Nikon A1+ Standard Operating Procedure

A1 controller Laser on/off key Laser buttons (2) Turning on/off the system 1. Turn on computer 2. Power on A1 controller and laser power supply. 3. Turn the key and select the needed lasers by pressing the buttons. 4. Turn on stage power supply and DIA lamp if bright field is needed 5. Power on microscope, wait until startup sequence is finished. 6. Log in with your LBIC account and start NIS-Elements AR 7. Turn off system in reverse order 4

1. Put your sample in the appropriate holder, if you have slides make sure the coverslip is facing down. 2. Start with either 10x or 20x objective 3. Navigate to a suitable place in your sample with the joystick. 4. Press “Eye port” to change the light path to the eye piece, if needed press the Sola button (on Sola pad) to activate the LED lights. 5. Use the coarse focusing mode and find focus (for slides around Z= 2100-2200, for 30mm petri dishes around Z=2500-2700) 6. Press “Eye port” again to switch to confocal mode (the first time the system is started do this twice, this is a bug in the software). 2 4 4

1. Setup your channel requirements 2. HV (Gain), start at 100 for 405/640 and 25 for 488/561 PMTs 3. Offset adjustment, leave at 0 4. Laser Power, start at 2% 5. Start with a scan size of 512x512 and scan speed of 2.2 µsec. 6. Select “Normal” and “Ch Series” if using more than one laser 7. Select 1.2 AU for Pinhole 8. Start “SCAN” 9. Adjust detector and lasers settings if needed 10. Change pixel size by manually changing zoom factor in A1+ Scan Area” dialog (View>Acquisition Controls>A1 Scan Area) or pressing Nyquist for optimal pixel size for the selected objective and pinhole size. 11. Quickly Capture an image with the current settings. Image must be saved manually afterwards. Save as .nd2. *If you need to go back to the eyepieces to check your slides, click “Eye Port”, click it again to return to the confocal! 2-4 1 5 6 7 8 10 11

Very useful for setting up advanced imaging protocols 1. Enter file save path (O:\ is recommended) 2. Combine function tabs as needed by clicking the boxes • Time is for time lapse videos • XY saves coordinates (Z can be included with the include Z box) • XY points carry over with objective change (as long as the sample is not moved in the holder • Large image captures multiple fields of view and stitches them to one image • Very useful for creating maps of your sample with lower resolution (10x or 20x) that can be used to find points of interest and adding to XY (right click on the point and select ”add this point to ND acquisition”. • needs at least one XY point to work (this point will be the center point of the large image). • Z is used for capturing Z-stacks • Can be setup by either manually selecting top and bottom Z-values or in relative mode where the center Z-value is taken from XY (include Z needs to be selected). The latter is used when you want to capture Z-stacks in more than 1 XY point. 3. Contextual GUI for each function tab *XY points can be added also when viewing in the eye port. Useful for quickly adding points of interest before starting scanning. 1 2 3

(10) 1 2 The confocal sample holders: 1. Multi-holder (slides and small petri dishes) 2. Multiwell plate holder (for all standard multiwell plates) 3. Oko-labs stage top incubator (see next page) with fittings for two 35mm petri dishes and one 75mm x 25mm slide (standard slides and chamber slides) Exchanging between them is simple 1. Remove plastic screws with screwdriver located on top of shelf. 2. Remove holder and put on shelf. 3. Change to the desired sample holder (they only fit one way) 4. Fasten plastic screws gently. DO NOT OVER TIGHTEN! The screws break quite easily. 5. Put back screwdriver on shelf.

(11) C02 tube for incubator Using the stage top incubator 1. Please ask Sebastian the first time you want to use the stage top incubator/holder for more in- depth instructions 2. Open C02 valve on tube 3. Turn on incubator 4. Change to the incubator sample holder (see previous page for instructions) 5. Wait until the temperature and C02 levels reach the desired values (see display). *If you want to use the 60x objective with the objective heater you need to connect the loose connector from the objective with the matching connector from the incubator controller. 1 2 3 4

• Check that the Sola is activated • Focus is wrong • Start at around Z: 2000 and go up slowly • Sample upside down? • Always put the sample with the coverslip facing the objective • Black image when scanning? • Are the lasers on? • Check the laser box and key and start the lasers that are needed. • Remember to press the “Remove interlock” • Lasers can be turned on/off in NIS On Off • Lightpath is set to “Eye port” • Press “eye port” to switch to confocal mode (remember to do this twice when you have first started the system.) • Did you select the correct channels? • Click the settings button to set up the channels you need (13)

• As a shortcut these can also be found by right clicking in the back open space of elements • NIS-Elements obeys most Windows-based functions, shift/control and Alt are used often; right-clicks are used to display options/properties • NIS-Elements uses “drag and drop” for overlaying windows/datasets • ND2 Files can be opened/viewed outside of NIS-Elements with a free download on: https://www.nikoninstruments.com/Products/Software/NIS- Elements-Advanced-Research/NIS-Elements-Viewer • There is also a plugin (Bio-formats) for ImageJ that enables reading of most image formats. • https://docs.openmicroscopy.org/bio-formats/5.8.2/users/imagej/index.html • You can also download Fiji (ImageJ with a lot of pre-installed plugins, including Bio-formats) from here: • https://fiji.sc/ • The easiest way to export images to .tiff files is by doing it through the Edit -> Create montage (8bit RGB) function and creating your desired montage. Remember that you lose bit depth so any form of analysis must not be done on the resulting files. Always keep a safe copy of the raw image files (.nd2) and save as new .nd2 files when you modify the original data, for example removing background or cropping. (14) Tips and tricks

• Create Optical configurations (OC) to save clicks • When creating new OC, uncheck the objective box in the bottom left margin to avoid unwanted objective changes • Always use the Pixel Saturation indication tool to avoid saturation • The help section is a good resource to search and covers almost everything in NIS-elements • Ask Sebastian (0731536005) if you get stuck. (15) Tips and tricks cont.