The Substage Diaphragm

I fear owning a digital microscope camera may require turning in my old-man card. -K

Every transmitted light microscope has some sort of diaphragm. On the simplest stands it is fixed and unchanging, represented more by the size of the hole in the stage than any proper apparatus. Traditional students microscopes featured a wheel perforated by holes of varying size that could be turned beneath the stage as a diaphragm. More complex models might feature an iris diaphragm standing alone or in series with a condenser. In every case the diaphragm affects the light sent into the specimen placed above its aperture on the microscopes stage.

It’s a common thing, and greatly lamentable, that some operators fail to properly operate the variable diaphragm and in so doing do not obtain the full resolution boasted by their objectives; the preceding however, is not the subject of todays wall o’ text. The discourse today concerns that which is intended to be manipulated by the diaphragm, light, more specifically, contrast.

Naturally one can expect that an opening of variable size located between light source and object will affect the lighting of the specimen. When gazing through the ocular and manipulating the diaphragm it soon becomes apparent that the size of the diaphragm opening affects enormously quality of light entering the objective. At issue is the tendency of new and enthusiastic microscopists to use the diaphragm as a means of regulating the intensity of illumination without appreciating the alterations in the image caused by their actions.

The required intensity of illumination should always be achieved by varying the source of light by the use of a variably transformer in the case of electric lights, the orientation of the flame in the case of paraffin lamps, and the use of filters of all sorts in any case. Using a small diaphragm aperture will of course result in a decrease of apparent illumination, but what may not be immediately apparent (depending on the specimen observed) is the alteration in contrast affected as well.

It’s all well and good to read that one should employ a diaphragm opening roughly the size of the objective front (object) lens. Or that in looking down the body tube with the ocular removed one should vary the diaphragm until one third of the objective back (eye) lens is lit. It’s quite an other thing entirely to see the effect of diaphragm manipulation when looking upon an object having a refractive index very near to that of the mounting medium. Below is an image of several diatoms taken using a 30mm objective with the diaphragm expanded to light the entire back (eye) lens of the objective.

Image

Poor contrast

Never mind the low quality of the image, it’s the fault of the cameraman who had a heck of the time figuring out where to put the film… One can see the diatoms and make out something of the structure of the silica composing the frustules. One diatoms at the top right shows a hint of color, they are all however, sort of washed out though certainly not dazzlingly illuminated. Observe the image below taken with the same light source and objective only varying the aperture of the diaphragm so that just less than one quarter of the back (eye) lens of the objective is lit.

Image

Proper contrast

With the diaphragm properly dispositioned the degree of contrast in objects of varying refractive index is sufficient to provide not only greater detail, but a range of color as well. Consider the world as it appears at night, dimly lit and nearly devoid of color. In the dark of night the iris in ones eye expands to let in as much light as possible so that some vision may be had from what light there is. The cost of having the eyes diaphragm allow in all available light is very poor color perception, very poor recognition of contrast. It is just the same principle with the diaphragm in the substage of ones microscope. Provided light the intensity of which is regulated by appropriate means the diaphragm may be kept small enough to provide optimum contrast.

Alright, that’s enough for tonight, time to go scowl and shake my fists at the young!

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