When consulting on an inverted microscope purchase, one question I always ask is whether the lab intends to use coverslip bottom dishes or plastic vessels. In approximately 9 of the 10 conversations the investigator says, “both.” How silly of me to ask.
Many companies would simply provide a long working distance lens that was designed for plastic, but worked just fine for glass as well. However, many years ago, Leica designed a lens with a correction collar that would allow the user to account for the different properties of glass or plastic – particularly thickness. The user simply needs to twist the lens housing to adjust internal lenses up and down. Whether the lenses are “up” or “down” will align the lenses to provide the sharpest image possible for the vessel being used. Thankfully there are even handy little markings on the lens that give the user reference points for lens adjustment.
Inevitably I will visit the lab months, or in many cases years, later and users complain that the 40X lens is bad. Other users say, “We don’t use the Leica because the 40X lens doesn’t produce the images we need.” Comments like this leave the 40X microscope lens feeling sad and forlorn. Fear not, the 40X lens has great resolve-ing power. If you listen closely, you can hear it whispering, “What you perceive as my biggest weakness is actually my greatest strength.”
I certainly hear the whisper when I walk into the lab, which is why I stand up in support for the 40X correction collar lens: “You Sir, were designed for a greater purpose!” Once the new users are educated on the virtues of adjusting the 40X lens, they see the difference in a properly adjusted lens in all lighting techniques – even low-resolution techniques such as phase contrast. Among the many benefits the user will notice are increased resolution, improved flatness correction, and increased brightness. In fact, once the lens is correctly adjusted, many people can’t help but say, “WOW!” This is uplifting for the lens and motivates it to perform at its best!
The difference is critical for techniques such as Differential Interference Contrast (DIC) and fluorescence. DIC depends on correct optical adjustment because of the light sheer that takes place in the prisms and polarizers, which are required for the technique. If the lenses are out of alignment, the angle of the light sheer is not optimal; the image looks fuzzy, and lacks contrast.
For fluorescence microscopy, light is at a premium and lenses that are out of alignment do not transfer light efficiently. They also take the single points of light that are generated by fluorescent labels and turn them into giant fuzz balls – great for stuffed animals, not so great for your eight hour experiment.
Making the correction collar adjustment is simple: adjust the correction collar clockwise, focus the microscope on your sample, adjust the correction collar counter clockwise slightly and refocus the microscope. Repeat this process until you are satisfied with the image. There is no “focus indicator” with this process, simply trust your eyes. They are one of nature’s best detectors! If you use the same vessels for your experiments you may consider making a small mark on the lens housing to make adjusting the lens faster. It will also help you show your less technically inclined colleagues how to adjust the lens properly.
So before you blame your Leica microscope for behaving badly, double check the correction collar on your 40X lens for proper adjustment. And if it’s the 20X lens that you are mad at, check that lens too. Leica makes both 20X and 40X long working distance lenses with correction collars. So don’t get mad, just get smart and look under your microscope stage to see if you have a correction collar on your rig.
Before starting your next experiment or snapping your next picture, do not forget to adjust your lens, it could be the difference you were looking for in your experimental data. Only then will thank Leica for making a lens that gives you the flexibility you need in your lab.