CAPTURING IMAGES ON THE HIGH-MAGNIFICATION MICROSCOPE

Transcription

1 University of Virginia ITC Academic Computing Health Sciences CAPTURING IMAGES ON THE HIGH-MAGNIFICATION MICROSCOPE Introduction The Olympus BH-2 microscope in ACHS s microscope lab has objectives from 2x to 100x. It is equipped with a digital camera connected to a computer. This setup makes it possible for researchers to capture digital images for analysis, manipulation, and publication. In order to obtain accurate, high-quality images, the microscope, condenser, and camera must be properly configured and adjusted. This document describes the setup and operation of the highmagnification microscope and digital camera when capturing images of specimens with unpolarized, visible light. Setup is divided into three sections: initial set up, condenser adjustment, and camera use. The exact method of condenser adjustment depends on the desired level of magnification. These instructions start with the lowmagnification setup and then add other steps to adjust for highmagnification viewing. Initial Setup These steps are necessary for viewing brightfield specimens at any magnification. Refer to the picture at right. A. Turn on the power supply for the light source (1), and set it to 9 Volts (2). B. Put the beam splitter rod (3) in the center (green) position. This divides the light between the eyepiece and the camera. C. Pull the fluorescent cube selector (4) all the way out. This removes the fluorescent filters from the light path. D. Place the light filter labeled LBD-2 (5) over the lower lens. This makes whites whiter and brights brighter. E. Rotate the field iris ring (6) clockwise to the fully open position. For steps F H, refer to the photo at left. F. Rotate the condenser selection ring so that notch 1 (7) is under the white mark. G. Rotate the polarizing selection ring so that the range labeled AS (8) is under the white mark. H. Rotate the condenser iris adjusting ring (9) to position 0.9 (fully open). For steps I, M, and O, refer to the photo at right. I. Raise the condenser lens lever (10) to move the lens out of the light path. J. Rotate the lens turret (11) to a low-power objective, either 2x or 4x. K. Place your microscope slide on the microscope stage (12) with the stain or cover slip on top. L. Move the eyepieces (13) to set the proper interpupillary distance. M. Close your left eye, and use the coarse (14C) and fine (14F) focus knobs to bring your specimen into focus in the right eyepiece. N. Close your right eye, and rotate the outer ring of the left eyepiece to bring the specimen into focus in that eyepiece as well. O. Use the stage translation knobs (15V,15H) to move your specimen in the field of view. The top knob moves the image vertically; the bottom knob moves the image horizontally. Adjustments for the 2x and 4x Objectives (and Sometimes 10x) At low magnification, the condenser does not have much effect on the lighting of the specimen. 1. Adjust the fine focus if you change objectives. 2. Contrast is improved by gradually closing the field iris (6). Resolution is improved by gradually opening the field iris. ACHS /28/03

2 Condenser adjustment for the 10x, 40x, and 100x Objectives At higher magnifications, the condenser is used to concentrate light on the specimen and attenuate unwanted light. (Use of the condenser with the 10x objective is a borderline case.) The following procedure describes standard Kohler illumination. Refer to the photo at right. 1. Swing the condenser lens lever (10) down to put the lens in the light path. 2. Rotate the lens turret to the desired objective. 3. Use the coarse and fine focusing knobs (14) to bring the specimen into focus. 4. Rotate the condenser iris adjusting ring (9) to position 0.2 (the smallest aperture). 5. Close the field iris (6) to the smallest aperture. The image should be a small circular region of white on a black background. 6. Turn the condenser focusing knob (16) until the boundary of the circular region is in sharp focus. 7. Gradually open the field iris (6) until its image nearly reaches the outer edge of the field of view. 8. Center the field iris image in the field of view by rotating the two condenser centering screws (17). 9. Open the field iris (6) just enough to illuminate the entire field of view. 10. Remove the right eyepiece from its socket. Use the condenser iris adjusting ring (9) to reduce the iris aperture to approximately 80% of the full field while looking into the empty eyepiece socket. Return the eyepiece to its socket. 11. To increase contrast, close the condenser iris (9) slightly. To improve resolution, open the condenser iris slightly. A tutorial on Kohler illumination can be found on the web at Focusing the Digital Camera 1. Focus the camera separately from the eyepieces by using the grooved ring near the top of the microscope column. (See photo at right.) Capturing an Image Digital photos are made by importing images into either Adobe Photoshop or Image Pro Plus. Save files on your own Zip disks or on the Meduser server. This computer is also equipped with a CD burner. Files may be saved on the computers E: drive but are subject to deletion at any time!!!. 1. Log in to the computer using the name and password listed on the monitor. 2. Click on the icon for Adobe Photoshop or ImagePro Plus on either the desktop or the Start Menu. 3. Follow these instructions: a) For Photoshop: Click on File Import Twain DPController Data Source. b) For ImagePro Plus: Click Acquire Scan 4. Click the Play button to activate the live preview. 5. Click on the User Settings tab. Capture Button User Settings Tab ACHS /28/03

3 6. Click the Load button. 7. A browse window will appear. The window should default to the Microscope Settings folder. Click on the file named Default.env and click the Open button. If the window does not open to this folder navigate to E:/Microscope Settings/. Selecting this file will reset the software to the recommended settings. Users may save their own settings in this folder as well. It is highly recommended that you save this settings file on a disk or CD as this drive could be erased at any time. 8. A yellow box labeled Spot will appear on your image. This is the area the software will use for White Balance. You can move it around the image to a lighter or darker area if you wish. This box will not appear on your captured image. Top Button Bar One Touch White Balance One Push White Balance Clip Region For Brightfield Microscopy Use these instructions 9. Click on the One Push White Balance button on the top button bar to balance the colors of your image. This should be done for each slide. If you wish to select the area of your image to use for white balance select the One Touch White Balance tool from the top button bar. The cursor will change to an eyedropper. Click on the area you wish to use for white balance. 10. Capture the image : Once the settings are satisfactory, click on the Capture button to capture the image. a) If you wish to capture an area smaller then the entire screen click on the Clip Region button on the top button bar. A yellow box will appear over your image. Only the area within that box will be captured. You can move and resize the box to your desired area. To turn off this feature simply click on the Clip Region button again. 11. Save the image : On Photoshop s top menu, click on File Save As. Then choose a folder and filename for the new file. Repeat steps 9, 10, and 11 for each subsequent photo. ACHS /28/03

4 For Florescent Microscopy Use these instructions It is recommended that you use a manual exposure for florescence. 9. Click on the Capture tab and select Manual from the Exposure Mode section. 10. Adjust the exposure by sliding the Exposure Time slider to the left or right until the desired image is obtained. 11. Capture the image : Once the settings are satisfactory, click on the Capture button to capture the image. a. If you wish to capture an area smaller then the entire screen click on the Clip Region button on the top button bar. A yellow box will appear over your image. Only the area within that box will be captured. You can move and resize the box to your desired area. To turn off this feature simply click on the Clip Region button again. 12. Save the image : On Photoshop s top menu, click on File Save As. Then choose a folder and filename for the new file. Repeat steps 10, 11, and 12 for each subsequent photo When You Are Finished Please clean up after yourself. 1. Close Photoshop or ImagePro. 2. Turn off the light source (and the fluorescent light source, if you used it). 3. Remove your specimen from the stage. 4. Clean up any immersion oil, if you used it. ACHS /28/03

5 Troubleshooting I can see the specimen in the eyepieces, but nothing appears on the monitor. Make sure of the following: The light source is set between 9V and 10V. The beam splitter selector is in the center (green) position. The image s colors are strange (yellow, red, pale, etc.), even in the eyepieces. There are many potential remedies: Make sure that the condenser is properly configured for the magnification you are using. The most frequent error is that the condenser lens has not been swung into the light path. Make sure that the purple light filter labeled LBD-2 (shown in photo at right) is in place over the field iris lens. Make sure that the fluorescent filters are removed from the light path (selector bar out). Turn the light source to 9 Volts. There is a noticeable bright spot near the center of the image. Try the following two remedies. If neither of these helps, find an ACHS staff member. Clean the objective lens and other exposed glass in the light path. If the problem persists, gradually open the condenser iris toward the 0.9 setting until the bright spot disappears. I am using the 40x objective, and I cannot bring the specimen into satisfactory focus, even after cleaning the objective and adjusting the camera focus ring. The 40x objective has an additional focusing ring (see photo at right) that corrects for the thickness of the cover slip. Turn this ring (without unscrewing the lens from the turret) until optimum focus is attained. There are ugly dark spots or smudges in the image. If the condenser is properly adjusted, then something is dirty. Cleaning implements abound. Glass cleaning solution (little pink bottles) for greasy surfaces Long-handled cotton swabs Lens paper and low-lint tissues Pressurized air in an aerosol can for dust (Hold the can LEVEL while spraying!) Here are the candidates for cleaning: Your slide. Clean it as you like. The condenser lenses. Blast them with pressurized air. The objective lens. These can be unscrewed, cleaned, and blasted with air. ACHS /28/03