Nikon E800 Manual de usuario

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Nikon E800 Operating Instructions

Note: These instructions were written for the microscope, equipped with a Diagnostic

Instruments SPOT camera, located in the Life Science Microscopy Facility at Purdue

University.

1. The Nikon E800 Microscope.

The Nikon E800 is a fixed-stage upright microscope configured to image in

transmitted light, DIC, and epifluorescence modes. The Nikon is equipped with the

following objectives:

2X/0.1 NA Plan Apo ------- ∞/- WD = 8.5mm

10x.0.45 NA Plan Apo DIC L ∞/0.17 WD = 4.0mm

20x/0.75 NA Plan Apo DIC M ∞/0.17 WD = 1.0mm

40x/0.9 NA Plan Apo DIC M ∞/0.17 WD = 0.11-0.23mm

100x/1.40 NA Plan Apo DIC H ∞/0.17 WD = 0.13mm Oil

The last objective (shaded) is an Oil immersion objective.

The condenser is: C-CU with 0.9 N.A. dry lens (swing-out) And contains the

following inserts: A(bright field) /DICL/ DICM/ DICH

DIC lens prisms are inserted above each individual objective lens.

The Nikon also has a filter cube slider with the following filter cubes (right to left):

1. empty (VIS)

2. Red (Rhodamine): 560±20/585/620 longpass

3. 11001 Blue (GFP longpass) 470±20/500/515 longpass

3. 41017 Green (GFP) 470±20/495/525±25 narrow band

4. 11000 Ultraviolet ( DAPI) 360±20/400/420 longpass

Camera: the microscope is equipped with a SPOT RT slider CCD camera from

Diagnostic Imaging. It can be used as a B/W camera with the color filter sets

removed or as a color camera with the filters inserted.

Turning on the Microscope:

1) Sign in on the log sheet.

2) Turn on the main power and the camera power.

3) Turn the microscope power to “on”.

4) Turn on the Fluorescence power only if you plan to use fluorescence imaging.

Please note: fluorescence bulb needs `30MIN to warm up to full illumination. It also

needs 30 MIN to cool after to has been turned off before it can be turned on again.

PLEASE OBSERVE THESE TIME REQUIREMENTS TO PRESERVE THE

BULB!!

5) The computer is normally left ON. Just wake up the monitor and open the SPOT

ADVANCED software.

Turning off the microscope

1) Power off all switches (except computer)

NOTE: make sure the next user does not plan to use it before turning of fluorescence

Burner. All users should put an “F” on the sign-up sheet if they need fluorescence so the

previous user knows to leave the burner on. You must be on time for your session, as the

previous user MUST turn off the microscope completely before they leave and they will

not wait for you. Once the burner if off, you must wait 30min before turning it on again.

2) Move objective turret to 2x or 10X position.

3) Leave condenser in position “A”.

4) Leave DIC upper filter in the OUT position.

5) Clean off immersion oil from objectives using lens paper if necessary.

6) Log off computer.

7) Make sure all your supplies

are removed and area is left clean.

8) Turn off all room lights.

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Parts of the microscope:

Neutral density filters to adjust illumination

if required. They can be used in any

combination to adjust the illumination so

that it is comfortable for your eyes..

Push lever down to engage filter. Filters are

marked ND2, ND8, & ND32. They can be

used in any combination.

Illumination intensity can be adjusted with

power dial on left side of microscope. Please

note that turning the dial changes the voltage

to the lamp. This also changes the wavelength

of the emitted light. Since wavelength

determines color, this should not be used for

any color samples. Instead adjust to “SET”

position (extreme counter-clockwise) and then

use the neutral density filters to adjust the

illumination intensity.

The condenser system consists of the

condenser turret and also the DIC lower filter.

Positions on the turret are marked “A” for

brightfield and DICL, DICM, and DICH for

Nomarski DIC contrast.

The microscope power selector is on the front

bottom and the condenser field aperture is on

the lower right.

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Adjusting the microscope for Koehler illumination.

1) Insert a sample and focus it using an objective that you typically use for imaging.

Tip: Focus on the edge of the coverslip to set the

course focus. Then focus up and down using fine

focus to locate sample.

2) Stop down (close) field aperture by rotating

dial on left side of microscope base. You may see a

round circle of light through the eyepieces that has

a blurry edge.

3) Adjust the condenser height (#1: black knob on left of condenser) until the circle of

light is smaller and the edges are sharp. This will appear as the octagon (8-sided) shape

of the aperture.

4) Open up the field aperture to fill ~half the area

with light.

5) Adjust the aperture centering with the two

silver knobs (#2 below the condenser turret). When

centered, open the field aperture until the entire field

is full of light.

Note: You may have to adjust the light for other

objectives. Lower magnification objectives have a

wider opening so will require opening the field

aperture more.

6) Close the condenser aperture by moving the

slider to the right. This is the position of maximum

diffraction. This will give additional contrast to the

sample but reduces resolution.

Open the condenser aperture until the light level does

not change. This is the position of maximum

resolution and minimum diffraction. Use this position

for all higher magnification objectives.

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You have now aligned the microscope for routine brightfield imaging.

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Taking an Image in Brightfield Mode:

1) Complete the Koehler alignment. Find an appropriate area of your sample and focus.

2) Open the SPOT Advanced program.

3) Select Brightfield-grayscale or brightfield-RGB in the lower right of the SPOT screen.

4) You need to do a color balance to show the camera what is white if you are doing color

imaging. The camera program will then assign the other color values.

a. Move to a clear area of your slide.

b. Click on the color balance icon on the menu bar (on the right).

c. Proceed to follow instructions to do the color balance. The values will be saved

in the Brightfield-Color dialogue box.

5) Move back to your sample, focus using the live window, and then capture an image using the

camera icon on the upper right.

6) Select “SETUP > Image Setup” and a dialog box will appear. Click on “Modify”. This will

open the main dialogue menu for brightfield

imaging.

a. Select Exposure dialogue box and make

sure “auto” is selected. It should also

be set to:

8-bit for grayscale

24-bit for color

no binning

b. Select “auto”. This brings up another

dialogue box.

Adjustment: use this to adjust image brightness: lower # is darker image.

Gain: This is usually set at “0” for BF or DIC imaging.

7) Modify adjustment setting to darken or lighten your image.

8) Go to Post-Processing. Modify “Gamma” setting to adjust contrast

in your image. Lower # gives more contrast.

9) Take another test exposure and repeat above until you have the desired image.

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Adjusting the microscope for Nomarski DIC imaging

1) Select DIC grayscale or DIC color on the lower left of the SPOT camera program window.

2) Make sure the camera filter selector is pushed in for grayscale or pulled out for color.

3) Insert and, using the LIVE mode on the camera, focus on a sample using the objective you

intend to use for your imaging.

4) Adjust the lower DIC filter to the extreme right position.

5) Move the condenser to the correct position to match the objective you plan to use.

DICL = 10x or 20x

DICM = 40x

DICH = 100x (oil immersion)

6) Insert the upper DIC filter. Adjust the microscope light using the neutral density filters.

7) Adjust the position of the lower DIC filter until you have the amount of DIC contrast you

want.

8) Remove your slide and select “camera > Get flatfield” from the camera menu. Make a

flatfield and save it as “ff”. Overwrite the previous flatfield. This procedure takes an image

of your light path through the microscope and will subtract it from your image. This will

remove any uneven light from the final image. A new flatfield must be made if you change

anything in your setup such as objective or position of lower

DIC filter.

9) Insert your slide and proceed with locating and focusing

your sample.

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10) From the Setup menu, use “Setup > Image setup” to get into the dialogue boxes to adjust

image.

a. Select “modify” in Setups dialogue box.

b. Select Exposure dialogue box and make sure “auto” is selected. It should also

be set to:

8-bit for

grayscale

24-bit for color

no binning

c. Select “auto”. This brings up

another dialogue box.

Adjustment: use this to adjust image

brightness: lower # is darker image.

Gain: This is usually set at “1” for BF or DIC imaging.

11) Select Post-Processing dialogue box. Adjust gamma (lower # adds contrast to image).

You should see the flatfield box checked.

12) Proceed to take your image. Modify Adjustment and Gamma if necessary to get a

satisfactory image.

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Fluorescent Imaging

1. Align the microscope for Koehler Illumination as described above.

2. Locate your sample using either brightfield or Nomarski DIC imaging.

3. In most cases, the fluorescence signal is not bright enough to use camera live imaging to

focus. Thus it is important that what you see through the eyepieces is the same as is seen by

the camera. Do this as follows:

a. Focus on the line pattern in the right eyepiece with your right eye. Adjust the

eyepiece so that this looks like 4 double lines.

b. Then pick a feature on your slide and bring the image into focus with the right eye.

c. Now adjust the left eyepiece so that the feature is still in focus with the left eye.

d. You should now have both eyepieces adjusted so that the focus through the eyepieces

is the same as that of the image taken with the camera.

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