How to View Microscope Images (part 1)

Over the years, it has become more apparent to me that a lot of people do not know how to look at photos from a microscope. One of the things I try to do in my consulting work is to ensure that I don’t zoom in to a feature at a high magnification without giving the viewer a reference point so they know what they are looking at. This works well for a lot of reasons. For images that I send electronically, it is impossible for me to know at what size the image will be viewed. Every computer screen is different and different viewing software products have different default settings. So, if my client has a problem with a small screw, and the first photo of the series shows the entire screw, then they can immediately understand the magnification.

Let’s say there’s some dirt in the threads that they want to identify. The next image might be zoomed in to the portion of the threads that has the dirt. But the magnification will be low enough to show the entire diameter of the threads and, hopefully, at least three or four threads worth of length. Maybe the dirt is not visible at that magnification or maybe it's not easily visible. If it’s visible but not easily, I might put a circle or arrow showing the location of the dirt that the next image zooms in to. Let’s look at a series of images I took of a threaded part to understand more of the difficulties of looking at optical microscope images.

Figure 1 shows a screw, and I have embedded both metric- and English-system marker bars in the image. If you sent me the screw, you presumably are familiar with it. A lot of labs would have labeled this image with the magnification “10x” in the name somewhere. Well, that is the setting I used on the zoom feature. But the image will only be 10x if you’re looking at is so that the double-headed white arrow in the upper left corner is exactly 1 inch long. I have a fairly big monitor for a desk computer, and it’s over 2 inches on my screen. That would make it over 20x. If you’re looking at it on a project image on a large wall screen in a conference room, it might well be 2000x or more.

In the old days, people had standard settings on microscopes, and we got used to how things looked at those settings. Those days are gone. I resisted. I kept using Polaroids until 2003 in my electron microscope work; 2005 in my optical microscope work. I resisted as long as I did because I knew it was going to be a pain in the neck to deal with the magnifications.

I encourage everyone who has not done this before to get out a ruler and measure the 0.1 inch or the 2 mm markers, and figure out what actual magnification you are looking at. By the way, the markers are of a glass scale made for the purpose, traceable to an NIST calibration source.

Now that you have done that, look at Figure 2. This image was obtained at twice the magnification as Figure 1. If you look carefully, you can see the reddish stain at the crest of the threads in the white box. At this point, it is still fairly easy to see which areas are the thread crests and which are the roots.

Now look at another doubling of the magnification. See Figure 3. If you look at the area in the white box, we have a reference and can still see the reddish stained thread crests. But if you cover the top of the image and only look at the lower portion in the red box, good luck to someone who only saw that portion of the image, without Figures 1 and 2, to even figure out what is the thread crest and what is the root.

The reason for this is that optical microscopes have very poor “depth of field.” This is a fact of life and not due to the poor quality of my Nikon SMZ 1000 microscope. It’s a natural effect of the way light and optical systems work. For example, if you take a photo of a landscape, you can get a whole mountain range and the moon in focus. A landscape is, in comparison, extremely low magnification. It’s anti-magnification. You’re getting a mountain range of perhaps hundreds of kilometers into a 10-cm-wide image.

Figure 4 shows an example. It’s somewhere in the Kumaun Region of India. Both of the leaves right in front of the camera are in focus and the crest of the Himalaya is in the background. So are the tips of the pine trees in the lower corner, obviously far away, and the other pine-covered slopes. I might have had plenty of time to stand still and rest my arm on something to get the leaves in focus as well as the far mountain. The range of focus, or “depth of field,” is greater when I close the aperture (hole) that lets the light into the camera.