Image acquisition:
The slide:
Clean the slide. Really. When we look through the scope, we unconsciously edit out dust and fingerprints and debris, but it is distracting in a photomicrograph. It will also be a lifesaver when getting the brightfield image discussed below.
Kohler illumination:
It’s a thing. Do it. You know what it is. If you don’t know, look it up. Ask your attending physician to show you. You need to have the light path working right.
Focus:
To get the best focus I can, I choose my field and then zoom the camera display to as high a magnification I can. It’s almost always fairly blurry, so I choose an area of high contrast and focus at this high magnification. Use the computer monitor for focus. Then I zoom back to no zoom and check my field. Take your photographs at a higher magnification than your plan to use for your presentation. I usually make a panorama image one or two objectives higher in mag, and then shrink it down after I’ve taken my photo.
Exposure:
I choose my exposure with postprocessing in mind. This is the one place where taking the best looking image is not my goal. Instead, I underexpose a little. The reason is that in my experience, when I make an image as bright as I like it, I max out a lot of the pixels and make them all white, but if I make it a little darker, I *don’t* tend to make too many pixels black. I don’t know if it’s generalizable, but when I stretch the histogram a little (I’ll talk about that later), it works better for me to lighten a slightly dark image than try to darken a too-bright image. So, for instance, here’s a typical raw image I take:
It looks dark, doesn’t it? But… here’s the histogram. For those of you who don’t know, the histogram of the image is a graph of the number of pixels at any given brightness level. For a red/blue/green color image, the histogram is usually that of a computed luminance or greyscale value. You can, of course, look at the histograms of the individual color components if you need to. In any case, here’s the histogram of this image, taken from the “curves” function in GIMP, my default image manipulation program:
Note that most of the pixels are just a bit higher than half the maximum intensity. That means that I can now stretch the pixels to make them both brighter and darker without losing information. A simple histogram stretch, for instance, results in this:
I won’t do that, of course, because the background illumination is still anisotropic. Instead, I’ll correct for the anisotropic illumination (in the next installment), and that will *also* take care of the dynamic range issues:
At this point, how much to stretch the dynamic range is a matter of aesthetics, which I’ll get into later. The point is that even though the original image is “underexposed,” it doesn’t end up that way.
Playing games with the field and condenser diaphragms:
Make sure the entire field is illuminated. I see a lot of photomicrographs where you can see the edges of the field diaphragm resulting in a circular image or image with the corners blacked out. Not only does it look bad, but it makes some of the postprocessing harder. Playing with the condenser diaphragm can increase refractivity of the image. Sometimes this makes the image aesthetically more pleasing, sometimes not.
Take a brightfield image:
It’s almost a given that illumination of the field will not be uniform. Traditionally, that means that the center of the field will be brighter than the edges. That might not be true for these new LED scopes, but I think it is. It’s certainly true for my scope. The human brain is surprisingly good at accommodating this, and it may not be obvious while looking through the eyepieces of the microscope, but it can be obvious in the resulting photomicrograph. You can get rid of this by postprocessing if you have a brightfield image.
To take a brightfield image, you just take a photo of a field where there is nothing but clear glass. Since you will be processing to remove problems with the illumination used for taking the photomicrograph, you need to take the brightfield image without changing the focus or illumination level.
This isn’t as easy as you might think. Almost all of my slides have copious scratches, dust, etc. so it’s hard to find a place where it really is nothing but clear glass. The easy near-fix is to just move the slide off the scope and take a picture without anything there at all, but it turns out that the slide and coverslip change the lighting just a little, and this is less of an exact solution. If you take a picture with a speck of dust in the field, you will have to remove it in postprocessing.
Maybe take a darkfield image:
A darkfield image is an image taken with the lights off. Mostly the purpose is to remove “hot” pixels in post processing. These are pixels that are not actually black when there’s no illumination. You can also have unwanted currents running around that provide uneven darkness. In my camera, it’s not a significant problem, and I often don’t do this. Many photomicrographs are pretty busy, and one slightly off pixel is not really noticeable. But if you’re a purist, then go for it.
Maybe take multiple exposures:
I’ll talk a bit later about high dynamic range imaging. I’ve had mixed results with this, but you might like to play with it.
Maybe take multiple focus planes:
At high magnification, you can focus on multiple levels in the tissue. We pathologists do this as a habit by focusing up and down as we look at the slide. It’s almost unconscious, but if you watch most pathologists, they are constantly focusing up and down *just a teeny bit*. This allows us to focus up and down through the tissue. Another thing you can play with is “focus stacking” where you take an image at different focus layers and then combine the in-focus parts of each level to create one image where everything is in focus. I’ve had mixed results with this, too, but it’s fun to play with it sometimes.
The table:
Put the microscope on a firm foundation. You don’t want vibration in longer exposures.
The shutter release:
Use a remote shutter release. Pressing the button on the camera will cause vibration.
Monitor:
Use a monitor to display the image that the camera will take rather than trying to see things through the tiny viewport. You can see how good or bad your focus is much better on a big screen.
ISO/Shutter speed/aperture issues:
In order to collect light, three things work together.
- The efficiency of the sensor. This is set using ISO. A “slow” ISO is not as sensitive to light as a “fast” ISO. The upside of the fast ISO in the real world is that it is fast, so you can take pictures of moving objects like thrown baseballs, and it will minimize the blur. The downside of a fast ISO is that it builds an image from fewer photons, and that means that there will be more noise in the image, which makes it look grainier. It turns out that for classical noise, the amount of noise decreases with the square root of the number of samples. Thus taking four samples will halve the noise. For microscopy, time is cheap, so use a slower ISO. How slow depends on the camera.
- The shutter speed. The shutter speed is how long you collect photons. For a “slow” ISO you need a longer shutter speed. For my photography, I set the ISO and adjust my shutter speed to get the exposure I want.
- Aperture: This is how big the pinhole is that lets light onto the sensor. Little holes allow fewer photons, bigger holes allow more. This is not an issue with me, since I don’t have a lens on the camera. Instead, it’s attached to the microscope optics directly. But if I did have a lens with an aperture on my camera, I would have to adjust it as well.
For me, the rule of thumb is slower ISO and longer exposure. YMMV, of course. A longer exposure may not work as well for you if you have a jiggly table and vibration artifact is an issue. The real bottom line, though, is that modern cameras are so fast that it’s hard to screw it up, and it’s not much of a consideration any more. If it looks good, it is good.