Operating Manual for the Olympus BX51 and Image Capture System Brightfield (transmitted light)………..…………………………..… 2 Brightfield (reflected light)………..…………………………..…….3 Fluorescence……………………………………………………….. 4 Diagram of the BX51 Microscope…………………………………. 5 Guide to switches, filters, knobs, etc………………………………..6 Image acquisition and measurements……………………………….8 Troubleshooting……………………………………………………. 10

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BRIGHTFIELD (transmitted light) Illumination transmits through a transparent sample from the bottom of the microscope. 1. Turn on the halogen light switch (1) for transmitted light to “│” (ON). 2. Engage the brightfield mirror unit (BF) (17). 3. Adjust the light path. The light path selector knob (2) should be pushed in all the way. 4. Select transmitted light (lower position) on the switch by the brightness indicator (4). 5. Adjust the light intensity using the brightness adjustment knob (3). The numerals by the lamp voltage indicator LEDs (4) indicate the voltage. Deselect the “PRESET” button (not illuminated green) to avoid using maximum illumination. 6. Adjust the interpupillar distance. While looking through the eyepieces, adjust the oculars (5) until the left and right fields of view coincide completely. 7. Put the 10x objective (6) in place. Place your specimen on the stage (7). 8. Find your specimen using the stage controls (9). 9. Focus specimen using fine/course focusing knobs (10). 10. Adjust the diopter: - Close your left eye and focus on the specimen using the fine focus knob. - Close your right eye and focus on the specimen using the diopter ring (11) on the left ocular. - Open both eyes and confirm that the focus is comfortable. 11. If desired switch to the next objective by rotating the nosepiece (12) and focus. Continue until you reach the desired magnification. 12. Establish Koehler illumination: - Close the field iris diaphragm (13) until you can see the edges. - Focus the image of the field iris diaphragm by raising or lowering the condenser using the condenser height adjustment knob (14). - Check if the circle of light is centered in the field of view. If not, use the two condenser centering screws (15) to move the field iris diaphragm image to the center of the field of view. - Open the field iris diaphragm until its image circumscribes the field of view. - Match the opening of the condenser aperture iris diaphragm (16) with the N.A. of the objective in use in order to achieve the optimum objective performance. 13. Examine specimen and document image if necessary: - To obtain still digitized images and movies, use the checklist on page _____ for the Sensicam Cooke Camera and ImagePro Plus software. 14. When finished: - Lower the stage by turning the focus knob (10) and remove your specimen. - Turn the nosepiece (12) until the 10x objective is into place. - Lower the light intensity to zero using the brightness adjustment knob (3). - Turn the halogen light switch (1) to “○” (OFF). - Return any slide filters (26)(31)(32) to their disengaged positions.

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BRIGHTFIELD (reflected light) Illumination is shone onto a reflective sample from above (the reflected light housing at the back of the microscope). 1. Turn on the halogen light switch (1) for reflected light to “│” (ON). Engage the brightfield mirror unit (BF) (17). 2. Adjust the light path.

- The light path selector knob (2) should be pushed in all the way. - The knob on the reflected light splitter (30) should be pushed in all the way.

- The shutter knob (20) should be slid to the position marked “○” (OPEN). 3. Select reflected light (upper position) on the switch by the brightness indicator (4). 4. Adjust the light intensity using the brightness adjustment knob (3). The numerals by the lamp voltage indicator LEDs (4) indicate the voltage. Deselect the“PRESET” button (not illuminated green) to avoid using maximum illumination. 5. Adjust the interpupillar distance. While looking through the eyepieces, adjust oculars (5) until the left and right fields of view coincide completely. 6. Put the 10x objective (6) in place. 7. Place your specimen on the stage (7). 8. Find your specimen using the stage controls (9). 9. Focus specimen using fine/course focusing knobs (10). 10. Adjust the diopter: - Close your left eye and focus on the specimen using the fine focus knob. - Close your right eye and focus on the specimen using the diopter ring (11) on the left ocular. - Open both eyes and confirm that the focus is comfortable. 11. If desired switch to the next objective by rotating the nosepiece (12) and focus. Continue until you reach the desired magnification. 12. Establish Koehler illumination: - Close the field iris diaphragm (13) until you can see the edges. - Focus the image of the field iris diaphragm by raising or lowering the condenser using the condenser height adjustment knob (14). - Check if the circle of light is centered in the field of view. If not, use the two condenser centering screws (15) to move the field iris diaphragm image to the center of the field of view. - Open the field iris diaphragm until its image circumscribes the field of view. - Match the opening of the condenser aperture iris diaphragm (16) with the N.A. of the objective in use in order to achieve the optimum objective performance. 13. Examine specimen and document image if necessary: - To obtain still digitized images and movies, use the checklist on page _____. 14. When finished: - Lower the stage by turning the focus knob (10) and remove your specimen. - Turn the nosepiece (12) until the 10x objective is into place. - Lower the light intensity to zero using the brightness adjustment knob (3). - Turn the halogen light switch (1) to “○” (OFF). - Return any slide filters (26)(31)(32) to their disengaged positions.

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FLUORESCENCE NOTE: The mercury bulb is fragile. It needs to be cool for at least 30 minutes before being turned on. And it needs to stay on for at least 15 minutes before being turned off. So check the usage log before turning it on and off. If you turn it on and off multiple times during the same session, please follow the guidelines. 1. Switch on the mercury bulb by setting the switch (18) on the Power Supply Unit to “I” (ON). The indicator (19) should light up. The mercury bulb will stabilize in 5-10 min. 2. Setup microscope for brightfield imaging (see instructions on ‘Brightfied reflected light’ page 2, steps 2-12). Use the reflected light splitter knob (30) to switch between the white and UV lamps that are located at the rear lamp housings. 3. Engage the fluorescence mirror unit (17) until the proper filter cube is in place: 1= Brightfield, 4 = TRITC, 5 = DAPI, 6 = FITC 4. When using the fluorescence mirrors, turn the light intensity down all the way using the brightness adjustment knob (3). 5. To allow light from the mercury bulb to reach the specimen, slide the shutter knob (20) to position marked “○” (OPEN). Close it whenever you wish to avoid photobleaching your sample. 6. To improve image contrast and to prevent color fading of fluorescent light in other part than observed region, pull out the field iris diaphragm knob (21) so that the image of the field iris diaphragm just circumscribes the field of view. 7. To help adjusting the brightness of the observed image and to improve the contrast, pull out the aperture iris diaphragm knob (22), the diaphragm will get smaller. 8. Examine specimen and document image if necessary: - To obtain still digitized images and movies, use the checklist on page _____ for the Sensicam Cooke Camera and ImagePro Plus software. 9. When finished:

- Slide the shutter knob (20) to “●” (CLOSED). - Push the knob on the reflected light splitter (30) in all the way. - If mercury bulb has burned for at least 15 min., turn it off by setting the main switch (18) on the Power Supply Unit to “O”. The light (19) should go out. - Lower the stage by turning the focus knob (10) and remove the specimen. - Turn the nosepiece (12) back until the 10x objective is into place. - Push the field iris diaphragm knob (21) and the aperture iris diaphragm knob (22) all the way back into the microscope. - Engage the fluorescence mirror unit (17) back to BF (brightfield). - Return any slide filters (26)(31)(32) to their disengaged positions.

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(See diagram in printed operating manual)

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BX51 Microscope Components (1) Lamp power switch – turns on both transmitted and reflected white light sources. (2) Light path selecter knob – switches image between eyepieces, camera or both. (3) Brightness adjustment knob – controls the brightness of transmitted and reflected white light lamps. (4) Lamp voltage indicator – shows the intensity of the light. The toggle switch selects the lower (transmitted light) or upper (reflected light) source. The ‘PRESET’ button engages the maximum illumination when pushed in (green). (5) Oculars – can be adjusted for interpupillar distance. Can also be adjusted for focus. (6) Objectives available: All flat field- and Fluorite-corrected

5x UMPlanFI (UV transmitting) 10x UMPlanFI (UV transmitting) 20x LMPlanFI (Long working dist.) 50x LMPlanFI (Long working dist.) 100x LMPlanFI (Long working dist.) (7) Stage (8) Stage clip (9) Stage controls (10) Fine and Coarse focusing knobs (11) Diopter ring – for focusing the ocular (12) Rotating nosepiece – for selecting objectives (13) Field iris diaphragm for transmitted light– for establishing Koehler illumination (14) Condenser height adjustment knob - for establishing Koehler illumination (15) Condenser centering screws - for establishing Koehler illumination (16) Condenser aperature iris diaphragm - for establishing Koehler illumination (17) Filter wheel holding mirrors and filters for reflected light sources Position 1 – BF, bright field (use for all white light sources). Position 2 – DF, darkfield filter Position 3 – DIC (Nomarski Differential Contrast filter Position 4 – TRITC, fluorescence filter for fluorophores, antibodies Position 5 – DAPI, fluorescence filter for fluorophores, nuclear stain Position 6 – FITC, fluorescence filter for fluorophores, antibodies (18) Power supply unit power switch – to turn the mercury bulb on and off. (19) Power light indicator – glows red when the mercury bulb is on. (20) Shutter knob – to open or close the shutter for reflected light (white or fluorescent) (21) Field iris diaphragm knob – restricts the diameter of the beam of light entering the objective (excludes extraneous light and improves image contrast). Also helps to prevent color fading of fluorescent light in other parts of the sample besides the observed region. (22) Aperture iris diaphragm knob – helps adjust the brightness of the observed image and improve contrast. Use a fine-tune after using the ND filters. (23) Not on this model. (24 bottom) Not on this model. (adjustment screw for condenser) (24 top) Not on this model (Reflected light analyzer (U-AN)). (25) Polarizer

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(26) DIC prism slide filter – to adjust the contrast when using DIC (30) Splitter control knob for rear lamp housings – selects between white light lamp and mercury lamp for reflected light mode. (31) Buttons for built-in filters ND6 decreases excitation light intensity to 6% (fluorescence applications) ND25 decreases excitation light intensity to 25% (fluorescence applications) LBD color conversion filter to simulate daylight (BF, image capture) OP dunno (32) Slide filters for reflected light U-25FR, frost slide filter - to eliminate uneven illumination U-25ND25, ND slide filter - to reduce the excitation light intensity. (33) Rear tungsten lamp housing – source of the reflected white light. Not on diagram.

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Image acquisition (with the Cooke Sensicam camera & Image ProPlus software, v.6.2) 1. Follow the instructions to visualize your sample on the stage. 2. Turn on the SensiCam camera (blue) at the power switch on the top of the camera.

Wait 2 -5 minutes for the camera to warm up. The indicator light by the power switch will go from flashing red (not ready) to constant green (ready).

3. Use the light path selector knob (2) to share the image with the camera. 4. Open the software and acquisition window

-Double-click the Image ProPlus Icon on the computer desktop. -Select Acquire:Video/Digital from the menu. The Sensicam Cooke Driver window appears.

5. Adjust settings while looking at a live, previewed image A) EXPOSURE

-Under the “Preview” tab, enter an estimated exposure time (milliseconds range). -Click the “Start Preview” button in the lower left corner of the window to open a live view of the sample.

-If the image is too bright (white), adjust the brightness of the light (3) to see a usable image in the live window. After this, try to use only the exposure time controls to adjust the image brightness.

-To adjust the exposure time on the preview, stop it by clicking the ‘Stop Preview’ button on the lower right corner of the window. Then change the exposure time. -Click “Start Preview” to see the adjusted exposure.

B) FOCUS

-Use the fine focus knob (10) to focus the onscreen image. C) ENHANCEMENTS (brightness, contrast and gamma corrections)

-Select Enhance:contrast enhancement from the menu to open the window -Use slide bars to adjust the brightness, contrast and gamma values for the previewed image.

-(More adjustments are also available under the Enhance and Process menu bars) 6. Adjust the exposure for the image to be acquired -Click the ‘Stop Preview’ button in the window.

-Select the ‘Acquire’ tab in the window. -Enter the desired exposure time.

7. Snap the picture and save it to your USER IMAGES file

-Click the ‘Snap’ button along the bottom of the window to take a picture. The image and a “Save File As” dialog window appear.

-If the exposure is sufficient, save the file to your folder in the USER

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IMAGES Folder that is on the desktop. -If the exposure is not right, click ‘Cancel’ in the “Save File As” window, and adjust the exposure time and snap another picture. -If multiple images are going to be captured using the same settings, save your settings under the ‘Setup’ tab by clicking on “Save settings… -Save them to the USER SETTINGS folder on the desktop.

Note: A settings file (sensicam default settings) on the desktop has default values. 8. Use the software to analyze your saved images.

There is a reference guide (blue/green and white wire-bound manual) that explains the full features. This includes some fairly neat measuring functions that can be found under ‘Measure’ menu section. Tutorials

are also available in the purple and white wire-bound manual. 9. Take the data with you on a flash drive. Sorry, but there is no internet access for the computer. Old files will be cleaned off the drive after 6 months. 10. Close the Image ProPlus software. 11. Leave the computer on. Extra Software Notes: Here are the commonly used features of the Sensicam Cooke Driver window Under the ‘Setup’ tab - Always select the “Sensicam Cooke Driver” as the current driver in the pop-up list. Use the “Load settings…” button to use any saved settings. Under the ‘Image’ tab – Check the “Enable multiple Image Capture” box and enter an interval and number of images to capture sequential images. Under the ‘Preview’ or “Acquire’ tabs – Click the “Set Area…” button to change the area of captured image. Use the current image or enter coordinates to change the size and resolution. To change the pixel resolution, change the Binning values. A value of 1 gives the greatest resolution which is indicated in the ‘Capture’ Area box as the “Final preview/capture resolution” values

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Troubleshooting (also see next page for company manual troubleshooting). 1. PROBLEM: White (brightfield) light isn’t making it to the sample. CAUSE & REMEDY Lamp not on.

Turn on the lamp (1) and look to see if both bulbs come on. One lamp is under the stage (13) and the other is in the rear lamp housing (33). Turn on the mercury lamp (18) (if it has been off for at least 30 minutes). Wait 5 10 minutes for the bulb to stabilize.

Wrong light source or incorrect light path. Select the ocular path (2) to allow the sample to be seen in the oculars. Select reflected (top-side) or transmitted (underneath) white light with switch (4). Select reflected white light vs. fluorescent light with slide knob (30). Open the shutter (20) for reflected light. Intensity is too low. Dial (3) up the intensity of white light sources. Disengage the ND filters (32). Open the field iris diaphragm (13). Open the aperature iris diaphragm (22) and field iris diaphragm (21).

Select BF, DF or DIC mirrors (17) when using white light. 2. PROBLEM: White light (brightfield) is too intense. CAUSE & REMEDY “Preset’ (4) is pushed in which automatically turns on the maximum light voltage.

Deselect the button (green light will go off). Intensity is dialed up.

Dial (3) it down. 3. PROBLEM: Fluorescent light is not hitting sample correctly. CAUSE & REMEDY Lamp not on.

Turn on the mercury lamp (18) (if it has been off for at least 30 minutes). Wait 5 to10 minutes for the bulb to stabilize.

Wrong light source or incorrect light path. Select the ocular path (2) to allow the sample to be seen in the oculars. Select reflected light (top-side) switch (4). Select fluorescent light with slide knob (30). Open the shutter (20) for reflected light. Incorrect filter cube and objectives are in place.

Select filters for fluorescence (TRITC, DAPI or TRITC) (17). Use the 5x and 10x objectives (6).

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4. PROBLEM: Can not capture a digital image. CAUSE & REMEDY Camera is not turned on or stabilized yet Turn on the camera (34) at the black power switch and wait 5 minutes. Incorrect light path for the camera to access the sample. Select the ocular path setting (2) to allow the sample to be seen by the camera. Wrong driver is selected for software image capture.

Select the Cooke Sensicam Driver. Select Acquire:Video/Digital from the menu. Click the ‘Setup’ tab in the window, and select the correct driver from the pull-down menu.