Nikon Eclipse TE200 microscope

Last updated: 7/31/12 By: Tim Starr

Equipment attached to microscope:

High Definition, cooled color camera: Nikon Digital Slight DS-Ri1 and Control unit for PC: Digital Sight DS-U3 unit

The DS-Ri1 realizes an image quality of 12.7 megapixels and 2200 TV lines with the pixel shift method (but is only 1 megapixel). Superior color reproduction capability allows recording of accurate specimen colors, and smooth live image display at a high frame rate of max. 19fps makes focusing easy. Because the CCD is cooled at 10 °C below its uncooled state, heat-induced noise is reduced, and this ensures clear fluorescence images.

The high-speed interface IEEE1394b emphasizes the total performance of the camera. Optional NIS-Elements imaging software enables the effective capturing, management, processing and analysis of images and movies, and data exporting. NIS-Elements also supports microscope and peripheral device control and it offers the option to build-up a microscope system.

http://www.nikon.com/products/instruments/lineup/bioscience/camera_microscopy/digital_sight/

Mercury Light Source for Fluorescent viewing: Nikon Intensilight C-HGFI

Precentered type—no lamp alignment required

Long life lamp—average lifetime as long as 2000 hours

Fiber connection—less heat and electrical noise conducted to microscope body. Ideal for time lapse and other lengthy observations

DC (direct current) lighting-constant, non-fluctuating light intensity

http://www.nikon.com/products/instruments/lineup/bioscience/biological-microscopes/accessory/fiber_illuminator/

Halogen Lamp and Power Source (Nikon Model TI-PS100W/A)

Lenses: 60x, 40x, 20x

Fluorescent filter box (see image below, our microscope has four filter blocks, open, DAPI, FITC and TRITC): UV-2E/C* DAPI (EX 340-380, DM 400, BA 435-485), FITC-HYQ (Ex 450-500, DM 505, BA 510-560), and TRITC HYQ (Ex 530-550, DM 565, BA 590-650).

Ex = excitation filter wavelengths. Only light within the wavelengths indicated will pass through the filter.

DM = Dichroic mirror wavelength. Dichromatic beamsplitters (dichroic mirrors) are specialized filters which are designed to efficiently reflect excitation wavelengths and pass emission wavelengths.

BA = Barrier filter emission wavelength. Barrier filters block (suppress) shorter wavelengths and have high transmission for longer wavelengths. When the filter type is also associated with a number, e.g. BA475, that designation refers to the wavelength (in nanometers) at 50% of its maximum transmission. Curves for barrier filters usually show a sharp edge at the left side, indicating the blocking of wavelengths to the left of that edge (see Figure 2(b)). Modern barrier filters are generally the interference type, many of which are band pass with sharp cut-offs at both the left and right sides of the transmission curve.

Instructions for using microscope:

• Turn on mercury (Intensilight C-HGFI) lamp for fluorescent viewing (first on, last off). Let lamp warm up for 15 minutes, otherwise you won’t get consistent images. This lamp is expensive and will wear out. If it is going to be used throughout the day, leave it on. IT IS NOT GOOD TO TURN ON THE LAMP IF IT HAS NOT COOLED TO ROOM TEMPERATURE. Do not kink the cord. Do not turn on and off repeatedly, if possible as one start is the equivalent of 3-4 hours of operation. The expected lifetime of the bulb is 2000 hours. Reset hour counter after replacing bulb. Open the shutter by turning shutter knob to open circle. The ND (neutral density) knob controls the percentage of light that comes through: 32 = 3% (1/32), 16 = 6.25% (1/16), 8 = 12.5% (1/18), 4 = 25% (1/4), 2 = 50% (1/2), 1 = 100% (Not reduced). Generally set at 100% (1) unless you want less light.
• Turn on power supply. Note: you must turn the switch on the power supply box and the switch on the microscope (lower left). This provides power to the Halogen lamp. Note, the Min/Max knob on the power source is not functional because control of the power source is given to the microscope because the external control cable is connected. To turn the light source up and down, use the Min/Max knob on the lower left side of the microscope. You can also turn the power on and off using this switch
• Turn on computer and monitor. Note: The Nikon camera (DS-Ri1) is powered by the computer through the cable.

Brightfield Imaging

• Select filter set (GIF, ND, NCB). GIF = green interference filter, ND = neutral density filter, NCB = neutral color balance filter. For most applications: GIF is out (not used) otherwise images look green, ND is out (not used) otherwise there will be less light, and NCB is in (used) to give more natural color
• Open the field diaphragm by pushing lever next to filters up.
• Set condenser ring (round turret just above the stage) to A (brightfield) and minimize the condenser aperture by pushing the small black bar just above the letter “A” all the way to the left.
• “C” stands for closed and will block the halogen light source.
• The condenser ring also contains PhL, Ph1, and Ph2. These are to be used for phase contrast illumination, but they only work if the lens matches. Read on if you want. The phase contrast annuli (annulus is the Latin term for ring, with the plural being annuli) utilized in the substage condenser on an upright microscope, or within the condenser turret on the illumination pillar of an inverted microscope, must be specifically matched to a particular objective equipped with a corresponding phase plate. For example, a 20x objective and a 100x objective, both of which contain a phase plate near the diffraction plane (rear focal plane), will require condenser annuli having different diameters that correspond to the objective magnification and numerical aperture. By matching the condenser annulus to the objective phase plate, the microscope can be aligned to superimpose illuminating light rays passed through the annulus onto the objective phase ring to achieve phase contrast illumination. The Ph1 (or equivalent) condenser annulus, which contains the smallest aperture, is designed to be employed with the lower power 10x and 20x objectives. Intermediate magnification objectives (40x and 60x) utilize the Ph2 annulus, while the highest power (and numerical aperture) 100x objectives require the Ph3 annulus, which contains the largest aperture. A specialized condenser annulus (PhL) is utilized with low-magnification objectives (4x and 5x) when the condenser swing-lens is removed from the optical pathway or in long working distance condensers designed for inverted tissue culture microscopes.
• The eyepiece wheel (O, M, B, C). Normally set at O for open. Can use M setting to magnify through eyepiece, but this will not magnify the actual image in the camera. The B setting is for “Bertram” lens used to set up DIC images. The C stands for “closed” and shuts off light to eyepiece
• Place your speciment on the stage
• Select the desired lens (20x, 40x, 60x), by manually turning the lens turret under the stage. Make sure the lenses are screwed in tight. BE VERY CAREFUL USING THE 60X LENS and it requires oil
• Flip the eye-camera lever (lower right side of microscope) to the "eye" setting. Otherwise you won't see anything in the eyepiece.
• Slide the lever on the fluorescent filter box all the way to the left (no filter).
• Focus image

Fluorescent Imaging

• Turn on the Nikon Intensilight C-HGFI power source. Open the shutter by turning shutter knob to open circle. The ND (neutral density) knob controls the percentage of light that comes through: 32 = 3% (1/32), 16 = 6.25% (1/16), 8 = 12.5% (1/18), 4 = 25% (1/4), 2 = 50% (1/2), 1 = 100% (Not reduced). Generally set at 100% (1) unless you want less light.
• Focus image using brightfield light first, find field of view that you want.
• Turn off halogen lamp (button on lower left of microscope).
• Slide lever on fluorescent filter box to either DAPI (uv), FITC (green), or TRITC (red).
• Refocus image.

Capturing image using the computer program NIS-Elements

• Turn on computer and monitor
• Open the program "NIS-Elements"
• Set the working folder: File -> Options -> Select your working folder. I usually leave the other settings as they are, but you can change the file format for autocapture if you want.
• Use the Tools menu to set the scale bar depending on the lens you are using, and toggle the Scale button to on.
• Use the DS-Ri1 Settings menu on right to set resolution and exposure. Generally set FAST to 1280x1024 Fine and Normal, and set QUALITY to 1280x1024 Fine - 8bit.
• For brightfield generally want to use Autoexposure.
• For red & green fluorescence, generally want to manually set the exposure (normally around 1 s), especially if you are comparing different images for intensity, you need to keep both the exposure time and analog gain constant.
• For brightfeld press the Auto White button.
• For Scene Mode, it usually doesn't make much of a difference, but you can play around with the difference Scene Modes if you want.
• I don't usually touch the Commands button.
• Flip the Eye - Camera lever (lower left of microscope) to "Camera" (towards you).
• From the Camera menu on the right side of the computer screen, click on "Live".
• You should see the image on the computer. If it is what you want, click on Freeze, then click on Capture. Then Click on Save As on the menu at the top, name the file and click save.
• Be sure to name your files with a name that will make sense (e.g. Pten860-lng-20x-bf-1, which is the mouse #, tissue, lens used, brightfield, first picture). Also save the pictures in a folder with a meaningful name, and the folder should be in your personal folder!