RT Slider SPOT Camera Instructions: Brightfield

Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
RT Slider SPOT Camera Instructions: Brightfield
1. Sign In your name on the Sign-Up Sheet.
2. If you are using fluorescence later, turn on X-Cite Lamp. If the lamp is ON and
someone else is signed up after you to image fluorescence, leave the lamp ON.
LEAVE THE LAMP ON FOR AT LEAST 30 MIN.
reduces the life of the bulb.
Turning the lamp on and off
3. Turn on Halogen Lamp Power Supply and set voltage to comfortable level
(e.g., 7 volts). THIS VOLTAGE SHOULD BE USED FOR YOUR IMAGE SETUP and
should not be changed.
4. Turn on Spot Camera Power Supply.
5. Turn on Computer and Monitor.
6. Log on to EMORYUNIVAD using your Emory server Login and Password.
7. Click on Spot Advanced on the desktop.
Initialize
If you turned on the SPOT power supply after you opened the program, you need to
initialize the camera in order to activate all of the capture and editing options. To do so,
select the Initialize option from the Camera menu.
Brightfield Imaging (e.g., H+E slides)
PULL OUT LEVER (A) ON RT SLIDER SPOT CAMERA TO SET CAMERA TO
COLOR. Pull out the Beam Splitter on the microscope to route the light to the
eyepieces.
Three adjustments are ABSOLUTELY necessary to obtain good brightfield
images:
1. Koehler illumination (for each objective)
2. Flatfield Correction (for each objective)
3. White Balance Correction (for each objective)
1. Koehler illumination
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Make sure to turn on the Halogen Lamp Power Supply for transmitted light.
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Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
2
Slide the filter block to position II for brightfield viewing. Pull out Beam Splitter to view specimen.
3
Make sure the moveable lens under the specimen stage is swung IN when using all
objectives higher than 5X. There is a push lever on each side near the top of the condenser;
when the lens is swung in the lever on the right is forward,
when swung out the left one is forward.
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Place a specimen on the stage (H&E is good), and focus with the objective you wish to use.
Set image to a comfortable brightness by turning the light intensity control knob located on the halogen lamp
power supply. RECORD THIS INTENSITY and use this intensity for Image Setup. DO NOT CHANGE
INTENSITY for any given Image Setup. If the voltage (intensity) is changed, the Image Setup parameters will
need to be changed.
5
Raise the condenser (under the stage) to the upper stop position by turning either knob,
the left or the right, located beneath and behind the stage.
6
Close the luminous field diaphragm (the large round lens at the base of the microscope
beneath the stage with all the light coming through it) by turning the outer ring until
the diaphragm is visible in the field of view as you look at your specimen.
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Lower the condenser (you raised it up in step 6) until the circle of light in darkness
(the luminous field diaphragm) becomes a 6 or 12 sided figure with clear edges in focus.
9
Open the luminous field diaphragm ( closed in step 7) until it almost fills the field of view
but the edges are still visible.
10
Center the luminous field diaphragm by using the two metal centering screws on the
condenser carrier (on each side oriented diagonally towards the front of the microscope).
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Open the luminous field diaphragm until the edges exactly disappear behind the field of view.
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Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
Open diaphragm
until edges disappear
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Set the aperture diaphragm:
First remove one eyepiece from the binocular tube,
then look down the eyepiece opening
while you use the sliding knob on the front of the condenser (marked from
MAX to MIN in increments) until the aperture diaphragm fills 2/3 to 4/5
of the diameter of the objective exit pupils (the field of light that you see).
If you have done all the previous steps correctly, the light will be exactly centered and
your specimen will be evenly illuminated.
Because each objective has a different field size and objective aperture, you will have to
repeat the steps 6 through 12 to set the Koehler Illumination for each change of objective.
Open Live Image
1. Click on Live image.
Click on Controls to open Live Image Controls.
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Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
Making an Image Setup for the RT Spot Slider Camera v4.0.9
Set the voltage (intensity) on the halogen lamp power supply and keep the same
intensity for each Image Setup. You will need a separate setup for each objective and
color (fluorescence or RGB). Each Image Setup that you create will also have associated
Flatfield Correction files and White Balance settings after you complete the setup.
Therefore, the next time you use the microscope, you can select your own Image Setups
from the tab at the lower right of the Spot Advanced window.
1. Double click on Spot Advanced.
2. From the menu at the top of the screen, select “Setup.”
3. Select “Image Setups” in the Setup window.
4. In the Image Setup window, choose “Add” to start a new Setup from the templates. If
you already have an Image Setup, be sure to click on Current to activate the setup you
want.
5. Or choose “Modify” to change an existing setup from the list.
6. For brightfield, choose <brightfield-transmitted light>.
7. For each color of fluorescence, choose one of these templates:
• < fluorescence>
• <fluorescence-red>
• <fluorescence-green>
• <fluorescence-blue>
8. Click “OK.”
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Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
9. In Image Setup window, choose Exposure Type: Auto or Manual for brightfield,
Manual for fluorescence.
• “Auto” for brightfield works well.
10. Bits per Pixel must be selected. 24(RGB) is the industry standard and usually selected.
11. Binning may be used for dim samples; this increases the light sensitivity, but decreases
the resolution. 2x2 combines 4 pixels into one pixel, 3x3 combines 9 into one, 4x4
combines 16 into one, thereby decreasing the download time.
12. Choose color(s) to be used. Red, green, and blue, or only one of these.
13. Click on the Auto Exposure window. Select image type, and leave adjustment factor on
“1” and Gain Limit on “1.” The White Balance numbers will be filled in here when you do
the white balance. For “Spot Metering Area” choose “Same as Imaging Area.”
14. For manual exposure, in the Live Image Controls
window, click Manual. If you have RGB images,
make Unlock the Exposure Times. fill in the time of
exposure for the colors being imaged and check
either milliseconds or seconds. Leave the gain at “1”
unless the staining is very dim.
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Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
15. In “Chip Imaging Area" window select “Full Chip." In Image Orientation, you may
select to rotate image by 90 degrees right or left, or flip image vertically or horizontally
here.
16. FLATFIELD CORRECTION:
Camera> Get Flatfield Image. The Get Flatfield Dialog box appears.
Follow the instructions in the Dialog box. Remove the slide from the field
of view.
Click Begin.
Enter a name in the File Name box.
Specify the image setup that will be associated with this flatfield image file when
using the Flatfield Correct option.
17. Click OK.
18. WHITE BALANCE:
White balance is the ratio of red, green and blue necessary to achieve the proper
color rendition for an image. You should perform a new white balance
calculation when:
Starting a new image capture session
Changing the lamp voltage
Switching to a different objective on the microscope
To compute a new white balance:
Set the illumination and the objective that you will use for the image capture.
Show the camera a sample of white light by positioning the slide so that
the specimen is not in the field of view, but light is going through the slide near
the specimen.
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Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
Camera>Compute White Balance Values or click on the icon
Click on Begin to start the calculation.
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Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
To change the setup that the new white balance values are saved to,
select a setup from the drop-down box.
19. Click OK.
20. To edit the white balance values that you saved, open the image setup (i.e.,
Brightfield). The values should appear in the Red, Green and Blue selection
bars. Edit the values as needed.
Once you have made an Image Setup, there are three ways to
take an image:
Be sure that the image is focused. You may want to view the image in full screen
mode
to aid in focusing.
1. Live Image Pause/Transfer
After pausing a live image, pressing the Transfer button saves this image to a
window.
2. Snap
Snap image captures avoid the time dedicated to computing exposure by using
the exposure time and gain
calculated for the live image.
3. Get Image (Toolbar button)
This allows you to use the live image window purely as an aid to framing and
focusing the image. Adjustments made to the live image have no impact on the
image capture
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Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
Calibration Marks
1. Edit>Add/Edit Calibration Mark.
Select a calibration setup from the drop-down box for the objective that you are
using (e.g., 10x)
2. Calibration Mark dialog box:
Set Orientation, line thickness, show text, decimals, color font.
Enter the size of the calibration mark. You can modify the size either by entering
the number in the Size box, or by using the cursor to stretch the on-screen
calibration bar.
3. To save the calibration mark that you have entered on the image,
Edit>Merge>Merge calibration mark.
To Save an Image
File>Save As
Save your images on the Emory server--NOWHERE ELSE.
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Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
Postprocessing
In the “Post Processing” window, you should check “Color Enhancement” and “Chip
Defect Correction” (for quantitative measurements of pixel brightness values you must
uncheck the defect correction.) You may want to select “Noise Filter” (default value is 50%)
to eliminate pixel values that are out of place due to electrical or thermal noise, or “Gamma
Adjust” with choices of “HSL” (hue, saturation, and luminance), HSV (hue, saturation, and
value), and RGB (gamma is applied separately to the red, green, and blue pixel values). Do
not select this in the setup. It is best to not do this until after you have saved your original
image. Then try these adjustments out in editing mode. This window is also the place where
the Flatfield Correction data are stored.
Below are images with no postprocessing adjustment (left) and histogram
adjustment (right).
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Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
At the end of your session:
a. Always File>Exit the SPOT Advanced program.
b. Always log off the computer.
c. Choose Shutdown from the START menu. Shutdown turns off the
computer.
d. Turn off the monitor.
e. Turn off the Halogen Lamp Power Supply and the X-Cite lamp (if used).
f. Turn off the RT Slider SPOT Camera Power Supply.
g. Clean up the working area.
h. If you have used the 40x, 63X or the 100X oil lenses (available from Dr.
Hilenski), clean the lens with LENS PAPER and Sparkle glass cleaner
ONLY.
i. Log the time out in the Log Book.
REMEMBER!
IMAGES SAVED ON ANY LOCAL COMPUTER DRIVES
WILL BE PERIODICALLY DELETED BY THE MiM
CORE STAFF WITHOUT NOTIFICATION. YOU ARE
RESPONSIBLE FOR SAVING YOUR IMAGES.
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Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
12
Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
13
Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
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Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
15
Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
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Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
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Hilenski 2013
Microscopy in Medicine (MiM) Core website:
http://medicine.emory.edu/MIMCore
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