Using The Amscope Microscope Cameras

Transcript

Using the AmScope Microscope Cameras Part 1 – Setup. In order to use the camera, you will need: a) the camera system; b) a computer running the camera software. The camera system is contained in a Pelican Case (see image to right). In the case there should be the camera, the camera lens cover, two adapter rings, and a USB cable. The camera is designed to replace the ocular lens (see image below). It can also be placed in a trinocular tube, or can replace the eyepiece in a dissecting scope or spotting scope. Steps in setting up the camera. 1. Install software. The software, called ToupView, is installed on several of the department’s laptops. If it is not installed on the computer you are using, install it. The software is located on the intranet, in the Biology/General/Programs/Toupe directory. Run the autorun.exe file, and install the Camera Drivers and ToupView software. 2. Set up the microscope. Set up the microscope as you normally would. Pay particular attention to making sure all parts of the scope are clean of dust, oil, fingerprints, etc. If you have not been trained (microbiology, cell biology) in routine cleaning of the microscope, get help from a faculty member if the scope is dirty. Carefully remove the ocular from the microscope and place it in the Pelican case where the tube of the camera was. Remove the cap from the end of the camera, and place the camera into the eyepiece tube of the microscope. Do this quickly, so there is no opportunity for dust to get into the microscope or into the camera. Place the camera end cap in the Pelican case so it doesn’t get lost. The camera tube should move freely in the microscope ocular tube – if it rattles or there is excessive play (i.e. the camera tube is way too small) add one or the other of the adapter rings from the Pelican case. Using the AmScope Microscope Cameras.docx 1 3/7/2012 10:07:00 AM 3. Connect the camera and start the software. Connect the camera to the USB port of the computer using the USB cable from the Pelican Case. This should cause the computer to begin installing the driver for the camera (unless the software had already been installed and the camera used with the computer in the past). Locate the ToupView software on the computer (usually an icon on the desktop) and start it. ToupView software and important icons: 1. 2. 3. 4. Live Capture – selects, starts camera Video source properties – adjust most exposure settings here. Video stream format – adjust video image size here. Capture still image – take still images here. Note: Do NOT use the camera icon; this takes lower-resolution views. 5. ROI – allows you to select a different Region of Interest 6. IF – manual fusion – allows you to stack focus several images. 7. Live Capture window – shows a live view from the camera. Using the AmScope Microscope Cameras.docx 2 3/7/2012 10:07:00 AM Setting up the slide: 1. Put a slide on the stage and set the scope to the lowest possible magnification. 2. In the ToupView software, click on the Live Capture Icon and select the camera (it should be the only camera there). 3. The Live Capture window will now open. 4. Adjust light levels – set the microscope rheostat (light source) to 5 or 6 and close the iris diaphragm fully. The camera should automatically adjust. a. If there is a pattern of horizontal or vertical bars you are getting too much light, and the camera is taking the image so quickly that it is capturing the flickering of the light source. Reduce the light, either with the rheostat or the iris diaphragm. b. If you have too little light you will get noise  5. Focus the scope and scan for an area of interest. Center the subject and move to a higher power if needed. Readjust light if needed. Adjusting the software: 1. Be sure the video size is maximized (2592 x 1944) using the Video Stream Format icon (#3, above) 2. Open the video source properties using the icon (#2, above). 3. On the ROI tab (below) move the ROI to a blank area of the slide. Using the AmScope Microscope Cameras.docx 3 3/7/2012 10:07:00 AM 4. On the color tab, click the “One Push” button to get an approximate color temperature setting. 5. Fine tune the color setting by sliding the Hue/Saturation and Brightness controls (if needed) 6. Normally, you should not adjust the contrast or Gamma controls. 7. Move to the exposure tab. a. Adjust the Target slider to get a good overall exposure. Don’t worry too much about pure whites or blacks at this point. b. Note that as you adjust the target slider, the exposure time is adjusted. If it gets too big (above 100ms or so) then the camera delay will be very long and the camera will be slow to track moving objects (or allow you to follow the slide as you move it). If this is the case, add light via either the microscope rheostat or the iris diaphragm, being careful not to get any horizontal or vertical bars. Readjust the target slider. c. In extreme cases you may need to unclick Auto Exposure and set the exposure time manually. 8. Go to the Histogram tab. If you need to get pure whites or blacks you can adjust the histogram via the boxes below the histograms or by dragging the pink lines. Watch the image as you adjust the histogram. Using the AmScope Microscope Cameras.docx 4 3/7/2012 10:07:00 AM 9. On the Misc. tab you can make several other adjustments: a. If the video does not run or is choppy, you can adjust the frame speed level. On slower computers you may need to slide it to the right. If the computer is really slow, you may need to decrease the size of the video (use the Video Stream Format icon). b. You can try adjusting the Light Frequency if you are getting noticeable flicker. In theory, you should set it to 60 Hz. c. If you can’t read a stage micrometer, try clicking one or both of the flip buttons. This might also help you move the slide in a predictable fashion. Take the picture: 1. Use the Capture Still Image (S), NOT the Capture Image (camera) icon. 2. Save the image (File:Save As), normally as a bmp or tiff file to avoid compression. 3. See the scale bar procedure below if your image will require a scale bar. Take a video: 1. Use the menu item Capture:Start Video Capture 2. You will have to answer several questions in a dialog, including the file name – this may take several seconds to do. 3. When you complete the dialog, video capture will start, and progress can be monitored on the status bar at the bottom of the window. 4. When you have captured what you want, use the Capture: Stop Video capture on the menu (you can also set a time limit in the dialog; if you do so the video will automatically stop at that point). Notes: I have not tested the various video settings, so I can give you no advice at this point. Also, in some cases you might want to use the time-lapse icon to capture frames less frequently than the video does. This will still give the appearance of motion for things that take minutes to noticeably change. The camera will take a series of still images which can later be combined into a video. Using the AmScope Microscope Cameras.docx 5 3/7/2012 10:07:00 AM Image Stacking: You can take a number of images at different focal points and stitch them together using the ToupView software or Photoshop. This allows you to get much greater depth of field. ToupView Technique: 1. Set up the slide as you normally would. 2. For capture, use the manual fusion icon (IF). 3. This brings up a dialog. Focus to the top of the object you are interested in, and click the capture button on the dialog. 4. Focus down slightly with the fine focus knob and capture another image 5. Continue this process until you have focused all the way through the object – this may be 3-10 or more images. 6. Click on the Fusion button. The software will take 10 seconds to several minutes to work its way through the images, combining the in-focus parts of each. 7. Save the file and add a scale bar as you normally would. You should add a letter to the file name, text to the image, or data to the file’s metadata to indicate that it is a fused image. Using the AmScope Microscope Cameras.docx 6 3/7/2012 10:07:00 AM Photoshop Technique: 1. Take multiple images, starting from the top down with slight focus changes each time (as done in the ToupView technique, but manually using the capture still image icon then saving the files). 2. Initial Processing a. Close all programs except for Bridge!!!!! b. Open the folder you saved the ToupView images to in Bridge c. Using Bridge, select all the photos you want to stack d. Use the Tools:Photoshop:Photomerge menu item e. Unclick “blend images together” and click OK f. When file is completely open in Photoshop close Bridge 3. Final Processing a. In Photoshop, crop and resize image. Make image as small as possible for your needs to avoid memory problems and to decrease processing times. b. Select all layers c. Use the Edit:Autoblend Layers command and blend images together option d. Save the resulting file as a PSD file e. Use the Layers:Flatten Image menu item to flatten the image f. Sharpen and save as a JPG or other file. Notes: At present I cannot recommend one technique over the other. At first glance, using the ToupView technique is much quicker, but the image quality does not appear to be as good. Using the AmScope Microscope Cameras.docx 7 3/7/2012 10:07:00 AM Below: Image stacked with Photoshop. The algal filament to the right moved between photos and the algorithm for stacking ghosted the image with a lot of color. On the other hand, image detail is good in the areas where there was no movement. Below: Image stacked with ToupView. This image is pretty good overall, and has almost as much detail as the Photoshop stack. However, it is harder to control details of exposure in this case. Notice that the ToupView handled the ghosting of the moving filament better (although to be fair, it didn’t move as much). Using the AmScope Microscope Cameras.docx 8 3/7/2012 10:07:00 AM Scale bar procedure: If you want a scale bar on your micrograph (you should) you need to photograph something of known scale. In most cases, this will be a stage micrometer, a microscope slide with a tiny scale, usually 1mm long graduated in 0.01mm units. On a dissecting scope, you could photograph a normal millimeter scale. 1. It is essential that you photograph the scale at the same magnification and quality (pixels) that your micrograph is photographed at. Be sure to use the same capture button and record the objective in use. If using a dissecting scope, DO NOT ADJUST THE ZOOM. 2. Photograph the scale at the same magnification as your micrograph. Save the file, ideally with a file name that includes the magnification (scale_100x.bmp). 3. 4. 5. 6. 7. 8. Open the scale bar file and use the Select:All command to select the entire image Use the Edit:Copy menu command to copy the scale bar to the clipboard Open your micrograph in Photoshop. Add a new layer (call it scale bar image) Use the Edit:Paste command to paste the scale bar from the clipboard to the new layer. If needed, use the Edit:Transform:Rotate command to rotate the scale bar until it is straight – do not worry about any background that shows, or parts of the scale bar that get clipped. 9. Add a third layer (name it scale bar) 10. Use the line tool, set to a reasonable thickness (weight) and with a color that will contrast with the background of your micrograph. Holding down the shift key (to get a straight, horizontal line) draw a line over a known, representative area of the scale (for instance, 0.1 mm of the one mm scale at 100x) (see image to right) 11. Hide the scale bar image by unclicking the eye icon to the left of the scale bar image layer on the layers palette. 12. Use the moving tool (at the top of the toolbar) to drag the drawn scale bar to a blank area on the micrograph (usually the lower right corner). Using the AmScope Microscope Cameras.docx 9 3/7/2012 10:07:00 AM 13. Use the Type tool to type in the length of the drawn scale bar. Remember to place a 0 in front of any decimal point (i.e. 0.1mm instead of .1mm). The thickness of the type should be approximately the thickness of the scale bar. 14. If the scale bar and/or legend doesn’t stand out from the background, you may want to doubleclick on the layer and adjust the layer styles, Adding a contrasting stroke color is often an effective tactic. Be sure to treat both the scale bar and the text to the same style. Using the AmScope Microscope Cameras.docx 10 3/7/2012 10:07:00 AM Alternate scale bar technique: 1. Using the Analysis:Set Measurement Scale:Custom dialgog, draw a line over the image of the scale and set the scale. Click OK. 2. Use the Analysis: Place Scale Marker menu item to bring up the next dialog. Set the length as needed and set the font size. Note: the bigger the image is, the bigger the font size will need to be. It is not unusual to use a size 48 or bigger font). 3. Hide the scale bar image layer by unclicking its eye and thus showing the new drawn scale bar on the micrograph instead. 4. After the scale bar is placed, use the move tool to relocate it as needed. Using the AmScope Microscope Cameras.docx 11 3/7/2012 10:07:00 AM Putting things away: 1. Computer: a. Close the ToupView Software. b. If you are done with the computer, shut it down. c. Neatly coil the power cords. d. Return the computer to its designated place. 2. Camera: a. Unplug the camera from the cord, and unplug the USB cord from the computer. b. Remove the camera from the microscope and immediately replace it with the eyepiece. c. Immediately cap the camera and place it in the Pelican Case. d. Replace any spacer tubes used in the Pelican Case. e. Close and lock the Pelican Case f. Carefully coil the USB cable and place it in the Pelican Case g. Return the case to its designated place. 3. Slides/specimens: a. Remove any slides from the microscope. Slides should be cleaned for re-use. Coverslips can be discarded in a SHARPS BOX ONLY. b. Be sure to replace any stage micrometers where they were obtained. c. Return any specimens to their designated place in the same condition you got them (in general, lids should NOT be screwed on tight). 4. The microscope should: a. Have the lowest power objective (or blank) in place over the stage b. Have the stage raised to its highest point c. Have the eyepiece directed back over the body of the scope (pointed towards the focus knobs) d. Have the power switch turned off e. Have the cover in place f. Have the power cord coiled neatly around the base g. Be replaced where it belongs. 5. Work area: a. The work area should be cleaned up and wiped down with disinfectant. b. All materials should be returned to their designated places. Using the AmScope Microscope Cameras.docx 12 3/7/2012 10:07:00 AM