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OPERATING INSTRUCTIONS FOR NIKON FLUORESCENT MICROSCOPE ECLIPSE E400

1. The microscope can operate as a bright field or as a fluorescent microscope. It has an epi fluorescent attachment housing 3 fluorescent cube filters that can be individually selected by moving the slider on the front of the microscope. The first filter cube is a multiband filter DFT for use with DAPI/FITC/Texas red stains. The second filter cube is for use with Texas red stains (excitation wavelength 540580nm, DM 575nm, BAbarrier filter 600660nm) The third position has no filter in place and is used when doing bright field microscopy. The fourth filter cube is for use with FITC stains (excitation wavelength 465495nm, DM 505nm, BA barrier filter515555nm) 2 of the objectives, 20X planfluor and 40X planfluor are for fluorescence microscopy; the 60X planapo objective is the oil immersion lens for bright field microscopy. If you need to use an oil immersion objective for fluorescent work, a 100X planfluor objective is mounted on the Nikon Eclipse E800 set up to operate DIC. Do not use oil with the 20X or 40X planfluor objectives. TO SET THE KOHLER ILLUMINATION DURING BRIGHTFIELD WORK Switch microscope on at rocker switch on the bottom right hand side of the base. Select the diascopic light source by moving the slider into position 3. Lock the slider into position by pulling it out. Put a slide onto the mechanical stage and focus using the low power (20X) objective in place. Turn the field diaphragm until the hexagonal light is in the center of the field of view. If not possible, center it with the centering rings attached to the condenser. Bring the hexagonal light into sharp focus by shifting the condenser up and down using the knob on the left hand side of the microscope body underneath the mechanical table. Once the condenser position is fixed, turn the field diaphragm until the hexagonal light just moves out of the field of view. Your microscope is now ready for standard bright field microscopy. The planflour objectives can be used for bright field microscopy, but the planapo objective cannot be used for fluorescent work.

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TO SET UP THE MICROSCOPE FOR FLUORESCENT WORK

Before starting, shield your samples from light and try and work in the dark as far as possible (dark room with red light on) Before starting, check the time the mercury lamp has been on. If the time exceeds the average operational life for the lamp (600 hours) replace the lamp. The replacement lamp is purchased from Osram. It is a mercury short arc photo optic lamp HBO 100W/2. Precautions and details for replacing the lamp are listed below. Always leave the UV lamp on for a minimum of 30 minutes and allow it to cool down for 30 minutes before starting up again. Always use nonfluorescent glass slides and coverslips. Always use nonfluorescent immersion oil (100X oil immersion objective for fluorescent work is mounted on the Nikon Eclipse E800 set up to operate DIC. Always keep the shutter closed whenever you are not actually looking through the binocular eyepiece. This helps prevent your specimen from fading. 1. Use the slider to select the filter cube with the desired excitation method into the optical path. (When using the Live/Dead Baclight kit the following filters are selected: For Syto9 use FITC filter cube; for Proponium Iodide use the Texas Red filter cube)

2. Close the shutter so that the specimen is shielded from the episcopic light source 3. Make sure the diascopic light source is switched off. 4. Turn on the episcopic light source at the transformer by pressing and holding the ignition button for ± 15 seconds. The orange light will come on briefly and turn off again. After ± 30 seconds the orange light should come on and indicate that the lamp is now ignited. 5. Center the lamp using the centering tool attached to the nosepiece next to the planapo 60x objective as well as the centering knobs on the right hand side of the lamp housing. 6. Switch off the room lights and allow only the red light to glow. 7. Prepare your specimen and place it on the stage and focus with the 20X objective. 8. Centre the field diaphragm first by lowering the field diaphragm lever. 9. Move the center of the field diaphragm image to the center of the field of view by turning the field diaphragm centering screws on the right hand side of the body in front of the lamp housing. Adjust the field diaphragm to the same size as the field of view by raising the field diaphragm lever. Once again move the center of the field diaphragm image to the center of the field of view. These steps are very important when doing photomicrography 10. Switch to the desired objective to view the specimen and open the shutter.. 11. Readjust the focus using the fine focus control 12. Use the ND filters located on the left hand side of the epifluorescent attachment to adjust the brightness of the incoming light from the mercury lamp. ND4 ND8 ND16 Brightness

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13. Apply oil if doing oil immersion. Remember to use both a planfluor objective as well as an oil immersion planfluor objective.

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TO SET UP THE CAMERA FOR FLUORESCENT PHOTOMICROGRAPHY Switch on the camera (Nikon Coolpix 990). Make sure the flash is switched off by pressing the thumbnail button marked 9. By default the flash automatically fires in poor light. Make sure you see the flash cancel sign on the LCD display. See attached figure. Adjust the image quality by pressing the thumbnail button marked 8. The options are normal, fine and basic. Adjust the image size by pressing the same thumbnail button and while keeping it depressed, turn the command dial marked 4. The options are Full, XGA, VGA and 3:2 Use the following table to calculate how many images are possible. Remember the higher the resolution, the fewer images can be stored on the memory stick. Full (2048X1536 pixels) 10 20 40 XGA (1024X768 pixels) 40 79 151 VGA (640X480 3:2 pixels) (2048X1360 pixels) 100 11 187 23 333 46

Fine Normal Basic

6. If the fluorescence emanating from the sample is not very bright, then you may need to increase your exposure time. 7. Depress the Function 1 key marked 18 on the top of the camera. 8. Keeping this key depressed, turn the black command dial marked 4 until the P on the LCD screen changes to M. 9. Release the Function 1 key. 10. Depress the function 1 key once to switch between the exposure time and F stop value. 11. Change the exposure time values by turning the command dial marked 4 from ¼ sec up to 1 sec or 2 sec depending on the level of fluorescence detected in your sample. 12. To improve the quality of you image the following settings can also be changed manually. 13. Select the mode dial marked 2 to M (manual) 14. Press Menu button marked 16 15. Select white balance and set this function up. 16. Select spot metering and set this function up 17. Once all your settings are satisfactory so that you have a good fluorescent picture in your field of view on the LCD display, (dark background bright fluorescence), open the beam splitter on the microscope, open the shutter on the epifluorescent attachment and take a photograph by depressing the shutter release button marked 1.

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OPERATING INSTRUCTIONS FOR NIKON FLUORESCENT MICROSCOPE ECLIPSE E400

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OPERATING INSTRUCTIONS FOR NIKON FLUORESCENT MICROSCOPE ECLIPSE E400