Condenser properties condensed

Clive’s Corner by Clive Bagshaw

A new feature to MicroNews, Clive’s Corner is a place created for the sharing of knowledge, tricks, and tools. The Corner is where you read about clever microscopical hacks - and submit your own. Clive’s Corner is the namesake of SFMS Member Clive Bagshaw, who has spent a lifetime looking into microscopes - including 50 years studying protein reactions. 

In this Corner: A Disclaimer and Condenser Properties Condensed.

Disclaimer: As a newcomer to the Society, I have yet to discover the full breadth of experience of its members, which I assume ranges from beginners to professionals. My own “professional” experience is limited to a very specialized branch of fluorescence microscopy, but over the last few years I have discovered tricks and hacks appropriate for amateur microscopists using basic equipment ($100-$300 price backet) which I thought would be fun to share. Most of these are rediscoveries which no doubt will be familiar to some of you, but a few are original (our Victorian predecessors did not have access to Talenti gelato!). Please feel free to provide feedback by email to cbagshaw@ucsc.edu.

Condenser properties condensed

The instructions for adjusting the condenser in the manuals for some student microscopes can be obscure. Some simply say adjust the iris to get a satisfactory image. The instructions which came with my Amscope 120 scope read:  The condenser-adjustment knob allows you to control the concentration of the light hitting your slide. By changing the aperture (hole size) of the iris diaphragm, you can adjust the background brightness. While this is true, it misses the key role of the condenser which is to control the angle of the light which impinges on the sample. Once this is set, the intensity of the image is then adjusted by changing the brightness of the light source. Reading the instructions for more advanced microscopes may not help the student microscopist. These protocols refer to Koehler illumination, which achieves even illumination across the field of view and first requires adjusting the condenser height to bring the field iris into focus at the sample plane. However, student microscopes usually lack a field iris. These microscopes rely on a translucent disk above the light source to even out the illumination and are generally preset to give optimal illumination when the condenser is raised to be close to the bottom of the sample slide. The condenser iris is then adjusted to match the objective aperture. Here’s where the fun begins!

The angle of the cone of light (usually defined by the half-angle, θ) which can be captured by the objective lens is defined by its numerical aperture (NA):

NA = n. Sine θ,

where n is the refractive index of the medium between the lens and the sample. So, for a 10x 0.25NA objective operating in air (n=1), the full angle is about 29 degrees. For optimal resolution in the sample plane, the condenser aperture should match that of the objective lens. This can be done by removing the eyepiece and looking down the barrel to view an image of the condenser iris at the back focal plane of the objective. As the condenser iris is closed below that of the objective NA, it will come into view. This procedure is also useful for checking that the condenser unit is centered on the optical axis. If the circle of light from the closed condenser is not in the center, the condenser needs to be shifted using the adjusting screws that hold the unit to the body of the scope. Research-grade microscopes are often equipped with a built-in Bertrand lens for examining the back focal plane at higher magnification. For microscopes lacking such a lens, a phase telescope can be inserted into the eyepiece tube. Don’t have a phase telescope? Well, we will be making one for free in a future Corner. For best resolution, the condenser iris should just be visible at the back focal plane. But this may not be optimal for your purposes. Closing the iris further gives better contrast and increased depth of field. Having the condenser iris set larger than that of the objective NA is definitely not appropriate, for bright field imaging at least, because light scattered by the sample will reduce the contrast of the image.

Schematic diagram showing the effect of the condenser iris on the angle of illumination.

The resolution of the microscope is defined by the minimum distance, d, between two objects that can be distinguished. A widely quoted equation to relate this to the NA and the wavelength of light, λ, for conditions where NAcond ≤ NAobj, is:

d = 1.22 λ/(NAobj + NAcond)  

although this theory has been modified by Hopkins and Barham 1950 Proc. Phys. Soc. B 63 737. From this formula, the resolution limit for a 1.25 NA oil-immersion objective is around 0.25 µm and that of a low power 0.25 NA objective about 1.2 µm. However, the gain in depth of field is from around 1 µm to 30 µm as the NA is reduced. 

For practical purposes, high resolution is not much use if the image is so lacking in contrast that structures cannot be discerned. There are other tricks to increase contrast, but when using standard bright field imaging, a compromise in the condenser aperture must be reached. This conundrum has been around since the early days of microscopy. Ernst Abbe, who was instrumental in elucidating the resolving power of the light microscope, was accused of being a wide-aperturist and invited to defend himself against a bunch of low-aperturists from the Royal Microscopical Society in London. He did so eloquently in a 1882 paper, where he pointed out the NA setting depends upon what you are trying to visualize. The introduction in this paper makes interesting reading and conjures up Victorian gentleman rolling up their sleeves to settle their arguments.

For near-transparent plankton swimming around in water, I find using a 10X objective with a minimal aperture is best. Typically, these organisms are around 20 to 50 µm in size and having sufficient depth of field to get an impression of the overall cell shape is important. Furthermore, the sample, being in water, is suboptimal for aberration correction, and hence resolution is worse than the theory indicates. And, if the plankton are motile, you don’t have time to accumulate images for focus stacking. On the other hand, if you are looking at cleaned, fixed and compressed specimens, embedded in a high refractive index medium, then setting NAcond to just a bit less than NAobj is best to resolve details.

So, the take home message is that you adjust the condenser iris to obtain a satisfactory image.…. errr, isn’t this where we started? Yes, but hopefully with a bit more understanding. Well, there is more to the role of the condenser than bright-field illuminator and this note will serve as an introduction for future topics. In the meantime, do not pass up a jar of Talenti gelato if you spot it on your food market shelves.