Work Instruction WI0008 Rev: D
Operation of the Nikon Eclipse TE300
1. Materials
1.1. Name: Nikon Eclipse TE300 Microscope
2. Procedure
2.1. Warm up
2.1.1. If active vibration dampening is desired, activate the air supply to the Micro-g vibration isolation table by turning on the wall air supply. The regulator should be set to provide 70 +/- 5 PSI of pressure.
2.1.2. Turn on and warm up the light source.
Fig. 1: Image by Keck Laboratory
2.1.3. Warm up the Hg arc lamp if fluorescent imaging will be used by turning on the power switch at the controller. Allow at least 10 min. to warm up prior to imaging. Do not turn off prior to 30 minutes after powering up since this would shorten the life of the bulb.
2.1.4. Connect the desired camera to C-mount side port. For most fluorescence imaging applications, the Cooke SensiCam Cooled CCD will be necessary for sufficient signal. Turn on the camera. You may wish to check the cleanliness of the camera by focusing on a clean coverslip and rotating the camera. Any objects on the screen that are fixed relative to the camera (do not rotate) are likely dirt.
2.2. Optics selection and calibration
2.2.1. Set the light path. For fluorescent imaging, it may be necessary to switch between “A” and “C” since “D” may not provide sufficient light for both the eye or camera. “A” or “D” will be necessary for the calibration steps.
Fig. 2: Image by Keck Laboratory
A: Eyepiece only
B: Not used
C: Camera only
D: Eyepiece (20%) and Camera (80%)
2.2.2. Set optics for calibration
2.2.2.1. Turn the condenser turret to bring the “A” module into place
2.2.2.2. Make sure the polarizer is not in the light path
Fig. 3: Images by Keck Laboratory
2.2.2.3. Make sure the GIF, ND, and NCB filters are not in the light path.
2.2.2.4. Make sure the analyzer is not in the light path. There should be two clicks as it is slid within the socket: the outer one is a holder place with the analyzer out of the light path.
Fig. 4: Image by Keck Laboratory
2.2.2.5. Make sure no fluorescent filter cube set is in the light path (“R” or “G” should not selected on the block above the analyzer).
2.2.2.6. Remove the DIC prism below the desired objective if the last user forgot. Exchange it for a dust cover from an unused turret spot.
Fig. 5: Image by Stephen Cody
2.2.2.7. Turn on the microscope power. This activates the brightfield illumination.
Fig. 6: Image by Keck Laboratory
2.2.3. Eyepiece adjustment
2.2.3.1. Turn down the reticule in/out lever to bring the reticle into view. The reticle is needed for eyepiece adjustment and helps when centering the condenser.
2.2.3.2. Focus each eyepiece by turning the diopter adjustment ring until the crosshairs are visible as double lines in each eyepiece.
Fig. 7: Images by Keck Laboratory
2.2.3.3. Remove the reticule (optional, it can be useful when viewing specimen).
2.2.4. Koehler illumination setup / condenser adjustment (for brightfield and DIC)
2.2.4.1. If the oil immersion (60X) objective will be used, a special condenser is available. Otherwise, make sure the long working distance (LWD) condenser is installed under the condenser turret. Also make sure the LWD correction lens is installed.
Fig 8: Images by Keck Laboratory
2.2.4.2. Turn the nosepiece to select the desired objective.
2.2.4.3. Mount the sample and focus on the desired plane, adjust the intensity control on the microscope and light source as necessary.
2.2.4.4. Open the condenser aperture diaphragm to 60-90% by turning it to the right
Fig. 9: Images by Keck Laboratory
2.2.4.5. Close the field aperture diaphragm by sliding the lever down all the way.
2.2.4.6. Adjust the height of the condenser using the condenser focusing knob until the image of the field aperture diaphragm is focused in the eyepieces.
Fig. 10: Images by Keck Laboratory
2.2.4.7. Center the image of the diaphragm using the condenser centering screws.
2.2.4.8. Open up field aperture diaphragm by sliding the lever up until illumination just fills the entire field of view.
2.2.4.9. The condenser is now set to evenly illuminate the sample, continue with the desired magnification and light path.
2.2.4.10. Adjust the correction collar on the objective lens to achieve the best optics for the surface thickness. One way to do this is to look at an imperfection in the sample, like a dust speck. When the proper correction collar setting is selected, it will appear similar when defocusing above and below with the fine adjustment knob. At the wrong setting, the object will defocus asymmetrically. Some recommended settings are (note that you will need to measure the point spread function with this material, correction collar setting, and camera if you plan to deconvolve the images):
Material / Correction Collar SettingGold Seal No. 1 Coverglass / 0.2 mm
Polystyrene Culture Dish (~1 mm thick, 100 X 15 mm) / 1.0 mm
2 Polystyrene Culture Dishes (~1 mm thick, 100 X 15 mm) / 1.8 mm
2.2.5. Setting Differential Interference Contrast (DIC) imaging mode (optional, may not work well with plastic surfaces due to polarization by plastic)
2.2.5.1. Insert the proper DIC prism below the desired objective.
2.2.5.2. Insert the polarizer.
2.2.5.3. Slide the analyzer in its inmost position, in the light path.
Fig. 11: Image by Stephen Cody
2.2.5.4. Select the Bertram lens by turning the eyepiece wheel to “B.”
Fig. 12: Image by Stephen Cody
2.2.5.5. Focus the Bertram lens by turning the silver knob on the eyepiece wheel next to the “B” until you can see the image of the field diaphragm clearly.
Fig 13: Image by Stephen Cody
2.2.5.6. Slide the condenser turret to select the condenser module with the letter matching that on the objective (e.g., for 20X, select “DIC L”).
2.2.5.7. Adjust the analyzer, and polarizer if necessary, for cross polarization. The image should have proper cross polarization when it is darkest.
Fig. 14: Image by Stephen Cody
2.2.5.8. Remove the Bertram lens by setting the nosepiece slider back to “O.”
Fig. 15: Image by Stephen Cody
2.2.6. Epifluorescent illumination setup
2.2.6.1. Centering the Hg lamp
2.2.6.1.1. The DIC prism will influence the point-spread-function: make sure it is not in the light path if not necessary.
2.2.6.1.2. Turn off the brightfield illumination with the power button on the microscope.
2.2.6.1.3. Make sure the HA filter of the epifluorescent attachment (near the Hg lamp house) is in the light path.
2.2.6.1.4. Remove both of the ND filter sliders on the eipfluorescent attachment from the light path.
2.2.6.1.5. Select one of the fluorescent excitation/emission cubes with the selector above the analyzer (“G” or “R”).
2.2.6.1.6. Set the fluorescent field diaphragm lever to the “O” position.
2.2.6.1.7. Replace the sample with a piece of paper fir this procedure if there is a danger of photobleaching and refocus.
2.2.6.1.8. Turn the collector lens focusing knob on the lamp house until the fluorescent illumination occupies the smallest area possible
2.2.6.1.9. Center the fluorescent dot by turning the vertical and horizontal centering screws on the lamp house to align with the center of the reticle.
2.2.6.1.10. Turn the collector lens focusing knob until the illuminated area is maximum.
2.2.6.2. Centering the fluorescent field diaphragm
2.2.6.2.1. Move the fluorescent field diaphragm lever toward the “C” position to get an image of the field diaphragm.
2.2.6.2.2. Turn the fluorescent field diaphragm centering screws to bring the field diaphragm image to the center.
2.2.6.2.3. Open the fluorescent field diaphragm so it is just larger than the view field.
Fig 16: Hg lamp house and fluorescent field diaphragm adjustment.
2.3. Operation
2.3.1. For brightfield or DIC applications, the GIF, NCB, or ND filters may be desirable, see the definitions section. Generally, it will be beneficial to use the GIF.
2.3.2. Depending on the epifluorescent application, the ND filters may be desirable, especially to reduce photobleaching, see the definitions section.
2.3.3. For brightfield applications, refer to the following table for assistance in condenser turret module and objective selection.
Mode / Objective / Condenser turret module / Polarizer / AnalyzerBrightfield / 20X / A / Out / Out
Brightfield / 40X / A / Out / Out
Brightfield / 60X / A / Out / Out
DIC / 20X / L / In / In
DIC / 40X / M / In / In
DIC / 60X / H / In / In
Table 1
2.4. Shut down
2.4.1. Turn off the illumination on the microscope with the power button by the focusing knob on the microscope.
2.4.2. If DIC optics were used, switch back to normal optics.
2.4.2.1. Remove the analyzer from the light path. It you should feel the holder click into the position without the analyzer as you slide it out.
2.4.2.2. Remove the polarizer from the light path.
2.4.2.3. Select module “A” on the condenser turret.
2.4.2.4. Remove the DIC prism from the light path.
2.4.3. Turn off all light sources. The Hg lamp should not be run for an interval shorter than 30 minutes.
2.4.4. Remove fluorescent filter blocks and ND filters from the light path.
2.4.5. Remove GIF, ND, and NCB filters from the light path.
2.4.6. If the vibration isolation table was used, turn off the air supply at the wall.
3. Definitions
3.1. Analyzer: One of the four special components of the DIC optical system. It is a polarizing filter oriented perpendicularly to the illumination polarizer that filters all but the light rays whose phase changed while traveling through the sample. The analyzer effectively transforms changes in polarization into changes in intensity, which the eye can detect.
3.2. Condenser: “An external auxiliary lens is used to condense the light from a light source so that the object is brightly and uniformly illuminated. The usual purpose of a condenser system is to make sure that as much light as possible coming from the object goes through an optical system [Answers.com].”
3.3. Condenser aperture diaphragm: “The aperture diaphragm acts essentially as a control for resolution and contrast in optical microscopy. By varying the size of the diaphragm opening, the illumination cone projected into the objective is changed. Opening the diaphragm too much results in glare and loss of contrast, while closing it too far results in increased diffraction and loss of resolution. An intermediate position is optimum, which corresponds to an opening size of between 60 and 90 percent [Olympusmicro.com].”
3.4. Condenser prism: One of the four special components of the DIC optical system. It splits polarized light into two perpendicularly polarized rays separated by a small distance. A second prism recombines the rays. If the rays encountered similar refractive indices, they are unaltered and recombine to the original ray. If two different refractive indices were encountered, the polarization of the recombined ray will change relative to the original.
3.5. Field diaphragm: “This aperture iris diaphragm controls how much light enters the microscope [Olympusmicro.com].”
3.6. Green interference filter (GIF): creates green monochromatic light by the destructive interference of other wavelengths. It narrows the illumination band of microscope and theoretically improves DIC optics, but this effect is generally small and it can be removed from the light path to improve illumination. They also remove UV wavelengths to improve cell viability while imaging for extended periods.
3.7. Heat absorbing (HA) filter: Mercury arc lamps produce a considerable amount of heat. Heat absorbing filters removes infrared radiation from the light to protect other optical components and the sample.
3.8. NCB filter: “Daylight filter” that matches the transmitted light to have a similar distribution of intensity a function of wavelength as daylight. This distribution is termed the color temperature; the term is derived from the distribution of light emitted by a black body radiator at a given temperature. Most color optical capture equipment requires the NCB filter be used so the color temperature of the illumination matches what the device was designed for.
3.9. Neutral density (ND) filter: Transmits a limited percentage of incident light equally across all wavelengths, effectively dimming the illumination. For example, ND-70 filters transmit 70% of the incident light at each wavelength.
3.10. Polarizer: One of the four special components of the DIC optical system. It polarizes the incident light.
4. References
4.1. Additional Nikon Eclipse TE300 Protocols (Images for this protocol obtained here):
4.1.1. http://sols.asu.edu/klab/pdf/videoscopeinstructions3.pdf
4.1.2. Stephen Cody. Setting up Normaski DIC. 2001. http://www.ludwig.edu.au/confocal/Local/FullDIC_TE300.PPS
4.2. Articles and books
4.2.1. Davie, J.T., M.H.P. Kole, J.J. Letzkus, E.A. Rancz, N. Spruston, G.J. Stuart, and M. Häusser, Dendritic patch-clamp recording, Nature Protocols 1(3) (2006) 1235-1247.
4.2.2. Lanni, F. & Keller, H.E. Microscopy and Microscope Optical Systems (Eds. Yuste, R., Lanni, F. & Konnerth, A.) Cold Spring Harbor Laboratory Press, 1999.
4.2.3. Inoué, S. and K.R. Spring. Video Microscopy: The Fundamentals. 1997, Plenum Publishing Co.: New York.
4.3. Web References
4.3.1. http://micro.magnet.fsu.edu/primer/
4.3.2. http://www.answers.com/topic/optical-microscope
4.3.3. http://www.olympusmicro.com/primer/anatomy/anatomyjava.html
4.3.4. http://www.fhcrc.org/science/shared_resources/imaging/microscopy_tutorial2.pdf
4.3.5. http://www.microscopy-uk.org.uk/mag/artnov04/pjtube.html
5. Revision History
Date Rev Description Revision Author
11/24/06 - Initial release Brian J. Schmidt
11/26/06 A Elaborated on DIC setup Brian J. Schmidt
2/10/07 B Revised steps in procedure for logical flow Brian J. Schmidt
2/12/07 C Added pictures of the Hg lamp house and Brian J. Schmidt fluorescent field diaphragm.
9/01/07 D Added information about correction collar Brian J. Schmidt settings. Updated format.
bme.virginia.edu/lawrence Date: 9/01/07 1/10