduce the digital resolution of the im- age while resampling the image. This does not add more detail (optical resolution), just more/less pixels. Histogram - The “Histogram” tool allows the user to view the different color and light channels for a previously captured image. Useful in attempting to recreate a comparison image to another sample. Resolution - The “Resolution” function opens the like named menu which allows a user to set a type of mea- surement per pixel, much like calibrating, but for a property of the image, instead of a recallable measurement at a given magnification. Tells another user when transferring the file what measurement scale was used. Mosaic - The “Mosaic” function is similar to the “Stitch” function, however, is used to process any open images into a single image--usually used to create side by side comparison images.

User’s Manual Top Navigation Bar Process Menu The “Process” menu contains a plethora of useful tools to create more deeply focused, larger viewable area, and better annotated images using digital enhancement procedures built right into the software. Stitch... - The Stitch tool allows users to take images taken at different points along the X and Y axes, and put them together into a single image. It is particularly useful when the field of view is not large enough to capture the entire region of interest in a single photo, allowing the images to be stitched together in a linear or grid format. Shift+T is the keyboard shortcut for this function. High Dynamic Range (HDR)... - The HDR tool allows users to capture multiple images at varying degrees of with a larger range between how dark and how bright an image can be in terms of contrast, creating a more deeply contrasted image. Extended Depth of Focus (EDF) - The EDF tool allows users to take a sequential series of photos from different focal points, and have the software digitally mesh them together, creating a more deeply focused image. This is useful when you need high magnification values, but, cannot get a large enough depth of field to have the whole area in focus at one time due to contrasting sample topographies. Shift+F is the keyboard shortcut for this tool. Note: This function is further explained in the “EDF” section of this manual. Deinterlace - The “Deinterlace” function allows a user to take an interlaced image (one which is processed in a series of rows, displaying a partial or blurred image until all rows have been processed) into a deinterlaced im- age (one which displays at the same time.)

User’s Manual Top Navigation Bar Process Menu (Continued) Color Composite - The “Color Composite” tool allows the user to select various color channels, and separate them into independently controlled elements. This allows a user to isolate specific colors of interest and elimi- nate others--particularly useful in fluorescent microscopy. This tool is very detailed, and will be covered later in the “Color Composite” section of this manual. Segmentation & Count - The “Segmentation & Count” function allows the user to use various histograms and methods to create segments of what one is interested in within an image, and exclude the rest, or to perform a count of objects of interest within the field of view. Denoise - The “Denoise” function allows the user to use several methods to remove noise from the image, cre- ating a cleaner and crisper image. This tool is covered in the “Denoise” section of this manual in greater detail. Sharpen - The “Sharpen” tool is a function that allows the user to command the software to create a sharper image than the original capture by combining an unsharp positive image mask with the image’s negative, ultimately providing a better image than before. This tool is covered in depth in the “Sharpen” section of the manual. Color Toning - The “Color Toning” function encompasses a number of tools used to calibrate the image based on the color representation of the monitor the image is being viewed with, as well as calibrating manipulation of the histogram for optimal results. “Color Toning” will be covered in depth in a later section. Filter - The “Filter” function contains a number of tools used to enhance the image based on a number of dif- ferent scenarios, such as using a high pass filter to enhance high-frequency information by adjusting contrast- ing pixels within a certain frequency. The filters here are complex but very powerful, and are covered in detail in the “Filters” section of the manual. Image Stacking- The “Image Stacking” function is used to take the average of multiple photos taken at the same point of reference, creating stronger photos with a greater signal to noise ratio (especially in low light mi- croscopy such as fluorescent and darkfield microscopy). “Image Stacking” will be covered in depth in its future section. Line Profile - The “Line Profile” function is used to create a graph illustrating the distribution of pixels along a line by color intensity. Surface Plot - The “Surface Plot” function works similarly to the “Line Profile” tool, however, instead of creat- ing a graph, it creates a 3D representation of the sample based on color intensity and color position of each pixel. “Surface Plot” will be covered in more detail in its own section. Pseudo Color - When a color is in grey scale, the “Pseudo Color” command can be used to color the image, useful in highlighting only specific details, or for temperature overlays.

User’s Manual Top Navigation Bar Process Menu (Continued) Range - The “Range” function allows the user to set a high and low limit for color intensity, allowing one to increase contrast, specifically in low light situations. The “Range” function will be covered in a later section. Binary - The “Binary” function sets a limit to the intensity of the grey pixel color, to where if the intensity is higher than the threshold, it is turned to white. This function is a key step in many other processes for ideal results, and will be covered in more detail in its own section.

User’s Manual Top Navigation Bar Layer Menu The “Layer” menu is used to create and manipulate various different levels within the image. Each layer can contain image data, measurement data, or other annotations, allowing a user to non-destructively create these notes on an image, and edit or delete one, without having to start over again, or take a new image if a change needs to be made. There is also a “Layers” sidebar that can be used for easier navigation. New... - The “New...” function creates a new layer in the current active image. Remove... - The “Remove...” command eliminates the currently active layer. Current... - The “Current...” command sets the selected layer as the active layer. Show/Hide... - The “Show/Hide...” command will either display or not display the currently active layer as a toggle. Rename... - The “Rename” command allows you to change the name of the layer for organizing data on mul- tiple layers easier. Export to Image - The “Export to Image” command exports the currently active image with all layers to an im- age file. Export to Microsoft Excel - The “Export to Image” command exports the currently active image with all layers to an Excel spreadsheet.

User’s Manual Top Navigation Bar Measurement Menu The “Measurement” menu contains the same tools as the top toolbar for drawing and measuring, with a few new features not found there, such as the “Scale Bar...” and “Z Order” features. The majority of the tools are covered in the top tool bar section, so in this section, we will only cover the “Ob- ject Select,” “Scale Bar...,” and the “Z Order” functions. Object Select - The “Object Select” tool allows the user to select a specific drawn or measured element in an image for viewing or manipulating with other tools. Scale Bar... - The “Scale Bar” function allows the user to designate a scale bar to be placed on an image, allow- ing quick reference of size when viewing a measured sample. Z Order... - The “Z Order...” function allows a user to move a drawn measurement up or down in the “Z” dimen- sion, which would be considered the layering order if the image was laid flat on a table, and the measurements were laid on top of it. A line can be placed on top of a circle, for example, to superimpose the image above the circle, or moved behind it.

User’s Manual Top Navigation Bar Options Menu The “Options” menu contains menus used to alter the way that the program behaves from the default settings, calibrate for measurement in real distances (instead of pixels), and other functions that have their own set of alterable options. Preferences - The “Preferences” menu brings up a menu containing settings that can be adjusted based on the core use of the software, such as default file locations, and languages. The keyboard shortcut for this menu is Shift+P. Measurements... - The “Measurements...” option opens the settings menu for measurements, allowing you to change parameters and behaviors of measurement tools. Magnification... - The “Magnification...” function allows you to edit magnification options inputted during the Calibration process, changing the list order, or importing previously exported settings. Calibrate - The “Calibrate...” function brings up the Calibrate menu, which allows a user to create various cali- brations for the software to translate pixels into real distance measurements at a given magnification value. Edit Dye List... - The “Edit Dye List...” function allows a user to create custom fluorescent dye parameters in the software, including extraction and emission wavelengths and dye names, useful when annotating fluorescent images. Auto Correction... - The “Auto Correction” menu brings up the “Auto Range” settings menu, which allows you to adjust how aggressively the limits on the image are when using auto correction functions, such as “Auto Level” and “Auto Contrast.”

User’s Manual Top Navigation Bar Window Menu The “Window” menu contains a few convenient but not often used features related to the viewing area’s tabs. Close All - The “Close All” function closes all open tabs simultaneously, preventing the user from having to close them individually. Reset Window Layout - The “Reset Window Layout” function will return the viewing area to it’s default set- tings upon a restart of the AmScope/ToupView software. Windows... - The “Windows...” function opens a menu allowing you to adjust or select an active tab as the main window, as well as saving a configuration of windows.

User’s Manual Top Navigation Bar Help Menu The “Help” menu contains the software’s manual as provided by the developer, as well as the “About” option. Help Contents - This menu options brings up this manual as a .pdf file within the software for review. About... - The “About...” function displays the current software version being used and developer information.

User’s Manual Process Menu Tools Stitch The “Stitch” tool is used when the user needs a larger field of view in a single image than the field of view the camera and microscope are capable of. This is done by “stitching” multiple captured images together at the edges, creating a single, larger image. This could be in a line or in a grid, depending on how many photos the user needs to capture, and how large of a field of view you ultimately need. 1. Begin by opening up the “Stitch” tool, which can be found under the “Process” menu, or opened with the keyboard shortcut “Shift+T.” 2. The new window that opens is the “Stitch” menu, which allows the user to capture an image, then move to the next area they’d like to capture, up to as many photos as needed to create the grid or line to cover the area needed. The “Snap” button will snap a shot of the area currently in view, and display it. You may use this in conjunction with the preview in the viewing area to line up the next snapshot to whichever direction you’d prefer. Continue until you have taken enough snapshots to cover the desired area of interest (in a grid or line). For best results, try to slightly overlap the image edges in whichever direction you are moving (horizontally or ver- tically). Press “Next” when ready to continue.

User’s Manual Process Menu Tools Stitch 3. The following menu allows you to designate a variety of settings to determine how the “Stitch” tool will be- have with the images you have taken. Panorama Straighten - This setting tells the “Stitch” tool which direction you are stitching in, either Horizontal, Vertical, or None. Choose None if performing a grid type operation, or choose the direction you are moving in. Sometimes it is easier to do multiple stitches if needing a large grid, by doing a horizontal line at a time, then stitching each element of a line together, then stitching all lines together. Projection Type - The “Projection Type” setting may seem confusing at first, but, it is both useful and simple. The “Projection Type” is a setting that will adjust the stitched image pattern to appear as if taken with a par- ticular type of a lens, by projecting the image over a specified image “map.” Options such as cylindrical, spheri- cal, fisheye, stereographic, plane, mercator, or transverse mercator can be selected here, while cylindrical is default. Seam Finder - The “Seam Finder” option allows you to set what method the “Stitch” tool uses to find seams to stitch the images together at. There are several options here as well, such as Voronai diagram, graph cut color, grapch cut color grad, and none. Graph cut color is the default option, and works for most samples. Exposure Compensation - The “Exposure Compensation” option is used to determine how the “Stitch” tool will handle differences in exposure between the various images to be stitched together. Options here include gain, blocks gain, or none. Default setting is blocks gain.

User’s Manual Process Menu Tools Stitch Bundle Adjust - The “Bundle Adjust” option is used to determine how the “Stitch” tool will compensate for nodal differences when capturing three dimensional images. Ray adjust and reprojection error are the avail- able options, with ray adjust as the default. When done, hit “Next” to continue to the next menu, or “Finish” if you want to immediately begin the stitching procedure. 4. This menu contains sliders to adjust parameters of the previously selected settings. Match Confidence - This slider adjusts the threshold used for creating seams and matching components between images to be stitched. The higher, the more precise, however, the more likely a failure to stitch error will occur, as this also requires greater precision when taking overlapping images. 65 is the default, and is the sweet spot between precision and leeway to create an image at all. Panorama Confidence - This slider adjusts the threshold match required to determine which direction to stitch the images together at. It is set to 100 by default since direction is defined in the previous menu, and recom- mended to leave at 100. Match Strength - The “Match Strength” slider determines how strict the requirement of pixel color and inten- sity must be before the software considers it a match. Moving it higher will result in more accurate stitching, provided the user takes photos with greater overlap in the same lighting conditions, otherwise it will fail. Blend Strength - The “Blend Strength” slider determines how much blending is used when aligning and stitch- ing images together. Raising this may cause blur, while lowering it may cause rough edges between stitches.

User’s Manual Process Menu Tools Stitch When you have finished adjusting these settings, click “Finish” to have the software engage the “Stitching” process and put the images together. 5. The software will then attempt to stitch together the images taken based on the settings provided. Note that if it fails to stitch, you may have to retake the photos, and/or lower the strictness of your settings to allow a greater margin of error to get a successful stitch.

User’s Manual Process Menu Tools High Dynamic Range “High Dynamic Range” is a post processing function that can fuse a sequence of multi-exposure images into a single image with a larger dynamic range (higher range between maximum and minimum contrast levels). The process requires the user to capture multiple images of the same point in their sample with different levels of exposure or different apertures on the microscope’s iris diaphragm for this to work. The HDR function can be used either in the browse window or the image viewing area window, provided the images are selected if in the browse window, or are open in the image viewing area. Both methods will provide the same result. 1. Much like the “Stitch” function, if you select the menu with photos captured in the viewing area, you will be taken to a menu where you can add images to a list of images for the function to use. Add any desired images taken at the same position with multiple types of exposure. Note: Unless multiple images are captured or selected in the browse mode, the HDR function will be grayed out. If unable to access the function, as shown above, please capture at least 2 images in the viewing area, or select at least 2 images in the browse menu.

User’s Manual Process Menu Tools High Dynamic Range 2. Once the images are added to the list, press “OK” to have the software begin processing of the images into a single image containing HDR.

User’s Manual Process Menu Tools Extended Depth of Focus (EDF) The “Extended Depth of Focus” function allows a user to capture multiple images from varying focal planes and assemble them into a single image with greater depth within focus than a single image. This is useful when viewing with high magnification, which has a limited depth of focus, or when capturing images of three dimen- sional figures (such as when doing circuit board work, or viewing minerals). Note: The EDF function can be used with previously captured images, or it can be used to capture images as part of the tool. We will display it as if capturing with the tool, however, the user has the choice to perform the image capture however desired. To use them from the browse menu, simply shift+left click all of the desired images to use in your stacked image, right click to bring up the context menu, and click the “EDF” tool from the list. 1. Clicking the “EDF...” tool will bring up the capture image option if no other images are currently captured in the viewing area. From here, move the focus of the microscope to the top of the range, so that it is focused on the topographical tallest plane of the sample you wish to focus on. In layman’s terms, focus it so that the tall- est items in your sample are within focus. Typically, this means moving the stage or microscope head (depend- ing on your microscope type) to the farthest point away from the sample while still focusing on a part of the sample.

User’s Manual Process Menu Tools Extended Depth of Focus (EDF) 2. Once there, click “Capture,” and move the focus a little closer to the next plane of focus desired, and repeat. Repeat this process until you have focused through the tallest plane to the shortest plane (the closest that your microscope stage or head is to the sample before losing focus entirely). Clicking “Clear” will remove all photos from the list, so please use it only when needing to restart from the beginning. Once finished capturing photos while adjusting focus, press “Next” to continue. Note: The more images captured in smaller increments of focus, the better the end image will be. Also, the reason we capture from tallest to shortest is because the images must be in sequence for the EDF tool to func- tion correctly. If the images are taken out of order, they will need to be reordered before the tool will work correctly. Out of order images will cause strange focusing artifacts to occur when rendering the final image.

User’s Manual Process Menu Tools Extended Depth of Focus (EDF) 3. The following menu allows the user to select the level of contrast used in the final image, and the rendering method for creating the final image. The software itself provides a fantastic detailed description of each func- tion and when to use it, which can be seen below. After selecting a method of rendering, please press “Next” to continue to the following menu.

User’s Manual Process Menu Tools Extended Depth of Focus (EDF) 4. Next, we have the “Options” menu, which has various sliders to adjust for final image filtering and reproduc- tion. Detail Clarity - The higher this setting, the sharper the fine details will be in the field. Too much will look un- natural, however. Total Brightness - This affects brightness through the whole field including the sample. Use if your exposure was dark during capture to compensate, or if your exposure was too bright. Background Brightness - This affects brightness only in the white or dark background of your image, detected based on contrast between color intensity and lack thereof for the background. Adjust this if your exposure’s background was too bright or too dark, but your sample itself was correctly illuminated. The software, too, provides a good description of each in the menu, as seen below. When ready, click “Next” to continue to the final menu.

User’s Manual Process Menu Tools Extended Depth of Focus (EDF) 5. The final menu for the EDF process is the alignment options menu. Since there can be small variations in X- and Y- dimensions even when moving only the focusing knob (based on how a rack and pinion system is machined when moving forcus can cause variations), the auto alignment process attempts to correct for these variations to avoid a blurred image, or a misaligned image when stacking the images together. As before, the menu has several notes as to this menu’s purpose and function, shown below. Setting it to “None” will assume that the images are perfectly align and will not perform correction processes. Setting it to “Shift + Scale” will attempt to translate directionally as well as increase/decrease in size for best alignment. Setting it to “Shift + Scale + Rotate” will do the same as above, however, will also rotate the image if it was rotationally skewed during focus translation of the stage. When finished, press “Finish” to begin the software processing of the image stack.

User’s Manual Process Menu Tools Extended Depth of Focus (EDF) 6. The software will process your image, and output an image that is deeply focused, providing a greater depth of focus.

User’s Manual Process Menu Tools Deinterlace The “Deinterlace” function allows a user to take an interlaced image (one which is processed in a series of rows, displaying a partial or blurred image until all rows have been processed) into a deinterlaced image (one which displays at the same time. Interlacing is used to allow incremenetal loading of large resolution images on slower connections, at the expense of knowing when the image is done loading (such as with a progressive scan image, which will enlarge row by row until the full image is seen). While the AmScope/ToupView software and cameras by default captured deinterlaced images, if a user wished to use our software to deinterlace ain image sent to them, this function can be performed here. The major benefit of a deinterlaced image is that it is processed row by row when displaying, and will enlarge until the full image is seen (easier to tell when it is fully loaded), and they are better to compress when using on the web or sending to other clients. When an image is open, deinterlacing is performed by clicking the tool under the “Process” menu. It will be grayed out if no image is available to deinterlace. There are no adjustment menus to this process--deinterlac- ing will be performed as soon as the tool is clicked.

User’s Manual Process Menu Tools Color Composite The “Color Composite” function is extremely useful for users wishing to highlight specific colors of their sample only, or, to view only specific color channels (such as in fluorescent light microscopy). Ultimately, it is a tool used for coloring and separating color channels in an image for highlighting areas of interest. 1. To use this function, a user will need to have captured an image in gray scale in the viewing area. This can be performed by capturing an image, then going to “Image” and “Mode,” then selecting “Gray Scale.” 2. Once the image is in gray scale, the user can then select “Color Composite” under the “Process” menu to bring up the first menu of the tool.

User’s Manual Process Menu Tools Color Composite 3. This menu contains the core functionality of the “Color Composite” tool, with various options to adjust, as well as a place to add source images to work with. To begin, select the name of the source image to be used, and click “Add.” This will add the image to the color composite list, and bring up a new dialog menu asking which color this composite is to be. Pick which color you wish to work with either from the slider, the palette, or the list of dyes/stains in the bot- tom drop down menu, and hit “OK” when complete.

User’s Manual Process Menu Tools Color Composite 4. Once the color is selected, the image will change with the selected color, and the slider options to the right will be enabled for adjustment. They are, left to right, positional adjustment (aligning multiple images is criti- cal when you are coloring the gray images of the same image in for different color channels), brightness (B), contrast (C), and gamma (G). Background allows an image to be set as a non mixed image, so not affected by settings selected here, allowing, for example, a red dot on a blue background won’t turn the dot magenta. The user may continue to add images, change colors, and turn layers on/off by clicking the color box next to the image name in the composite list. When finished, close the box, and the newly composited image will be displayed as its own new image file. To discard and start fresh, click the “New” button. Because of the flexible nature of the tool, experimentation by each user is necessary to provide best results for your stain/sample.

User’s Manual Process Menu Tools Segmentation & Count The “Segmentation & Count” tool set is a very powerful set of tools in that it allows a user to create segments within an image, as well as use segmented areas to perform counting procedures. Because of the complexity and flexibility of this tool, there are a variety of different methods used to perform these functions. The methods available to segment an image are shown in the “Segmentation & Count” menu expansion when hovering over the menu option under “Process.” Only one method can be used per image.

User’s Manual Process Menu Tools Segmentation & Count Watershed... - This segmentation method is a mathematical morphology segmentation based on topological theory. This is done by taking the gray value of each pixel to indicate the altitude, and the boundaries of each local minimum value, and its impact area. This can be visualized by thinking of a flat plane with objects of vary- ing height on it, and holes poked in the bottom, then immersed in a liquid. The liquid fills starting at the lowest point where a hole is, and fills up the basins between taller objects, where only the tallest show, like an island in an ocean. It is best for simple background objects with a large contrast between the background and the items of interest. Approximation - Assigns whether the segments will be estimated as circles, ellipses, or none (the actual shape and size of the segment). With Holes - This tick box enables the segmentation method to create holes within a segment for areas that do not fit the segment. Outline Style - Assigns whether the segments will be colored in, outlined, or none (simply numbered). Outline Color - Assigns which color the segments or outlines will be colored in. Label Method - Assigns how the segments will be labeled. XOR contrasts the numbers against the background, true allows a color to be selected for all, and false eliminates numbering the sections entirely. Label Color - When true is selected as the “Label Method,” will allow selection of the color to use for the num- bering. Label Type - When true is selected as the “Label Method,” allows selection between displaying counted seg- ments, the area of each segment, or the perimeter of each segment.

User’s Manual Process Menu Tools Segmentation & Count Area (px) (Minimum & Maximum) - Allows the user to create an upper and lower limit for what the software considers a segment. Anything below the minimum or above the maximum value in area will be excluded from being a segment (in pixels). Perimeter (px) (Minimum & Maximum) - Allows the user to create an upper and lower limit for what the soft- ware considers a segment. Anything below the minimum or above the maximum value in area will be excluded from being a segment (in pixels).

User’s Manual Process Menu Tools Segmentation & Count OTSU Dark... - OTSU is a method to determine the threshold of an adaptive based on the gradation charac- teristic of an image, then divide the image into the background and the targets of interest. The larger the difference between the background and the target, the large a difference is displayed between the two, while the smaller differences will be less displayed. Specifically, the dark method means the segment from the dark objects from the bright objects are determined using an OTSU algorithm, and is best used with a dark target object with a bright background. This method is best when you need accuracy, as it minimizes possibility for misclassification and missegmentation. Approximation - Assigns whether the segments will be estimated as circles, ellipses, or none (the actual shape and size of the segment). With Holes - This tick box enables the segmentation method to create holes within a segment for areas that do not fit the segment. Outline Style - Assigns whether the segments will be colored in, outlined, or none (simply numbered). Outline Color - Assigns which color the segments or outlines will be colored in. Label Method - Assigns how the segments will be labeled. XOR contrasts the numbers against the background, true allows a color to be selected for all, and false eliminates numbering the sections entirely. Label Color - When true is selected as the “Label Method,” will allow selection of the color to use for the num- bering. Label Type - When true is selected as the “Label Method,” allows selection between displaying counted seg- ments, the area of each segment, or the perimeter of each segment.

User’s Manual Process Menu Tools Segmentation & Count Area (px) (Minimum & Maximum) - Allows the user to create an upper and lower limit for what the software considers a segment. Anything below the minimum or above the maximum value in area will be excluded from being a segment (in pixels). Perimeter (px) (Minimum & Maximum) - Allows the user to create an upper and lower limit for what the soft- ware considers a segment. Anything below the minimum or above the maximum value in area will be excluded from being a segment (in pixels).

User’s Manual Process Menu Tools Segmentation & Count OTSU Bright... - The bright variant of OTSU is used to segment bright targets from dark backgrounds, such as in darkfield. Otherwise, it behaves the same as “OTSU Dark.” Approximation - Assigns whether the segments will be estimated as circles, ellipses, or none (the actual shape and size of the segment). With Holes - This tick box enables the segmentation method to create holes within a segment for areas that do not fit the segment. Outline Style - Assigns whether the segments will be colored in, outlined, or none (simply numbered). Outline Color - Assigns which color the segments or outlines will be colored in. Label Method - Assigns how the segments will be labeled. XOR contrasts the numbers against the background, true allows a color to be selected for all, and false eliminates numbering the sections entirely. Label Color - When true is selected as the “Label Method,” will allow selection of the color to use for the num- bering. Label Type - When true is selected as the “Label Method,” allows selection between displaying counted seg- ments, the area of each segment, or the perimeter of each segment. Area (px) (Minimum & Maximum) - Allows the user to create an upper and lower limit for what the software considers a segment. Anything below the minimum or above the maximum value in area will be excluded from being a segment (in pixels).

User’s Manual Process Menu Tools Segmentation & Count Perimeter (px) (Minimum & Maximum) - Allows the user to create an upper and lower limit for what the soft- ware considers a segment. Anything below the minimum or above the maximum value in area will be excluded from being a segment (in pixels).

User’s Manual Process Menu Tools Segmentation & Count RGB Histogram... - This method segments a sample based on the image histogram, where pixels between a specific color and intensity range can be segmented away from the rest, or processed into another color if within or outside of the color range designated by the user. Limits can either be dragged to be set, or specific values used through user input. The color method used here is the RGB (red, green, blue) style. The numerical fields in the lower left corner of the dialog box when using the RGB Histogram method are the minimum and maximum values of that color to filter out or include. The user can either input precise values in there, or simply drag the limit bars along the histogram while viewing the results on the image itself. The drop down menu next to it allows the user to select between red, green, and blue channels, each with their own minimum/maximum values. The “Show All” tick box toggles between displaying a single channel and all channels in the histogram. The drop down menu to the right allows the user to select what color the segments will be within the image. The options menu allows the user to adjust the count parameters as shown in the previous two sections. When complete, pressing “OK” will render the segmented image as shown below on the following page.

User’s Manual Process Menu Tools Segmentation & Count HSV Histogram... - The “HSV Histogram” segmentation method is the same as above, however, uses HSV (hue, saturation, and value) and style color values instead of RGB. The numerical fields in the lower left corner of the dialog box when using the HSV Histogram method are the minimum and maximum values of that variable to filter out or include. The user can either input precise values in there, or simply drag the limit bars along the histogram while viewing the results on the image itself. The drop down menu next to it allows the user to select between hue, saturation, and value channels, each with their own minimum/maximum values. The “Show All” tick box toggles between displaying a single channel and all channels in the histogram. The drop down menu to the right allows the user to select what color the segments will be within the image. The options menu allows the user to adjust the count parameters as shown in the previous two sections. When complete, pressing “OK” will render the segmented image as shown below on the following page.

User’s Manual Process Menu Tools Segmentation & Count Color Cube... - This segmentation method may prove to be the most complex and the most user friendly simul- taneously, as it allows a user to select specific pixel colors to be segmented out using a color pipette tool, and a +/- tolerance setting. While flexible, it does take a good deal of time and patience to manually select the color pixels the user wishes to work with. The + and _ icons with the pipette allow the user to add pixel colors or remove pixel colors from the segmenta- tion divisions. The drop down menu next to it selects how large the circle of pixels is selected by the pipette, with the average of the area selected being the actual color added to the segmentation. The tolerance section allows the user to create a margin of error used when selecting a color. A greater toler- ance will allow more variance in color of the pixels selected based on the color clicked, while less will mean only precise colors near what was picked will be segmented. The “Options” section will bring up the counting parameters as in the above sections. When finished clicking pixels to select your segmentation area, click “OK” to render the final image, as seen in the following page.

User’s Manual Process Menu Tools Segmentation & Count Split Objects - “Split Objects” is a tool to be used, rather than a segmentation method. If using a segmentation method results in a segment adhering to another segment (as in, they show up as a single segment when they should be two unique segments), the object select tool can be used to manually select a segment, and split them manually into two segments. Note that this will require a bit of drawing using the mouse, so, accuracy is dependant on the user and how steady their hand is for mouse manipulation. Count Result... - “Count Result...” is another tool used to display statistics of the counted data from the above methods. Some statistics it can cover are index, center, radius, area, and perimeter of the counted items within the field of view.

User’s Manual Process Menu Tools Denoise The “Denoise” function provides a method for a user to do just that--remove “noise” from an image. Noise, in terms of digital photography, is considered random variation of brightness or color information in digital images, usually caused by electronic device interference from the sensor or circuitry of the device itself. It ap- pears as extra information in the form of pixel colors that were not present in the object imaged. The “Denoise” menu contains three different methods of identifying and removing noise from an image. Adaptive Wiener Filter - This method preserves more detail than those of the median, mean, and Gauss style filters. This is the best method to select to remove Poisson and Gaussian noise (additive and multiplicative style noises). Noise levels are automatically calculated since they are difficult to estimate and precisely enter. This is also the most hands off method of noise removal. Once this method is selected, it automatically takes effect--no dialog menus will appear. Bilateral Filter - This method considers spatial proximity, pixel value similarity, image spatial information, and gray levels to preserve edges while performing the denoising process. If your sample has many edges that are critical to maintain for clarity, this is the ideal method to use.

User’s Manual Process Menu Tools Denoise The “Value Domain” slider refers to the range that the software uses to analyze the neighboring pixels to an area suspected of noise. The higher this is, the longer the image will take to process, as it averages a larger area per pixel. Too large may cause strange artifacting, so it’s best to keep it near the mid point of the slider and adjust in small increments as needed. The “Space Domain” slider refers to how strong the denoising effect will be. The higher this value, the more aggressively the software will detect rogue pixels as noise. Best kept to the lower end of the spectrum as too high, and many items in your image that are as pictured correctly may be averaged out, losing out on image quality. Non Local Means - This method uses a weighted average of pixels over a given area to extrapolate what is noise and what is not over a separate area not adjacent to the search area. This allows a user to denoise more accurately than only denoising over a local area via the adjacent pixels, and provides greater flexibility in repro- ducing accurate to object images. The “Strength” slider refers to the aggressiveness of the software’s noise removal. The higher this is, the more noise will be removed, however too high and detail will be lost beyond jut noise. The “Template Window” slider refers to the template block size, and is used to calculate the weight at which the pixels will be averaged when a noisy pixel is detected based on the template area. The “Search Window” slider is used to calculate how large of an area will be averaged when a noisy pixel is found in the target area for denoising. When finished adjusting the sliders, press the “OK” button to engage the software’s denoising process. The resulting image will be displayed once complete.

User’s Manual Process Menu Tools Sharpen The “Sharpen” function attempts to create a crisper and cleaner image with more defined lines, and is useful for when an image is just slightly out of focus and would benefit from a small adjustment. Note that sharpen does not clear up out of focus images, so it’s always better to take a correctly focused image (or use EDF if needing greater depth of focus) before looking to use the “Sharpen” function. The “Sharpen” function has two different methods. UnSharp Mark... - Contrary to it’s odd sounding name, the “UnSharp Mask...” method is actually a sharpen- ing method. The name comes from the process it uses to create a sharper image by taking an unsharp positive image with a negative of the original image and merging the two in order to create additional contrast in the positive image. The “Radius” slider refers to the size of the edges to be enhanced. Larger settings tend to cause halos around edges, so, use sparingly to ensure fine details are enhanced without ghosting. Also, threshold and radius are related in effect, and thus, lowering one will allow the other to be increased if needed.