Leica DMI3000B Instructions Manual

transmitted Light Lamps: 107 or 107/2 Lamp Housing........................ 45 6.11 Installing the Lamp Housing Mount and Mirror Housing) ................................... 46 6.12 Installation and Replacement of Incident Light Lamps .................................. 48 6.13 Equipping the Incident Light Turret Disk .................................................... 52 6.14 Inserting the Front Module Slider ............ 55 6.15 Installation of the Polarizer and Analyzer................................................ 55 6.16 Optional Accessories................................. 57 6.17 Connection to the Electronics Box .......... 58 6.18 Connection to the Computer ..................... 59 6.19 Connection to the Power Supply ............. 59 7. Start-up ........................................................ 60 7.1 Functional Principle .................................. 60 7.2 Switching on the Microscope .................. 64 7.3 The LeicaDisplay ........................................ 65 7.4 The Function Buttons on the Stand ......... 66 7.5 The SmartMove Remote Control Module ............................ 69 7.6 Illumination .................................................. 69 7.6.1 Transmitted light .............................. 69 7.6.2 Incident Light - Fluorescence ........ 73 7.7 Checking Phase Contrast Rings ............... 74 7.8 Checking modulation contrast slit diaphragms............................................ 77 7.9 Setting the Motorized Polarizer ............... 77 7.10 Adjusting the Light Sources ..................... 78 Contents 1. Important Notes about this Manual ........ 7 2. Intended Purpose of the Microscope ..... 8 3. Safety Notes ................................................ 9 3.1 General Safety Notes ................................ 9 3.2 Electrical Safety.......................................... 10 3.3 Safety Instructions for Handling the Light Sources ................ 12 3.4 Notes on handling laser devices ............. 12 3.5 Safety Instructions for Handling Acids and Bases .................. 12 3.6 Disposal........................................................ 13 4. Overview of the Leica DMI Series .......... 14 5. Unpacking the Microscope ..................... 27 6. Assembling the Microscope .................... 30 6.1 Assembly Tools ........................................... 30 6.2 Installation of the Transmitted Light Illumination Carrier (TL) ............................. 31 6.3 Installation of the DIC Module and DIC Objective Prisms ......................... 32 6.4 Installation of Specimen Stages .............. 33 6.5 Installation of Condensers ........................ 38 6.6 Installation of Eyepieces ........................... 43 6.7 Installation of Objectives .......................... 43 6.8 Installation of Filters in the Illumination Arm............................... 44 6.9 Installing the transmitted Light Lamp Housing ............................................. 44

8.1 Switching on................................................ 81 8.2 Contrast Methods ....................................... 83 8.2.1 Bright Field (TL) ................................ 83 8.2.2 Phase Contrast (TL) ....................... 85 8.2.3 Dark Field (TL) .................................. 86 8.2.4 Polarization (TL) ............................... 87 8.2.5 Differential Interference Contrast (TL) ............. 88 8.2.6 Integrated Phase Contrast (TL) ..... 89 8.2.7 Integrated Modulation Contrast (TL) ................ 90 8.3 Fluorescence............................................... 91 8.4 Combination Methods ............................... 93 8.5 Focusing ....................................................... 94 8.6 Tubes ............................................................ 96 8.7 Port selection ............................................. 96 8.8 Eyepieces..................................................... 97 8.9 Objectives .................................................... 98 8.10 Stages and Object Displacement ............ 101 8.11 Magnifi cation Changer .............................. 102 8.12 Light sources ............................................... 103 8.13 Aperture and Field Diaphragm ................ 104 9. Troubleshooting .......................................... 105 10. Care of the Microscope ............................ 109 10.1 Dust Cover ................................................... 109 10.2 Cleaning ....................................................... 109 10.3 Handling Acids and Bases ........................ 110 11. Major Consumable and Replacement Parts ............................. 111 12. Dimensions.................................................. 112 13. Abbreviations and Pictograms ................ 113 14. Index ............................................................ 115 15. EU Declaration of Conformity .................. 117

Fig. 1 Left side, Leica DMI4000 B and DMI6000 B 1 Eyepiece 2 Eyepiece tube 3 Top port 4 Intermediate pupil interface 5 LeicaScreen 6 Light intensity 7 Field diaphragm 8 TL/IL switching 9 Aperture diaphragm 10 Focus wheel (motorized Leica DMI6000 B, manual (fi ne and coarse) Leica DMI4000 B) 11 Variable function buttons 12 Left side port 13 Booster lens (Leica DMI6000 B fl uorescence microscopes only) 14 Lamp mount (fl uorescence microscopes only) 15 Condenser head 16 Condenser base 17 Field diaphragm 18 Transmitted light lamp housing 19 DIC objective prism disk 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 17 16 18 19

Fig. 2 Right side Leica DMI4000 B and DMI6000 B 1 E-Focus buttons (Leica DMI6000 B only) 2 Focus wheel (motorized Leica DMI6000 B, manual (fi ne) Leica DMI4000 B) 3 Variable function buttons 4 Opener for drawer (fl uorescence microscopes only) 5 Drawer (fl uorescence microscopes only) 6 Right side port 7 Analyzer slot 8 Centering window (fl uorescence microscopes only) 9 Field diaphragm centering (fl uorescence microscopes only) 10 Incident light lamp housing (fl uorescence microscopes only) 11 Objective turret 12 Stage with attachable mechanical stage 1 2 3 4 5 6 7 8 12 9 10 11

Fig. 3 Front view Leica DMI4000 B and Leica DMI6000 B 1 LeicaScreen 2 Front control panel 3 Port switching 4 Top port 5 Manual transmitted light fi lters 6 Bertrand lens centering 1 2 3 4 6 5

Fig. 4 General view Leica DMI4000 B and Leica DMI6000 B with SmartMove remote control module Fig. 3b SmartMove remote control module 1 Travel in x 2 Travel in y 3 Focus 4 Variable function buttons (pre assigned at factory) 1 2 3 4 Fig. 3a Front control panel 1 Fluorescence cube 2 Shutter 3 100% light to all eyepieces 4 Port selection 5 Magnifi cation selection 6 1x tube lens 1 1 2 5 4 3

1 . Intelligent Automation • Switching between contrast methods at the touch of a button. Light rings, DIC prisms, etc. are automatically positioned in the beam path. • The microscope recognizes the selected objective and associated contrast method. The intensity (INT), aperture diaphragm (AP) and fi eld diaphragm (FD) are always set to suit- able values. • The INT, AP and FD values are always based on the currently activated illumination axis (transmitted light or incident light). • The INT, AP, and FD values can be adjusted individually. Manual adjustments overwrite the previous settings. The current setting is stored and is retained from one session to the next when power is switched off. 2. Controls • SmartMove knobs for stage and focus control • Fixed function buttons on stand for INT, AP, and FD, as well as for switching between transmitted light and incident light axis • Variable function buttons on stand, SmartMove, STP6000 These function buttons have functions suitable to the confi guration of your microscope as- signed to them at the factory. The functions can be reprogrammed and/or adapted to your specifi c requirements, however. • Complete control of microscope and camera via software (Leica Application Suite (LAS/LAS AF)) Thanks to its intelligent automation, the Leica DMI4000 B and DMI6000 B can be controlled using a variety of control elements.

Function Function Function Rotary and buttons buttons buttons knobs STP6000 Stand Stand 4000 6000 4000 6000 4000 6000 4000 6000 4000/6000 Select contrast method - - + + + + - - + Change transmitted light/incident light axis + + - - - - - - + Change to objective - - - + - + - - + Teach-in parfocality - - - - - - - - + Change operating mode (dry/imm) - - + + + + - - + Illumination Manager + + + + + + - - + Magnifi cation changer (motorized) + + - - - - - - + Focusing - + - - - - - +1) + Set stops - + - - - - - - + Go to stop - + - - - - - - + Change step increment (coarse/fi ne) - - - + - + - - + XY stage positioning - - - + + Change speed - - - - - - - - + Stage positions (store/go to) - - - - - - - - + Change to fi lter/refl ector cube + + (+) + + - - + Side and bottom port (DMI6000 B only) + (+) + - + DIC fi ne adjustment + + - - - - - - + Adapted Focus Control (AFC) - + - + - + - - + + always possible (+) optional - not possible 1) Focusing alternatively via wheels

For Leica DMI4000 B and Leica DMI6000 B: Function button Function BF Bright fi eld transmitted light PH Phase contrast transmitted light ICT Interference contrast, transmitted light DF Dark fi eld transmitted light IMC Integrated modulation contrast POL Polarization transmitted light CHANGE TL Cycle through all contrast methods INT ↑ Increase intensity (transmitted light) INT ↓ Reduce intensity (transmitted light) AP ↑ Open aperture diaphragm (transmitted light) AP ↓ Close aperture diaphragm (transmitted light) FD ↑ Open fi eld diaphragm (transmitted light) FD ↓ Close fi eld diaphragm (transmitted light) SHUTTER TL Open/close TL shutter TL FLT 1 Enable/disable transmitted light fi lter at position 1 TL FLT 2 Enable/disable transmitted light fi lter at position 2 FLUO Fluorescence (last fi lter cube) CUBE 1-6 Select fi lter cube in position 1-6 CHANGE CUBE CW Change cube clockwise (1 → 4) CHANGE CUBE CCW Change cube counterclockwise (4 → 1) INT FLUO ↑ Increase intensity (fl uorescence) INT FLUO ↓ Reduce intensity (fl uorescence) FD FLUO ↑ Open fi eld diaphragm (fl uorescence) FD FLUO ↓ Close fi eld diaphragm (fl uorescence) CHG FW Toggle fi lter functions IFW Activate external fi lter wheel ExMan Activate Excitation Manager SHUTTER FL Open/close fl uoshutter COMBI Combination method (PH fl uorescence or ICT fl uorescence) CHANGE COMBI Cycle through all combination methods CHANGE OBJ CW Cycle through objectives clockwise CHANGE OBJ CCW Cycle through objectives counterclockwise Z FINE Activate fi ne focusing (Leica DMI6000 B only) Z COARSE Activate coarse focusing (Leica DMI6000 B only) XY PRECISE Activate precise stage XY FAST Activate fast stage BTP ON/OFF Bottom port on/off (Leica DMI6000 B only) DRY/IMM Switch dry/immersion CHANGE FLT Switch TL fi lter CHANGE CS Switch to confocal application OBJ 1-6 Select objective at position 1-6 MEM 1-6 Memory activated stored functions AFC ON/OFF Turns AFC on or off AFC HOLD Holds current position

the LeicaScreen will display an error message. See Troubleshooting chapter, → p. 105. Components such as diaphragms, condensers, light, and phase rings have been pre-centered at the factory. It may be necessary to correct the centering after the microscope has been trans- ported and assembled. Before performing the required steps, please fa- miliarize yourself with the LeicaScreen and the controls. Caution! After turning on the gas discharge lamp, the burner must be immediately adjusted. There- fore, do not turn on the power supply unit yet. First, work in transmitted light in order to familiarize yourself with the microscope’s controls. Fig. 81 LeicaScreen Initialization 7.2 Switching on the Microscope Leica DMI3000 B: • Switch on the microscope’s power at the On/ Off switch. The signal lamp is lit when the in- strument is ready. (For the Leica DMI3000 B please continue at 7.4. Function Buttons on the Stand) Leica DMI4000 B and Leica DMI6000 B: • Switch on the power of the electronics box at the On/Off switch (80.1). The signal lamp (80.2) is lit green when the unit is ready. All motor ized microscope components will then run through an initialization phase. Note: If a PC is connected, switch on the electronics box fi rst, and then the computer. After the initialization (Fig. 81) is complete, the LeicaScreen will display the microscope’s cur- rent settings (Fig. 82). Fig. 80 front side Leica CTR6000 1 On/Off switch 2 Signal lamp 1 2 Fig. 82 LeicaScreen after Initialization

(Leica DMI 4000 B and DMI 6000 B) The screen displays the microscope’s current settings. The content of the display depends on the features of the individual microscope. For information on the abbreviations used, please turn to the table of abbreviations → p. 113. The screen has a number of areas and lines. Line 1: contrast method Line 2: objective/ magnifi cation Line 3: illumination/ diaphragms Line 4: active ports Line 5: focus/ stops (DMI 6000 B only) The content of the display changes according to the active function. Contrast method Objective/ Magnifi cation Focus/stops (DMI6000 B only) Ports/Eyepiece Illumination Diaphragm Fig. 83 Arrangement of the function buttons – overview 1 Four variable function buttons 2 Illumination Manager 3 Front control panel 4 Focus buttons (DMI6000 B only) 5 Three variable function buttons 6 SmartMove knobs 7 SmartMove function buttons 5 4 3 3 2 1 6 7 7 6 Pictograms

and fi eld diaphragm settings are stored for the in- dividual objectives and contrast methods. Fig. 84 Fixed function buttons (left side of stand) 1 Toggle transmitted light/incident light 2 Aperture diaphragm 3 light intensity 4 fi eld diaphragm 4 1 3 2 7.4 The Function Buttons on the Stand Leica DMI3000 B: • Focus wheels: the left-hand focus wheels can be used for both coarse and fi ne focusing; the right-hand focus wheel for fi ne focusing only (a version of the Leica DMI3000 B with mir- rored focus controls is also available) • Light intensity: the transmitted light intensity can be adjusted continuously from 0 to 12 V using the potentiometer at the lower left of the front of the microscope stand. For the Leica DMI3000 B please continue at 7.6. Illumination. Leica DMI4000 B and Leica DMI6000 B: A number of function buttons are located on both sides of the stand. These can be broken down into fi xed and variable buttons. The variable func- tion buttons have different functions depending on the features of the individual microscope. Fixed function Buttons on the Left Side The TL/IL button (84.1) toggles between the inci- dent-light and transmitted light axis. The contrast method last used with a given axis is restored when switching. The INT buttons (84.3) adjust the light intensity. The adjustment can be made in coarse or fi ne steps. Pressing both INT buttons at the same time toggles between coarse and fi ne adjustment. The AP buttons (84.2) for the aperture diaphragm and FD (84.4) for the fi eld diaphragm open and close their respective diaphragms. The motorized DMI stands can be controlled using the function buttons on the stand, the re- mote control SmartMove or the STP6000.

The variable function buttons are assigned func- tions at the factory that are appropriate to the features of your microscope. They are labeled accordingly. For details on button assignments, please refer to the included identifi cation sheet. For information on the abbreviations used, please refer to the list → p. 63. Note: The Leica Application Suite (LAS) software is re- quired for changing the button assignments. Fig. 85 Function buttons (left side of stand) 1 Variable function buttons 2 Open/close aperture diaphragm 3 TL/IL switching 4 Open/close fi eld diaphragm 5 Increase/decrease light intensity Fig. 86 Function buttons (right side of stand) 1 Variable function buttons BF PH ICT DF IMC POL CHANGE TL INT ↑ INT ↓ AP ↑ AP ↓ FD ↑ FD ↓ SHUTTER TL TL FLT 1 TL FLT 2 FLUO CUBE 1 CUBE 2 CUBE 3 CUBE 4 CUBE 5 CUBE 6 AFC ON/OFF AFC HOLD CHANGE CUBE CW CHANGE CUBE CCW INT FLUO ↑ INT FLUO ↓ FD FLUO ↑ FD FLUO ↓ CHG FW IFW ExMan COMBI CHANGE COMBI CHANGE OBJ CW (only DMI6000 B) CHANGE OBJ CCW (only DMI6000 B) Z FINE (only DMI6000 B) Z COARSE (only DMI6000 B) XY PRECISE XY FAST BTP ON/OFF (only DMI6000 B) DRY/IMM CHANGE FLT CHANGE CS OBJ 1-6 MEM 1-6 Turns AFC on or off Holds current position Possible functions*: * See page 63 for abbreviations 2 3 4 5 1 1

100% of the light goes to the eye piece (87.1). Toggle function for the side ports (87.2). This function depends on the individual microscope confi guration. Note: Switching to the bottom port: via the variable function buttons (Lei- ca DMI6000 B only), switching to top port: manually. SHUTTER Opens and closes the shutter (87.3). Switches between the possible mag nifi cations of the magnifi cation changer (87.4). The magnifi cation changer is set to the magnifi cation 1x (87.5). Fig. 87 Front control panel 1 100% light to eyepiece 2 Toggle ports 3 Shutter 4 Switch between subsequent magnifi cations 5 Subsequent magnifi cation 1x 6 Selecting fi lter cubes Fig. 88 1 Focus control buttons 2 Open fi lter drawer CUBE The CUBE 1 to CUBE 6 (87.6) buttons permit the direct selection of indi- vidual fi lter cubes, provided the se- lected cube is valid for the selected method. Press the CUBE 3 and CUBE 4 but- tons at the same time to display the assignments of the variable function buttons. To reset the display, press the buttons again or wait 3 seconds. Focus buttons (Fig. 88) (DMI6000 B only) Moves the Z drive in the indicated di- rection. SET + Z↑ Sets the upper focus stop. SET + Z↓ Sets the lower stop. 1 Z↑ Z↓ 2 1x → → → → 1 3 4 6 6 2 5

1 travel in x 2 Travel in y 3 Focus 4 Individual adjustment of button height 5 Variable function buttons (factory preset) 1 2 3 4 5 Shutter button + Cube buttons 1-6 (Leica DMI4000 B and Leica DMI6000 B only) The selected cube is moved to the loading position for replacement. „Load“ appears on the screen. After pressing the button (88.2) the draw- er is opened and the cube can be changed. The next fi lter cube will be moved to the loading position after the drawer is closed. 7.5 The SmartMove Remote Control Module SmartMove knobs (Leica DMI4000 B and Leica DMI6000 B) Use the knobs 89.1 and 89.2 to move the stage in X and Y directions. The image is focused using the knob 89.3 (Leica DMI6000 B only). The height of the knobs can be adjusted to a comfortable working position by turning 89.4. Variable function buttons on SmartMove The variable function buttons are assigned func- tions at the factory that are appropriate to the features of your microscope. They are labeled accordingly. For details on button assignments, please refer to the included identifi cation sheet. For information on the abbreviations used, please refer to the list → p. 63. Note: The Leica Application Suite (LAS) software is re- quired for changing the button assignments. 7.6 Illumination 7.6.1 Transmitted light If your microscope has not yet been set up for Koehler illumination, please continue with the „Koehler Illumination“ section. Leica DMI3000 B: • If necessary, adjust the TL bright fi eld position at the fi lter slider. • Select an objective with moderate magnifi ca- tion (10x–20x). • Set the condenser to the bright fi eld position. • Place a specimen on the stage. • Focus on the specimen using the focus wheels. • Adjust the light intensity. • Close the fi eld diaphragm manually until the edge of the diaphragm appears in the fi eld of view.

All of the contrast methods of the Leica DMI4000 B and Leica DMI6000 B can be selected and con- trolled via the variable function buttons and the Leica Application Suite (LAS). The only excep- tions are methods that involve com ponents re- quiring manual control (e.g. systems with manual analyzers). The following section describes the use of the function buttons on the stand. For in- structions on the use of the software, please re- fer to the separate manual. Contrast methods for the Leica DMI3000 B are controlled via the manual condenser, the manual objective turret, as well as turning knobs and sliders at the microscope. 8.2.1 Bright Field (TL) Leica DMI3000 B: • If necessary, adjust the TL bright fi eld position at the fi lter slider. • Set the condenser to the bright fi eld position. • Remove all other optical components such as analyzers, polarizers or IC prisms from the beam path. • Insert a transmitted light specimen. • Select your objective • Set the brightness at the light potentiometer • Focus the image with the focus wheels. Leica DMI4000 B and Leica DMI6000B: Note: If all positions of the fi lter turret are occupied, fi l- ter cube „A“ can be swapped for fi lter cube „A- TL“ using the Leica Application Suite (LAS/LAS AF). TL contrast methods are possible with that fi lter cube.

transmitted light (TL). • Select the BF (bright fi eld) contrast method by pressing the variable button BF. Alternatively: press the variable button CHANGE TL . (For details on button assignments, please see the identifi cation sheet.) BF will appear on the LeicaScreen. Motorized condensers will now move to the bright fi eld position. Coded condensers must be switched manually. The fl uorescence fi lter turret will automati cally go to an empty position or to the „A-TL“ fi lter cube. Fig. 108 Function buttons (left side of stand) 1 variable function buttons 2 Open/close aperture diaphragm 3 TL/IL switching 4 Open/close fi eld diaphragm 5 Increase/decrease light intensity Fig. 109 Function buttons (right side of stand) 1 variable function buttons 5 3 4 2 1 1 • Insert a transmitted light specimen. • Rotate an appropriate objective into place. • Focus the image with the knob on the Smart- Move or the focusing wheel and adjust the in- tensity with the INT function buttons.

(integrated phase contrast, see 8.2.6) Leica DMI3000 B: • If necessary, adjust the TL bright fi eld position at the fi lter slider. • Select a phase contrast objective. • Select the suitable light ring on the condenser. • Open the aperture of the condenser com- pletely. • Remove all other optical components such as analyzers, polarizers or IC prisms from the beam path. • Insert a phase contrast specimen. • Set the brightness at the light potentiometer • Focus the image with the focus wheels. Leica DMI4000 B and Leica DMI6000 B: • Use the TL/IL function button to switch to transmitted light (TL). • Select the PH (phase contrast) contrast meth- od by pressing the variable button PH. Alternatively: press the variable button CHANGE TL . (For details on button assignments, please see the identifi cation sheet.) PH will appear on the LeicaScreen. Motorized condensers will now switch to the correct light ring. Coded condensers must be switched manually. • Insert a transmitted light specimen. • Rotate an appropriate objective into place. Objectives that are suitable for phase contrast are engraved with PH. • Focus the image with the knob on the Smart- Move or the focusing wheel and adjust the in- tensity with the INT function buttons. Note: When selecting the phase contrast method, the aperture diaphragm is opened fully and can not be adjusted.

The fi lter slider (5a.9, S.24) is used to operate the fl uorescence module. • Pull the fi lter slider out completely to open the beam path. • Push the fi lter slider into the middle position (1st detent) to bring the blue fi lter into the beam path. • Insert the fi lter slider fully in order to block the beam path (shutter position). • The fl uorescence illumination is controlled by the rotary knob (5a.10, S.24). • The fi lter cubes are swiveled manually into the beam path by turning the incident light turret disk. Leica DMI4000 B and Leica DMI6000 B: • Use the TL/IL function button to switch to fl uo- rescence FLUO. • Place a specimen on the stage and select a suitable objective. • The current fl uorescence fi lter cube will be displayed on the LeicaScreen. • You may protect your specimen from fading by closing the incident light shutter. To do so, press the SHUTTER button (87.3) on the front panel. The following pictogram will appear on th Lei- caScreen: �Changing the fl uorescence fi lter cube �Fixed function buttons on the front panel: CUBE 1 to CUBE 6 or Cube CCW �Variable function buttons on the front panel and SmartMove: CUBE CW or CUBE CCW �Leica Application Suite (LAS) Software • Focus the image with the knob on the Smart- Move or the focusing wheel and adjust the in- tensity with the INT function buttons.

creased by using the booster lens (Fig. 112) on the left rear side of the stand (Fig. 113). If bright fl uorescence is required in the center of the fi eld of view, slide the booster lens into the receptacle with the marking facing the user. If a homogeneous distribution over the entire fi eld of view is required, turn the booster lens 180° so that the marking is facing forward. • For multiple fl uorescence, we recommend us- ing the Excitation Manager and/or the ultrafast internal fi lter wheel. Excitation wavelengths can thus be changed in milliseconds. They are controlled by the function buttons. Fig. 112 Booster lens Fig. 113 Booster lens in stand 1 • 1.4x 0.7x

(Leica DMI4000 B and DMI6000 B) Up to two combination methods are possible de- pending on the features of the individual micro- scope: FLUO/PH and FLUO/DIC • Select the combination method by pressing the variable button COMBI . Alternatively: press the variable button CHANGE COMBI . (For details on button assignments, please see the identifi cation sheet.) The content of the display changes accord- ingly. • Place a specimen on the stage and select a suitable objective. • Select the desired fi lter cube using the fi xed function buttons on the front panel. • The illumination settings for the fl uorescence and transmitted light axes can be adjusted separately. • Toggle the illumination axes with the TL/IL function button. The content of the LeicaS- creen changes accordingly. FLUO > DIC The transmitted illumination is activated. FLUO < DIC The fl uorescence illumination is activated. Note: The manual analyzer (Fig. 110) must be used for the FLUO/DIC method as described in Chapter 8.2.5, p. 88.

The left-hand focus wheels can be used for both coarse and fi ne focusing; the right-hand focus wheel for fi ne focusing only (a version of the Leica DMI3000 B with mirrored focus controls is also available) Leica DMI6000 B: Note: The parfocality teach-in has already been per- formed at the factory. However, it may be neces- sary to perform another teach-in after installing the objectives when setting the microscope up. We recommend checking parfocality before set- ting the stops and performing a teach-in with the Leica Application Suite (LAS) if necessary. Focusing the image The focusing is controlled by the knobs (116.3, p. 101) on the SmartMove remote control module. � � � Alternatively, use the focus wheels on either side of the stand. The current Z position is shown on the LeicaS- creen. In the case of motorized stages, the Z drive will travel to its lowest position prior to the stage initialization when switching the micro- scope on. The focus buttons Z↑ and Z↓ on the right side of the stand (Fig. 114) permit fast focusing or lower- ing of the objectives. Setting stops Set the lower focus stop by pressing and hold- ing the SET button and pressing the Z↓ button as well. The display will show . Pressing the button combination again will delete the stop. The display will show . The lower focus stop can also be set using the Leica Application Suite (LAS). The lower stop is the same for all objectives and can not be traversed. In addition, a focus position that can not be tra- versed can also be set. To do so, press and hold the SET button and press the Z button as well. The display will show . Pressing the button combination again will delete the stop. The display will show . The focus position can also be set using the Leica Application Suite (LAS). Set the focus position for the dry objective at the highest magnifi cation. The focus positions will then be set automatically for all other objectives, taking parfocality and working distances into ac- count. Abb. 114 1 Focus operating keys 2 Open fi lter drawer 1 2 � � �

�fi xed function buttons on stand �Leica Application Suite (LAS) Software Summary of pictograms lower focus stop not set lower focus stop set focus position not set focus position set Going to the stops Go to the lower stop by pressing and holding the Z↓ button. Go to the focus position by pressing and holding the Z↑ button. These functions can be assigned to variable function buttons on the stand or SmartMove, or they can be controlled via software. �Go to stops via �fi xed function buttons on stand �variable function buttons on stand and SmartMove �Leica Application Suite (LAS) Software Note: When going to the stops with the Z↑ and Z↓ buttons, hold the button until the stop has been reached. Setting the step increments It is possible to toggle between Fine and Coarse step increments. The Fine value varies to suit the current objective. Suit- able values have been predefi ned. The assignments can be changed with the Leica Application Suite (LAS). When selecting Coarse, the positioning speed is the same for all objectives. Coarse corresponds to the maximum speed. Note: The assignment of a specifi c step increment to an objective not only applies to the Z drive, but also to the step increments assigned to the stage when Precise (→ p. 101) is selected. �Switch between Fine and Coarse via �variable function buttons on stand and SmartMove �Leica Application Suite (LAS) Software Only for DMI6000 B with AFC (Adaptive Focus Control) AFC actively holds a pre-defi ned focus position over time. This feature is especially useful, if e.g. during a time-lapse experiment at 37°C the climate chamber has to be opened and a drop of the temperature may occur. Activate the AFC function, focus on your speci- men either with the hand wheel on the micro- scope stand or at the SmartMove and store the current focus position as hold position. AFC can be controlled either by. • Variable function keys at the stand or SmartMove • STP6000 • Software (Leica LASAF) � � � � � �

Leica DMI 3000 B and Leica DMI4000 B: Manual shifter rod activates and deactivates the left-hand photo port. VIS LEFT 100% 0% 20% 80% alternatively: 0% 100% Leica DMI 6000 B: The button on the front control panel switches 100% of the light to the eyepieces. Use the button, also on the front control panel, to select the side ports. 8.6 Tubes Note: Close any unused tube openings, as otherwise stray light can interfere with observation. Adjusting the viewing distance • Adjust the viewing distance of the eyepiec- es so that a congruent total image is seen (Fig. 115). Adjusting the viewing angle • Ergotubes feature a tilting binocular section for a 30–45° viewing angle adjustment range. Beam splitting in photo tubes The beam splitting is set manually by pulling out a control bar. Observation photo 100% 0% 0% 100% alternatively 50% 50% BL activation of Bertrand lens* �Light distribution via �manual control bar Fig. 115 Tube setting ♦→ → →

now display - the active port (right or left) and - the percentage of light going to the port (100%, 80%, 50%). Optional Leica DMI 6000 B: The bottom port selection function can be as- signed to one of the variable function buttons on the stand or the SmartMove. The top port can only be selected manually. �Select ports via �fi xed function buttons on stand (side ports) �variable function buttons on stand and SmartMove (bottom port) �manual action (top port) 8.8 Eyepieces Note: The eyepiece’s aperture protector must be re- moved or folded back, during microscopy while wearing eyeglasses. We recommend removing bifocals and spectacles with progressive-addi- tion lenses when using the microscope. • For the adjustable tubes with documentation output, choose the 100% VIS position. Eyepieces with inlaid reticle • Focus the reticle by adjusting the eyelens. • Focus on the object through this eyepiece. • Then, close that eye and focus on the object by adjusting only the second ocular. Correction for Vision Problems • With your right eye, look through the right eye- piece and bring the specimen into sharp focus. • Then, with your left eye, view the same speci- men and rotate the left eyepiece tube until the object is brought into sharp focus. Do not change the Z position in the process!

Select objectives manually with the objective tur- ret. The objective turret of the DMI4000 B is coded so that the selected objective is shown on the display. Leica DMI6000 B: The objectives can be selected with the func- tion buttons on the stand or the SmartMove, or by manually turning the objective turret. When changing objectives manually, please ensure that the turret clicks into position. The positions of the objectives in the objective turret have been specifi ed at the factory and must be observed when installing the objectives. (→ also see Objectives, p. 43). When selecting an objective, the microscope au- tomatically selects: • the optimal setting for the fi eld diaphragm • the optimal setting for the aperture diaphragm • the light intensity for the current contrast method The objective magnifi cation and total magnifi ca- tion are displayed on the LeicaScreen. Note: We recommend running a teach-in via the Leica Application Suite (LAS) software when using eyepieces not included in the scope of deliv- ery. This will ensure that the total magnifi cation shown in the LeicaScreen is correct.

immersion medium. OIL: only use optical immersion oil according to DIN/ISO standards. Cleaning → p. 109. W: Water immersion. IMM: Universal objective for water, glycerol, oil immersion. Caution! Follow safety instructions for immersion oil! Color coding of objectives The magnifi cation of each objective is indicated by a color ring in accordance with DIN/ISO stand- ards: Immersion objectives are marked by an addition- al, lower color ring. black oil or Imm (universal objective for oil, water or glycerin) white water orange glycerin The various engraved markings of the objectives provide information on their applications: black or bright fi eld objectives, dark blue strain-free green phase contrast objectives, strain-free 100x 63x 40x 25x 16x 10x 6.3x 4x 2.5x 1.6x 125x 50x 32x 20x 5x 150x 160x white dark- light- dark- light- yellow orange red brown gray blue blue green green

�variable function buttons on stand and SmartMove �Leica Application Suite (LAS) Software �Manual selection Changing the operating modes „dry“ ( DRY) and „immersion“ ( IMM) Each objective is assigned to a specifi c objective category: 1) Dry objectives (DRY) 2) Immersion objectives (IMM) Note: It is possible to use objectives for both operating modes. The mode can be assigned in the Leica Applica- tion Suite (LAS). Changing the operating mode • First, select the operating mode (Imm or Dry) using the function buttons. The operating mode may also be selected in the Leica Application Suite (LAS). • The objective turret is lowered to its bottom stop. This is to permit the application of the immersion liquid when changing from a dry to an immersion objective. It also permits the re- moval of the liquid when changing to dry mode. The current objective remains in the beam path. • Next, press the but on for the objective you in- tend to use. Note: If the Imm or Dry operating mode buttons are pressed accidentally, the original mode can be restored by pressing the appropriate button. �Change operating mode via �variable function buttons on stand and SmartMove �Leica Application Suite (LAS) Software Note: When replacing objectives, you must perform a teach-in for the new objectives in the Leica Application Suite (LAS). A parfocality teach-in should also be performed. Note: For lockable immersion objectives lock these by pushing the front part upwards until it stops (ap- prox. 2 mm). Then, after a gentle turning motion to the right, the objective is locked. For objectives with corrective mounts turn the knurl to adjust the objective to the thickness of the cover glass.

Leica DMI3000 B and Leica DMI4000 B: The motorized stages are controlled via a sepa- rate control unit. Leica DMI6000 B: Object displacement using SmartMove The positioning of the stage is controlled by the knobs (116.1, 116.2) on the SmartMove remote control module. Setting the step increments The positioning speed of the stage can be varied by switching between the Fast and Precise step increments. When selecting Fast, the positioning speed is the same for all objectives. The Precise speed varies to suit the current ob- jective. � Switch between Precise and Fast via �variable function buttons on stand and SmartMove �Leica Application Suite (LAS) Software Fig. 116 SmartMove remote control module 1 travel in x 2 Travel in y 3 Focus 4 Individual adjustment of button height 5 Variable function buttons (factory preset) 1 3 4 5 2 Storing and restoring stage positions A variety of stage positions can be stored tempo- rarily in the Leica Application Suite (LAS). The XY position is stored, not the Z position. In addition to a loading position (Load), 5 stage positions can be set temporarily. When switch- ing the microscope on, the stage will travel to a previously-defi ned starting position. �Temporarily store and restore stage positions via �Leica Application Suite (LAS) Software

Leica DMI3000 B: A mechanical magnifi cation changer can be used optionally. Magnifi cation factor: 1.6x. A slider switches between 1x and the magnifi ca- tion factor. The mechanical magnifi cation chang- er affects the eyepieces and the top port. Leica DMI4000 B and Leica DMI6000 B: A mechanical magnifi cation changer can be used optionally. The following magnifi cation fac- tors are available: 1.5x, 1.6x and 2x A slider switches between 1x and the magnifi ca- tion factor. The mechanical magnifi cation changer affects the eyepieces and the top port. The selected factor is shown in the LeicaDisplay or the relevant window of the Leica Application Suite (LAS) and taken into account when calcu- lating the total magnifi cation. Leica DMI4000 B and Leica DMI6000 B: A motorized magnifi cation changer can be used optionally. The following magnifi cation factors may be selected: 1.5x, 1.6x, or 2x The selected factor is displayed on the LeicaS- creen and in the relevant fi eld of the Leica Ap- plication Suite (LAS), and is taken into account when calculating the total magnifi cation. The motorized magnifi cation changer affects all ports. Pressing the left button (117.1) switches between the possible magnifi cation factors; pressing the right button selects the factor 1x. Note: a microscope can not have both types (manual and motorized) of magnifi cation changers. Fig. 117 Front control panel 1 Function buttons for magnifi cation changer 1 1 �Change magnifi cation via �fi xed function buttons on stand �Leica Application Suite (LAS) Software

�fi xed function buttons on stand �variable function buttons on stand and SmartMove �Leica Application Suite (LAS) Software Fig. 118 Fixed function buttons, left side of stand 1 Aperture diaphragm 2 Transmitted light/incident light 3 Field diaphragm 4 Light intensity 1 2 3 4 8.12 Light sources Leica DMI3000 B: • Light intensity: the transmitted light intensity can be adjusted continuously from 0 to 12V us- ing the potentiometer at the lower left of the front of the microscope stand. • FLUO: The intensity can be adjusted in 5 fi xed levels. 100% / 55% / 30% / 17% / 10% ( FIM=Fluorescence Intensity Manager) If you continue turning to the left at 100% or to the right after 10%, a 0% position (shutter posi- tion) is set. Leica DMI4000 B and Leica DMI6000 B: • Adjust the intensity with the function buttons (118.4). The INT function buttons are always assigned to the currently active transmitted light (TL) or incident light (IL) axis. • For TL and IL: The setting can be made in coarse and fi ne steps. Pressing both INT (118.2) buttons as the same time toggles between coarse and fi ne adjustment. The light intensity displayed on the LeicaScreen changes accordingly. Coarse adjustment: 0–20 Fine adjustment: 0–255 • The intensity is individually adjusted and stored for each objective and contrast method. • FLUO: The intensity can be adjusted in 5 fi xed levels. 100% / 55% / 30% / 17% / 10% ( FIM=Fluorescence Intensity Manager)

Leica DMI3000 B: Transmitted light: • The manual aperture diaphragm is adjusted on the condenser. • The manual fi eld diaphragm is adjusted on the illumination arm. Incident light: • The fi eld diaphragm is set via the rotary knob (5a.11, S. 24). Diaphragm apertures of different sizes (round or angled) can be selected. (See the labeling on the rotary knob.) Round apertures are suitable for observations through the eyepieces; angled apertures are suitable for observations with CCD cameras. Leica DMI4000 B and Leica DMI6000 B: Both diaphragms have been set to suitable val- ues for the current objective and contrast meth- od at the factory. The aperture diaphragm is controlled manually when using the manual condenser. The fi eld diaphragm is controlled manually when using the manual illumination arm. • The motorized diaphragms can be adjusted at any time with the AP (aperture diaphragm) (118.1) and FD ( fi eld diaphragm) (118.3) function buttons. The values displayed on the Lei- caScreen change accordingly. The function buttons are assigned to the cur- rently active transmitted light (TL) or incident light (IL) axis. Microscopes with structured illumination are not equipped with a fi eld diaphragm. Caution: The old values will be over- written by the current ones! Caution: When using PH or DF, the aperture diaphragm is fully open and can not be closed. �Adjust diaphragms via �fi xed function buttons on stand �variable function buttons on stand and SmartMove �Leica Application Suite (LAS/LAS AF) Software

9. Trouble Shooting Problem Stand The microscope does not respond. Illumination The image is completely dark. The image is unevenly or not uniformly illumi- nated. The illumination fl ickers. The lamp does not illuminate immediately upon being switched on. Cause/Remedy �Ensure that the AC outlet has power. �Ensure that the electronics box is connected to an AC outlet. �Check the cable connections. �Inform Service and have the supply unit fuse checked. �Open the shutter (→ p. 68). �Check the connections of the lamp housings on the microscope (transmitted light/fl uores- cence) �Ensure that the lamps are connected to the power supply and are not defective. �Inform Service and have the ebq 100 supply unit fuse checked. �Remove all unneeded fi lters from the light path. �Center the lamp (→ p. 78ff) �Replace the old lamp (→ p. 45, 49ff). �Be sure that there is no loose connection at the power supply. �Replace the old lamp (→ p. 45, 49ff). �The ebq 100 must be switched-on repeatedly. �Hot Hg lamps should cool down before switch- ing on again.

Problem Bright fi eld The specimen can not be brought into focus. Dark Field No defi nite DF contrast is possible. The image is unevenly or not uniformly illumi- nated. Undesirable stray light. Cause/Remedy � Use the correct immersion medium. �Place the specimen on the stage with the cov- erslip facing down. �Make sure that the cover glass thickness is correct and that it suits the indication on the objective. �Ensure that you are using an objective with coverslip correction. �Adjust the correction ring on the objective if present. �Be sure that a DF objective is being used. �The objective aperture is too high: maximum 0.7 for condenser S1 maximum 0.4 for condenser S23/28 If necessary, reduce the objective aperture us- ing the iris diaphragm on the objective. �Check the condenser centering. �The magnifi cation is too weak. Use a higher magnifi cation. �Remove the condenser head or condenser lenses. �Clean the specimen and neighboring lenses (→ p. 109f).

Problem Phase contrast No phase contrast is possible. Polarization No polarization contrast is possible. Transmitted light interference contrast No transmitted light interference contrast is pos- sible Cause/Remedy �The specimen is too thick. �The cover glass is not placed evenly. �Check the centering of the light rings (→ p. 74). �Bring the polarizer and analyzer into cross position until they reach maximum darkness (without specimen). (→ p. 87). �The specimen is too thick or too thin. �Embedding medium or specimen are of bire- fringent material. Rotate the specimen. �The difference in the refractive indices of the specimen and the embedding medium is too small. �The cover glass is too thick. �Check the Koehler illumination (→ p. 70). �Bring the polarizer and analyzer into cross position until they reach maximum darkness (without specimen). (→ p. 87). �Check whether the suitable condenser prism and corresponding objective prism are select- ed (manual alternative → p. 87). �Ensure that the IC prisms are correctly seated (→ p. 40).

Problem Fluorescence The image is completely dark (no fl uorescence). The fl uorescence is too weak. LeicaScreen Init Error! Cause/Remedy �Open the shutter (→ p. 68). �Select the incident light axis (IL) (→ p. 66). �Check your specimen, e.g. its antibody binding. �Insert a new lamp (→ p. 45, p.49ff). �Insert the booster (→ p. 92). �Center the lamp (→ p. 78ff) �Insert a new lamp (→ p. 45, p.49ff). �Check the cable connections. �Check whether the cover of the fi lter disk has clicked into position. �Check the installed objectives, fi lter cubes, etc. �Switch the microscope off and back on.

Contrasting method Magnifi cation Illumination Ports/Eyepiece Focus Lower focus stop not set Lower focus stop set Focus position not set Focus position set Shutter open Shutter closed Transmitted light fi lter Field diaphragm, rectangular Field diaphragm, round Aperture diaphragm Light distribution 13. Abbreviations and Pictograms → → � � � � � �

AP Aperture diaphragm BF Bright fi eld COMBI Combination method CUBE Fluo cube DF Dark fi eld incident/transmitted light DIC Differential Interference Contrast FD Field diaphragm FLUO Fluorescence axis (incident light) ICR Interference contrast, incident light ICT Interference contrast, transmitted light IL Incident light INT Intensity IMC Integrated modulation contrast IPH Integrated phase contrast PH Phase contrast POL Polarization, incident/transmitted light TL Transmitted light

AFC Adaptive Focus Control 17, 62, 95 Allen key 30 Ambient temperature: 10 Analyzer 55, 56 Analyzer slot 21, 25 Aperture diaphragm 20, 66, 84, 104 Arc 80 Attachable mechanical stage 21, 25 Attachable mechanical stage for fi xed micromanipul 34 Beam splitting 96 Bertrand lens 75, 96 Booster lens 20, 47, 92 Bright fi eld (TL) 106 C-mount 0.5x/0.63x 57 Camera 57 Centering key 30 Centering the mercury lamps 79 Centering window 21 Changing objectives 98 Cleaning glass surfaces 110 Cleaning objectives 110 Cleaning the stage 109 Coarse 95 Collector 50, 51 Color coding (objectives) 99 Combination Methods 93 Computer 64 Condenser 17, 38, 41, 55, 64 Condenser base 20 Condenser base S1-S28 38 Condenser cable 58 Condenser centering 72, 75 Condenser head 20, 42 Condenser head S1 38 Condenser head S28 38 Condenser prisms 39 Condenser tool 40 Consumable parts 111 Contrast settings 72 Contrasting method 14, 65, 83 Controls 18, 60 Correction for vision problems 97 Corrective mount 100 CUBE 68, 91 Dark Field (TL) 86, 106 Diaphragm 65 DIC module 32 DIC objective prism 32 DIC objective prism disk 20 DIC specimen 88 Differential Interference Contrast (TL) 88 Digital camera 57 DIN VDE 8 Direct interface 58 Disposal 13 Drawer 21, 24 DRY 100 E-focus control buttons 21 ebq 100 supply unit* 59, 81 Electromagnetic compatibility 8 Electronics box 58, 64, 81 EU directive 8 EXT1-EXT4 sockets 30 Eyebase 96 Eyepiece tubes 20, 24 Eyepieces 20, 24, 43 Fast 101 Field diaphragm 20, 66, 73, 84, 104 Field diaphragm adjustment 73 Field diaphragm centering 21, 22, 26 Filter 44 Filter block 53 Filter cube 52, 68 Filter drawer 68, 94 Filter/refl ector cube 53 FIM 103 Fine 95 Fixed function buttons 62, 66, 103 Fixed micromanipulation stage 34 Fixed stage 35 Fixed stage (normal) 33 Fluorescence 91, 108 Fluorescence fi lter cubes 91 Fluorescence illumination 93 Focus 65 Focus control buttons 68, 94 Focus position 94 Focus stop 68 Focus wheel 20, 21, 24, 25 Focusing 94 Focusing telescope 74 Frequency 10 Front control panel 22, 23, 68, 102 Function button 63 Function button assignment 63, 67 Function buttons 67, 68 Fuses 10 Gas discharge lamps 81 General view 23 Glass insert 36 Graticule 97 Halogen lamp 12V 100W 45 Heating Insert P 36 Height compensation plate 112 Hg 100W lamp 48 Hg mercury burner 47 High-pressure mercury burner 49 High-pressure mercury burner 100W 49 High-pressure xenon burner 75 W 49 IC prisms 32 ICT condenser prisms 40 Illumination 65, 105 Image centering 72 IMM 100 Immersion objectives 99, 100 Immersion oil 111 Incident light - fl uorescence. 73 Incident light axis 14 Incident light lamp housing 21 Incident light turret disk 52

Inserting the lamp 46 Inserts for attachable mechanical stage 35 Installation location 28 Intelligent automation 60 Intensity control 103 Interfaces 18 Intermediate pupil interface 20 Knobs 62, 69 Koehler Illumination 70, 72 Lamp bases 49 Lamp housing 106 z 46, 48, 50, 78 Lamp housing 106 z L 48 Lamp housing 107 L 78 Lamp housing 107 or 107/2 45 Lamp housing mount 46 Lamp mount 20 Lamp power cable 58 Lamp replacement 44 Leica CTR6000 electronics box 10, 18 LeicaScreen 20, 22, 61, 64, 65, 82, 108 Light guide 12 Light intensity 20, 66 Light rings 38, 64 Light source adjustment 78 Light sources 103 Load 53, 69 Lower focus stop 68, 94 Magnifi cation 65 Magnifi cation changer 15, 68, 102 Manual method 87 Mechanical 3-plate stage 33 Medical instrument 8 Micromanipulation stage with attachable mechanical 33 Motorized 3-plate or scanning stages 37 Motorized method 87 Motorized polarizer 77 Multiple fl uorescence 92 Object displacement 101 Objective turret 16, 21, 24, 43 Objectives 65, 98 Observation ports 17, 18 Opener for drawer 21 Operating mode 100 Operating temperature 11 Overvoltage category: 10 Parfocality compensation 43 PCI card (PC) 57 Phase contrast 107 Phase contrast centering procedure 74 Phase contrast rings 74 Phase rings 39, 64 Phillips screwdriver 30 Pictograms 113 Polarization 107 Polarization (TL) 87 Polarizer 55, 75 Polarizer holder 55 Pollution degree 10 Port 96 Port selection 96 Port switching 22, 26 Power input 10 Power supply 59 Precise 101 Protective gloves 48 Protective mask 48 Refl ector cube 78 Relative humidity 10 Removing Immersion Oil 110 Replacement eyecup 111 Replacement lamp 111 Replacement of incident-light lamps 48 Reset function 61, 82 Right side port 20, 21, 24 RS232 ports 58 Safety class 9 Safety regulations 9 Screw lengths 34 Setting stops 94 Shutter 68, 91 Side-Port 21 SmartMove 23, 60, 62 SmartMove remote control module 23 Software 62 Software tools 19 Spare parts 111 Specifi cations 10 Stage 21, 25 Stage positions 101 Stages 16, 33, 101 Stand 105 Stand package 27 Step increments 95, 101 Stops 65 Supply unit 10, 50, 51, 59 Supply voltage: 10 System package 27 Three-plate micromanipulation stage 33 TL/IL switching 20 Toggling transmitted light/incident light 66 Top-Port 20, 22, 24, 26 Transmitted illumination 93 Transmitted Light Axis 14 Transmitted light interference contrast 107 Transmitted light lamp housing 24 Transmitted-light 69 Transmitted-light fi lter 22, 26 Transmitted-light illumination carrier 31 Transmitted-light lamp housing 20, 44 Transmitted-light specimen 84, 85 Transport 29 Tube 15 Tube setting 96 Turret disk 53 USB 58 Useful life 49 Variable function buttons 20, 21, 23, 62, 67 Variable function buttons on SmartMove 69 Viewing angle 96 Xe 75 burner 50 XYZ-control 58 Z focus 17