For many scientists, pictures taken at a light microscope make up a significant portion of their figures. Do you know that spending a little extra time to adjust the microscope and computer settings will guarantee the quality of the pictures so that you can easily meet any journal’s requirements? Let’s review some simple steps that you can take to make sure you have the best pictures possible.
1. Before putting your slides on the microscope stage, clean the parts that light passes. Many of the microscope parts are concealed and should only be cleaned by professionals, but you should make sure parts that you can easily see and access are clean, including your slides and cover slip. Take off the objective that you are going to use and inspect the lens. It is tiny at the end of the objective, but if you look through the other end (the end where it is screwed onto the microscope), you can tell if it has dirt on it. Use compressed air to remove loose dirt and a clean cotton swab soaked with distilled water to remove stubborn dirt. If water doesn’t work, use an appropriate lens cleaning solvent next. Dry the lens and put it back onto the microscope and place a slide on the stage.
2. Don’t rush to take the pictures yet; time to set up Köhler illumination, which ensures even illumination in the microscopic field and proper contrast. For a detailed article with pictures explaining Köhler illumination set up and an interactive tutorial, go to Leica’s Science Lab (http://www.leica-microsystems.com/science-lab/topics/basics-in-microscopy/). Here we summarize the steps as follows: turn on the lamp and focus the specimen; close the field diaphragm so the field of light is at the smallest; adjust the height of the condenser till the edges of the circle of light come to focus; turn the centering screws until the circle of light is at the center of the microscopic field; open the field diaphragm till the edges of the circle of light just disappear out of view; and finally, open the aperture diaphragm fully and then slowly close it until the field of view just turns darker. The last step is subjective, so you will need to make a judgment of the best contrast for your specimen. Note that if you switch to another objective, these steps should be repeated. If you come back to the microscope another day, Köhler illumination should be set up again.
3. Just a couple more steps till we can take that picture. As we are in a digital world, chances are your microscope is equipped with a digital camera and the pictures are shown on a computer monitor. The monitor needs to be configured properly. You should be able to adjust the brightness and contrast and calibrate the color of the display in the advanced settings of your computer by following the on-screen instructions. It is very likely that the default setting is perfect and you don’t need to change anything, but checking this will make sure the pictures appear as close as across different computer screens (think the computer connected to the microscope, the one you use at your desk, the one used by the journal editor, and more). Good thing is you only need to check this once unless someone deliberately messes up the display settings.
4. The last step before we can start taking pictures is to adjust the settings of the imaging program. Take a test picture or use the live view of the program. Is the background (empty space in the slide) white? Does the color of the staining match what you see from the eyepiece? The program might have a white balance correction feature that you can use. If the color still looks off, you will need to manually adjust the color balance by altering the numbers for red, blue, and green. This will take some time and a lot of trial and errors until the colors in the pictures match what you see from the eyepiece as close as possible, but it’s worth the effort (remember our goal? Best picture possible). The program might have a few different sizes you can choose for you picture, usually given in pixels. Always choose the largest size. This way, you can easily resize your figure to meet the resolution requirement by journals (typically 300 dpi minimum for a figure with micrographs only and 600 dpi minimum for a figure with both texts and micrographs). For example, if the largest size is 1600 x 1280 pixels, and you set it to a resolution of 600 dpi, the picture is 2.67 inches x 2.13 inches in physical size when printed. That’s a good size and allows a journal to reduce its size without affecting quality. However, if you choose a smaller size, 800 x 640 pixels, the picture will be only 1.33 inches x 1.07 inches big at 600 dpi; that might be too small when printed.
5. Finally, we are ready to take pictures! A few things to consider while doing this: always center the region of interest; avoid defective regions (a fold in the section, a tear in the tissue, etc); take as many pictures as possible so you have plenty of choices for your figure; and if the region of interest you want to show the readers only occupies a small portion of the picture, you should switch to a higher-magnification objective. You likely have control specimens and experimental specimens that you know will always appear side by side in a figure. Take pictures of them using the same equipment and settings so they look comparable. Record the magnification of every picture you take. However, noting the magnification in a figure is not very meaningful and most journals will ask you to add scale bars to micrographs. The imaging program might have the function of adding a scale bar to your picture, but I don’t suggest adding scale bars to all the pictures. The line is often so thin and lettering so tiny that when you resize the pictures, it’s impossible to see it clearly. I suggest taking one picture with the built in scale bar and all other pictures without scale bars. It’s easy to add a line of the same length to any picture of the same magnification during the figure-making process.
6. Save the pictures in TIFF format, not JPEG, if possible. You might lose information if the JPEG format is used. If you do any post processing, save as a new file so that you always have the original picture if you have to start over.
Keep these steps in mind next time you take microscopic pictures, and you never need to worry that you don't have publication-quality pictures.
(Please retain the reference in reprint: http://www.letpub.com/index.php?page=author_education_light_microscopy)