Brightfield Microscopy and Image Acquisition on Spotcam1 by Ryan Taylor/Nancy Kleene Last modified 10/02/05 by Birgit Ehmer
Log onto the computer. Enter your username and password to log onto the server. If you do not have an account, go to http://microscopy.uc.edu/ choose light microscopy and fill out the new user application some days BEFORE you want to use the scope. Click OK.
Sign Up for Spotcam1 Go to www.calendar.yahoo.com On the computer at the Spotcam station, you can go directly to this website by selecting the Signup for Spotcam icon on the desktop. Enter spotcam as YahooID and signup as Password. Select Sign In. This can be done from any computer that has access to the internet. Enlargement of part of website
Sign up for Spotcam1 If today is not desired, click on the desired date using the calendar on the left. Then, click on the desired time. Enlargement of part of website
Sign up for Spotcam1 For Title, enter name, phone # Next to Timed event, select starting time. Choose Duration. Click on Save button. Enlargement of part of website
Sign up for the Spotcam1 When you are done, select Sign out at the top of the of the website.
Sign up for the Spotcam1 If your plans change, PLEASE return to the website and delete your reservation by clicking on [delete]. Then confirm by clicking on the Delete button.
Turn on Nitrogen Turn CCW Turn the large silver knob counterclockwise (CCW) to turn on the nitrogen. The nitrogen will float the table on which the scope sits. This will isolate the scope from vibrations. Do not touch the table during image acquisition. Do not turn
Turn on the Power Supply Flip the rocker switch on the PSM-4A power supply.
Turn on the Scope Turn on the microscope by flipping the rocker switch on the left side of the scope. Flipping this rocker switch forward increases intensity, backward decreases intensity.
Screen Indicates Intensity The number to the right of Lamp indicates the intensity of the lamp.
Filters Light intensity can also be adjusted by pushing in the neutral density (ND) filters on the right side of the base of the scope. The greater the value of the ND filter, the more light that will be blocked. The Green Interference Filter (GIF) enhances contrast for phase contrast microscopy. The HE filter is used with Hematoxylin and Eosin. The Neutral Color Balance (NCB11) filter is used when taking color FILM photos.
Remove Fluorescent Filter Cube The dial that can remove filter cubes is on the front of the scope, just under the ocular lenses. Rotate the silver dial to select the OPEN position (#1); it does not contain a fluorescent filter cube.
Position the Specimen Slide holder Place the specimen in the slide holder. Slide holder lacks spring. Turn the stage control knobs to move to a part of the slide that contains the specimen. X stage control knob Y stage control knob
Select Light Path and Extra Magnification Look on the upper right of the scope. Make sure the photo/ob selector knob is pulled out, so the image is sent to the ocular lenses ( OB ) instead of to the camera. Make sure the zoom dial is at the desired magnification. Increasing the magnification here does not produce as good of an image as increasing magnification by using a higher power objective. Using the 1x position (no extra magnification) is recommended.
Select Objective The objectives are in the following CW order: 2x air, 4x air, 10x air, 20x air, 40x air/oil, 60x oil. The 40x air/oil objective has a collar that should be set to 1.3 for oil and 0.8 for air.
Select Objective The 10x and 20x objectives on spotcam2 are not as good for brightfield as those objectives on Spotcam1. Sign up for spotcam1 if you need better images at these magnifications. A 1x objective is available on the shelf above the computer. Please feel free to ask for help when changing the objective. Do NOT get oil on the air objectives. Please clean oil off all objectives when done. When rotating the turret from which the objectives hang, it is very easy to inadvertently coat an unused objective with the oil that is on top of your slide. This is the number one way oil gets on air objectives!
Focus Safely Focus knobs are on either side of the scope just below the level of the stage If you raise the stage too high while focusing, you may damage the objective lens and specimen. Focus with a low power objective before using an objective with a higher power. To focus, look at the stage and turn the focus knob until the lens and slide almost touch. Then, while looking through the ocular lenses, turn the focus knobs the opposite direction until the specimen is in focus. coarse focus medium focus fine focus
Fix Fuzzy Image The number one cause of a fuzzy image is a dirty objective. See the Clean Up slide near end of lesson to find out how to clean lenses. The number two cause of a fuzzy image is a dirty slide. The number three cause of a fuzzy image is failure to use oil with an oil objective.
Set Condenser Dial You must set the condenser for the correct type of microscopy. For brightfield microscopy, make sure the condenser dial is in the O position.
If you use 1X -2X objectives, use a low power condenser The lowest power objectives do not work with the multipurpose condenser that is usually on the scope. If you are going to use only low power (1-2x), use the low power condenser. If you are going to use both low and high power, use the condenser with the swing out lens. Swing the top lens out for low power (1-4X) and swing the top lens into the light path for 10X and higher. Please feel free to ask for help when changing condensers.
If you use low power (1X-2X) - Change the Condenser Lower the condenser as far as possible using the the condenser focus knob. Loosen the condenser with the silver knob.
If you use low power (1X-2X) - Replace Condenser Make sure that the condenser lens does not hit the underside of the stage. Remove multipurpose condenser and place in its box on the shelf that is above the monitor. Put in the low power condenser by reversing the steps. Always return the multipurpose condenser at the end of your session.
Rotate Stage if desired If you loosen the silver knobs that are on the front and rear edges of the stage, you can rotate the stage. The rear knob is not visible in this picture. When done, please return the stage to the center position and tighten the knobs.
Koehler Illumination Koehler illumination occurs when the condenser and field iris are positioned to optimally illuminate the specimen. First step: Choose the appropriate objective and focus on the specimen. If the next steps are done without the specimen being focused, then Koehler illumination will not be achieved. Koehler illumination must be rechecked if a different objective is used.
Koehler Illumination The condenser is located under the stage. Adjust the condenser to its highest position using the condenser focus knob on the left side of the microscope.
Koehler Illumination - Close Field Iris To adjust the field iris, turn the ring that encircles the lens that is under the stage. Close the field iris. Most of the field of view will be blocked. Open Iris Closed Iris
Koehler Illumination - Focus Condenser Adjust the condenser focus knob to make the edges of the iris as sharp as possible.
Koehler Illumination - Center the Condenser The iris may be off center when view through the ocular lenses. Centering is easier if you open the iris so that its edge almost reaches the edge of the field of view. Center the image of the iris with the condenser centering screws.
Koehler Illumination - Set Iris Just Outside Viewing Area Adjust the field iris again so that the edges of the field iris are just outside the field of view.
Brightness and Contrast Adjust the slider on the condenser to change the size of the condenser iris. The size of the condenser iris affects resolution (best open) and contrast (best closed). View the image through the ocular lenses while changing the slider to choose the best balance between resolution and contrast for your specimen. If the specimen is well stained it is best to leave the iris open. increase resolution increase contrast
Turn On the Camera Turn on the camera by flipping the rocker switch on the SPOT box.
Set Camera for Color or B&W Pull the knob on the camera out for color images. Push it in for B&W images. For Color For B&W
Start the Software Double-click the Spot Advanced icon on the desktop to start the program If you start the software before you turn the camera on, you will get an error message.
Choose a Setup File Select a setup file from the menu in the lower right hand corner of the screen, choose brightfieldchangeablecolor for color images. (The additional instructions for taking B&W brightfield images are given at the end of this tutorial.) Modify the setup file by right clicking the setup file name and selecting Modify.
Modify Color Setup File Check the settings under Exposure : 24 (RGB) for Bits per Pixel Exposure: Manual Binning: None Use: Red, Green, Blue RGB - Color Order Check the settings unter Auto- Exposure : Adjustment factor: 1.00 Image type: brightfield-transmitted light Gain limit: 1
Modify Color Setup File Check the settings under Chip Imaging Area: Chip Imaging Area: Full Chip Check the settings under Post Processing Chip Defect Correction on Flatfield Correction on if wanted Exposure times will be set later
Send the Image to the Camera To send the image to the camera, pull out the Photo/TV slider that is on the upper right side of the scope. You want the TV position. And push in the Photo/OB slider. Forgetting to push in the Photo/OB slider is one of the most common errors in acquiring an image. Some light does get to the ocular lenses with the Photo/OB slider pushed in. Especially for brightfield photography, this may be enough to view the specimen without pulling out the Photo/OB slider.
Set the White Balance Click the White Balance icon that is on the right menu bar of the Spot window. Move to a white or specimenfree area of the slide. You may need to pull out the Photo/OB slider to get enough light to the ocular lenses. Remember to push it back in before the next step. Pull the Photo/TV slider out Click Begin.
Exposure time too short Error If you get an Exposure time too short error, decrease the light and try again. Pushing in one of the ND filters is a quick way to decrease light. These buttons are on the right base of the scope. You may also lower the bulb voltage.
Set the White Balance When the software is finished calculating, you will see a dialog box that contains the calculated white balance values. Blue will always be higher than the others. Click OK
Calculate the Exposure Time Move the specimen back into view. (Push in Photo/TV slider to view through eye pieces, if necessary pull Photo/OB slider; remember to put them back for the next step) Select Camera on the top menu. Choose Compute Exposure or use the shortcut key F10 instead. If it takes more than 20 s, check that you have pulled the Photo/TV slider out and have the Photo/OB slider in.
Calculate the Exposure Time Once the exposure is calculated, click OK. If you get an error message: Exposure time too short for camera, then you should decrease the light intensity. If you do decrease the light intensity, you must recalculate both the white balance and the exposure time.
Acquire the Image Click the Image Acquisition button on the right bar of the Spot window. Allow the camera time to take the image.
Capture Flatfield Image Your image will have dark corners. If you are not bothered by the dark corners, you may skip the following steps for capturing a flatfield image. To get rid of the dark corners, you will acquire an image without the specimen (a flatfield image) and use it to correct the image of the specimen. Before flatfield After flatfield
Capture Flatfield Image Select Camera from the main menu. Select Get Flatfield Image from the dropdown menu. Choose 1 frame to average. Remove the slide from stage without moving the stage. Select Begin. The camera will compute exposure and then expose a red, green and blue image. Save the file and click OK. If you want to check on previously saved files or location click on the
Capture Flatfield Image The flatfield image will be automatically noted in the setup file. Reacquire the image.
Focus If the picture seems out of focus, click the Focus icon Position the cursor inside the black box. Press and hold the left mouse button while moving the box to the desired location. Choose a place with detail that you want in sharp focus. Double left mouse click inside the box.
Focus Select the Filter Color that most helps you see what you are trying to focus. Often that will be green. Select Binning: None and Speed:Fast. Click Begin
Focus Allow the camera time to acquire an initial image, then begin to focus your specimen. After each focal change, wait a few seconds for the image to be updated. If image is too bright or dark, move the Exposure Adjustment slider to fix the problem. Once focused, click Close.
Focus Your previous image does not change! You must re-acquire the image unfocused focused
When to Recalculate You may use the same white balance values, exposure times, and flatfield image for other specimens as long as you do not change the amount of light reaching the specimen. Changing the field iris diameter, the condenser iris diameter, the objective, the magnification dial, the neutral density filters, or the lamp voltage are some of the things that will change the amount of light reaching the specimen.
Save the Image Select File:Save As. Or select the save icon on the right side of the spot window. Tiff Uncompressed is the best format for saving images. You may also use JPEG.but make sure you also select quality 100 Do not use Tiff JPEG
Save the Image Select a folder. Please save in a folder under my documents if you want to save them on this computer If you put files on this computer, please remove them at the end of your session. if you want to use a ZIP disk ask us
Save the Image Enter in a file name Click the Save button The following slides will tell you how to improve your image. It is important that you save an original image before making postacquisition improvements.
Add a Calibration Bar Select Edit:Add Calibration Mark from the main menu. Select the calibration setup file that matches the objective and extra magnification that you used. Note that there are two 40x objectives: plane/air and oil.
Add a Calibration Bar Choose appearance and size of bar (Color, Font, Size [um]) Click on the image where you would like the bar to be. You can move the bar around. Click Stamp when you are done. If the bar covers part you want of the image, you need to have saved an original file not to loose any detail behind the bar.
Adjust Contrast There are many ways to adjust the appearance of your image. Here s one of the most common. Select Edit:Adjust RGB:Adjust Histogram.
Adjust Contrast The X axis indicates the intensity on a scale of 0 (low) to 255 (high). The Y axis is the number of pixels with each intensity. Choose log scale.
Adjust Contrast Slide the triangles along the x axis and observe the preview image. You may need to record the new pixel values so that you can do the same thing for all your images. Click OK when done. If you would like to know more, check the Help section or ask Nancy Kleene. Preview image
Save the Setup File If you plan to use the same settings on later visits, you may save them with your name. Open the setup file by right clicking the setup file name and selecting Modify. Enter the new name and click OK.
Save the Setup File Click on Save as New. Now your setup file will appear in the list of setup files that can be accessed from the lower right corner of the screen. There is a limitation to the number of setup files that can be saved. If you are having trouble getting a change to a setup file, there may be too many of them. Let me know and I will see if some of the setup files can be removed. You can help by deleting your old setup files which you don t need any more as described in the next slide.
Delete a Setup File If you don t t need a setup file created by you any more please delete it Goto top menu Setup: Image setups Select the file you want to delete, choose the button delete and confirm
Print the Image Select file:print from the main menu. Or, click on the print icon on the right side of the spot window.
Print the Image The Tektronic Phaser is the default printer. It make prints that are good enough for lab meetings. The printer is located outside of Vontz 3332. Click Setup to get to a dialog box that will let you select landscape or portrait layout. Click Add to select all the images that you would like on a single page. Click Print to print.
Clean Up - Objectives DO NOT use Kimwipes on the scope lenses. Use lens paper or cotton swabs. Clean oil from all objective lenses. First wipe with dry lens paper. Then, wipe with lens paper that is moistened with glass cleaner. Finally, dry with lens paper. Remove any oil from your slides and from the stage with Kimwipes and glass cleaner.
Clean Up Dimpled Objectives To clean the 20x and 40x objectives, you must unscrew the objective from the turret. Call me if you feel uncomfortable doing this. Hold the objective near the table top in case you drop it. Wipe the dimple with a Q-tip that has been moistened with glass cleaner. Then wipe the dimpled lens with a dry Q- tip. Use a different Q-tip to wipe any metal areas that require cleaning.
Do Not Wipe Lenses With Dry Lens Paper The scratches on the field iris lens resulted from being wiped with dry paper. Moisten the lens paper with glass cleaner. Our objective lenses cost up to $10,000.
Clean Up Saved Images Do not leave your images on the hard drive. Move them to the server, to a zip, or burn them to a CD. Icon to start CD burning software
Clean Up OFF or ON Turn the following things OFF: Camera Scope Power supply Nitrogen Tank Desk Lamp Leave the following things ON: Computer Monitor
Shoot a B&W Brightfield Image - Choose a Setup File The following slides will explain the steps that are different for taking B&W images. Push the slider in on the camera s left side. Choose brightfieldchangableb &W. Right mouse click on the setup name and select modify.
Check Default Settings Confirm the following in the Exposure Menu: Bits per pixel Palette: Grayscale Exposure: Manual Birnning none Any filter color (does not matter) Confirm the following in the Auto-Exposure Menu: Image Type: Brightfieldtransmitted light Adjustment factor: 1 Gain level: 1
Modify Setup File Check that Full Chip is selected in the Chip Imaging Area Menu Check in the Post- Processing Menu Chip Defect Correction is selected Flatfield Correct if you would like to apply this. You will need to acquire a new flatfield image as described earlier Exposure Times will be set later.
Calculate Exposure Time You do not need to calculate a white balance. Click the F10 to calculate exposure time. Make sure the Photo/OB slider is pushed in and the Photo/TV slider out Click Begin, allow it time to calculate.
Calculate Exposure Time Once it is finished, click OK. The camera is much more sensitive in the B&W mode than in the color mode. You are likely to get the error message: Exposure time too short for camera. If so, you should decrease the light intensity. The remaining steps are similar to those already described for color images.
Got Questions? If you have any questions or suggestion about using spotcam2, please call Birgit Ehmer (300-3801; email ehmerbe@uc.edu ) or Nancy Kleene (300-480;email Nancy.koster@uc.edu )