Don’t follow my advice on developing, I know microscopes; not photography. -K

Of all the books on photomicrography perhaps the most in depth is that written by Dr. Roy M. Allen. As technical as it is the book is not without its own charm. Buried in a footnote, Dr. Allen relates his first attempt at photomicrography as a child in the late 19th century. He made his images with a box camera, using the sun for illumination.

Having a box camera on hand, I thought I might give the process a try. The camera is an Agfa Ansco Shur-Shot B2 manufactured a few towns over from me in Binghamton, New York back in the 1930’s. It’s a wonderful camera for this, shooting widely available 120 film and featuring a shutter that provides for both short (approximately 1/50th) and long (B style) exposures. Unfortunately, the cameras lens is a hyperfocal meniscus lens that is less than optimum for photomicrography. A quick examination showed that the lens was held in by a simple tension ring and could be easily removed without causing any damage.

Using a lab jack made elevating the camera to the microscopes eyepiece very simple, though a stack of books could do just as well. A bright illuminator (a halogen lamp by B&L) that dates to the same period as the microscope and the camera, rounds out the setup. By using such a bright illuminator I hoped to keep the exposures short and avoid the vibration of operating a manual shutter.

The low power magnification didn’t require a condenser so a stand with a simple disc substage was used.

The depth of the box is less than 147mm, so photo and projection eyepieces are out, and a standard 5x Huygenian eyepiece was used. Thankfully, spherical aberration was less than I might have expected using achromatic objectives and a bellows length of less than 125mm. As box cameras have ground-glass view finders that are independent of the lens, (the two circular lenses seen on the right of the camera above) focusing can not be done through the camera once film is loaded. Prior to loading one may hold a paper or ground glass at the rear of the camera to determine the size of the image formed. A simple paper tube covered at one end and having a length equal to the distance from the cameras shutter to the film, serves as a focusing screen. Exposures are made by focusing with the tube and then replacing it with the camera and depressing the shutter.

An image as seen though the focusing tube.

With the film loaded into the camera and the intended slides at hand it is only a few minutes before I have a roll of B&W 120 film ready for developing.

After loading the film onto a reel and placing it into a developing tank in a changing bag, I processed it for 15 minutes with Pyro-Metol Kodalk followed by a plain water stop bath. Next the developer was poured into a beaker and the fixer went into the tank for six minutes. I used an alkali rapid fixer known as TF-4. Following the fixer, the film (now safe to expose to light) went into the used developer for two minutes prior to a 30 minutes wash.

Presto! A roll of 8 photomicrographic negatives having the classic circular aspect and clarity of medium format. Each negative image measures 45mm in diameter. All in all, the results seem rather nice considering the film I used was some Kodak T-Max 100 that expired back in the mid 1980’s and came to me via eBay for just a few dollars.

The question now is whether to get a negative scanner or a darkroom for making prints.

The digital photo does not do the clarity of this negative justice.

Preventing Obscurity

Once a photomicrograph is produced, one may desire to know some basic information regarding it; for example the scale of reproduction, or degree of magnification. To calculate most information only a little simple math is required. The formula for determining the scale of reproduction when using a camera with an integral-lens as follows:

Scale of reproduction = Objective magnification x Eyepiece magnification x Focal length of lens / 250

The formula works because the product of objective and eyepiece provides the initial base magnification, while the second portion of the equation provides the degree of reduction effected by the cameras optics. The lens will provide some level of reduction, unless it has a focal length equal to or greater than 250mm, which is expressed as the quotient of its focal length (in millimeters) and 250. The divisor in this case comes from the distance at which the virtual image is produced by the microscope expressed in millimeters.

The authors lens has a fixed focal length of 55mm, (which will provide a reduction of approximately 1/5) meaning that a 10x objective and ocular will produce a photomicrograph presenting with a magnification of 22x. To determine the accuracy of the calculation [or when using cameras with lenses of unknown focal length] one may measure the field of view visually with a stage micrometer (in this case measured at 150μ), and again as reproduced on the photomicrograph (measured at 63μ) and calculate the practical factor of reduction or effective magnification of the photomicrograph (23x which bears out the accuracy of the previous calculation).

Many consumer digital camera carry a lens with a variable focal length of from 2-25mm and this may be set by the user in manual mode, or determined after the fact by reading the exif data of the digital image.

For most focal lengths one will discover that the entire photosensitive surface is filled, be it 35mm film or a digital sensor. As a result only a portion of the microscopes field of view will be recorded. This effect will surprise some technicians who may expect to capture photomicrographs that are circular, just as the field of view is. Theoretically, to capture a more complete image of the field of view presented by the eyepiece, one may employ a lens having a shorter focal length. To determine the lens focal length that will provide the ability to capture the entire field of view one must know the nominal size of the field of view provided by the microscope eyepiece in use as well as the size of the imaging surface.

The rectangular sensor of the authors Nikon 1 J1 digital camera is 13.2mm by 8.8mm giving it a hypotenuse of 15.8mm. To entirely fill the sensor area, and produce a rectangular photomicrograph, while still imaging the largest portion of the field one will use the sensors longest dimension in the following formula. To image the majority of the field use the smallest dimension. The field of view index may be determined in a general way by measuring the field diaphragm of the ocular employed, it may be inscribed on modern oculars as a number following the inscribed power.

The imaged areas hypotenuse is equal to the sensors hypotenuse divided by the index of the field of view provided by the eyepiece.

Sensor dimension / Field of view index = Visible image diameter

This equation should well illustrate that a significant change in the scale of reproduction as effected by the cameras lens, will in essence, spread the photosensitive surface over a larger portion of the field of view. Doing so, will serve to reduce the resolution of the photomicrograph. So it is best then to use the above equation only to describe the power of magnification inherent in the image. With the above information one may mark a photomicrograph with a line and appropriately label its length with ease. Micrographs are best provided with this marking as it then acurately provides information on the size of imaged structures and magnification independent of the size at which the image is produced as a print.

Suffice to say that if one desires to image the whole of the visual field without introducing significant aberration or image degradation the simplest and best method is to operate a camera with a very large photosensitive area. One may consider that most of the historic photomicrographs (especially those presenting a circular field) were produced on plates or films considerably larger than 2.25 by 3.25 inches which is quite a bit larger than most widely available film or digital sensors.

What is the take away from all of this? Consider alternatives to the use of an integral-lens camera if one needs to produce an image having particular magnification or field coverage. Use the above to determine the characteristics of the photomicrographs one does produce and strive to take superior images with the equipment one possesses.

Don’t forget to renew (or begin) your membership in the Royal Microscopical Society before the 31st to ensure an uninterrupted full-year of membership. -K

Many of the primary faults of compromise photomicrography can be eliminated by simple means. Various apparatus may be purchased or crafted that contribute to success, and because of the ingenuity of microscopists the apparatus will not be expounded in any detail. Instead, the topic will be methods of producing optimum images when using a consumer grade digital or film camera which has a lens, but first permit an explanation of the chief faults of the method: vignetting, and obscurity.

A vignetted image is caused by the obstruction of a portion of the image by an aperture. The aperture may be within the cameras lens, the microscopes ocular, the body tube, or even condenser of the microscope. Most vignetting may be eliminated by ensuring the camera (and its lens) are aligned with the optical axis of the microscope, and the remainder by setting the lens of the camera properly as regards focus and the size of its aperture.

By obscurity one means the lack of precision regarding the photomicrographers knowledge of the image captured. When using a commercial apparatus one is aware of many things regarding the image because the manufacturer provides that information. When using a compromise apparatus one may be at a loss in determining the scale of the photomicrograph or even its degree of magnification, for example.

Preventing Vignetting

For optimum results one must position the camera so that the lens takes the place that would be occupied in normal operation by the eye of the microscopist. This means that not only should the camera share an optical axis with the microscope but that the initial optical component of the lens must be placed at the eye-point (exit pupil) of the microscopes ocular. In normal visual operation one finds this point instinctively by simply moving their head until the best image is observed by the eye. For photomicrographic use one may find the position of the exit pupil by holding a piece of index card over the eyepiece of the focused microscope. Move the card towards or away from eyepiece until the point of light seen on the card is smallest and then measure the height of the card over the eyepiece. The writers 10x B&L ocular produces its exit pupil at 5mm.

In operation a camera is focused on an object based on the distance of that object from the camera. In photomicrography, the object photographed is at a constant and known distance regardless of the ocular or objective in use. By design this distance is that of the eye at relaxed focus on an object as seen at 25cm (or 10 inches) distant-relaxed focus of the eye being equivalent to an optical system focused at infinity. Hence the oft made admonition that one should look through the microscope with eyes relaxed, rather than into the microscope with eyes strained. As such one should place an auto-focus camera in manual and set the focus to infinity (100feet or 30.5meters on some cameras). A manual focus lens should be set at infinity as well.

If the lens of the camera is then placed at the exit pupil of the focused microscope, no additional focusing of the microscope should be necessary to produce a sharp photomicrograph. This is particularly relevant if one is using a film camera (or digital camera without an LCD) as the cameras prism is unlikely to prove operational in this context. It is also relevant as the display of a digital camera will generally lack the resolution to ensure sharp focus. Following the above recommendation some microscopists may discover that they are in the habit of accommodating their vision for different focal distances when using the microscope. Young individuals in particular are likely to find this condition and can determine the degree of accommodation by focusing the microscope and then manipulating the focus of the cameras lens until a sharp photomicrograph is produced. A similar situation is apt to be experienced by those who wear eye-glasses and are in the habit of removing them when operating the microscope-one may discover if this is the case by focusing with eye-glasses on and then observing the sharpness of the image with eye-glasses removed.

Following the above methods, one is unlikely to experience vignetting. If it is still observed and can not be eliminated by altering the alignment of the camera to the microscope, it is apt to be attributable to the size of the camera lenses aperture. One must ensure that the camera lenses aperture is equal in size to, or larger than, the exit pupil produced by the ocular employed. With film cameras one should always employ the largest aperture opening available both to eliminate vignetting and to ensure that the image captured on film is illuminated consistently and does not dim towards the borders of the field of view.

This is getting rather long so eliminating some of the unknowns inherent in compromise photomicrography will be discussed in the next post. -K

There is no reason to run out and buy anything, for the below I used only what was already on hand. -K

Provided one has a microscope and a camera, no additional equipment is absolutely required. In a pinch a photomicrograph may be produced by holding it to the eyepiece and hoping for the best. Some simple steps may be taken to ensure the best possible result of this slipshod technique. The chief argument against holding a camera to the eyepiece is that the stability of ones hands is not so reliable as one might prefer. Overcoming this difficulty is not so complex as it may first seem.

The microscopists eye replaced with a consumer grade 35mm camera.

In a slim text titled Photomicrography put out by the Eastman Kodak Company an excellent method of positioning a camera at the eyepiece is illustrated. In the text it is recommended that one fabricate a simple wooden board with an appropriately sized hole to accommodate the lens of ones camera. Once placed through the board the body of the camera is held firmly in place by rubber bands. The whole is then suspended over the microscope on a standard laboratory ring stand so that the lens of the camera corresponds with the eye-point of the objective.

Once the microscope is focused normally, the camera may be swung into position. On some varieties of camera the regular sight would then be used to verify focus, or a ground glass focusing plate might be substituted for the regular camera back. The large digital displays of modern cameras provide an added convenience when it comes to focusing, and widely available software for tethered camera control as well as wireless remotes eliminate the risk of misalignment during exposure. Additionally, the absence of any mechanical shutter eliminates a common source of vibration which was the bane of many an amateur photomicrographer.

With a ring stand it was the work of a moment to position a camera over the eyepiece of a microscope oriented fully vertical. If one did not have a ring stand or similar support on hand, one might easily utilize a standard photographic tripod towards the same end. Adapting the method for use with a microscope with a permanently inclined eyepiece (such as the B&L Dynoptic, pictured below right) required no more effort than trading the ring support for a utility clamp mounted on an intermediate boss-head.

Vertical microscope with ring supported digital camera.

Considering briefly, one might see how this same general method could be adapted for use by an individual possessed of nothing but a standard microscope and camera. Manipulating the microscope so that it is fully horizontal permits simply sitting ones camera on any convenient support that will elevate it to the height of the eyepiece.

Fixed angle microscope with camera held at appropriate angle.

Presently, all manner of custom mounts and accessories have been manufactured and marketed to the amateur that simplify arranging and aligning photographic apparatus-from cellular phones to expensive DSLR’s-at the eyepiece of any optical equipment from microscope to telescope. Such equipment is largely unnecessary as has been demonstrated above.

But are the images produced by this method acceptable? Without specialized equipment or complex software the below image was produced. In all it took longer to walk to the shelf on which the ring stand was stored than it did to arrange the equipment and take the photomicrograph. In the image below one can see that ample room for improvement exist. Having lately been playing around with an old Russian range-finder lens which was mounted to a Nikon 1 J1 body, it was the camera used. Looking at the lens afterwards I note that it was focused to about 10.5 meters and the aperture was fully open. In any case below is the image produced of a traditional test object for low powers, the scales of a butterfly, as seen with a 10x objective and 10x ocular.

Be warned the full size image is ~4MB.

In the next post methods of achieving optimum image formation with the above method will be discussed.

Photography is as allied with microscopy as could be, so do forgive this. -K

There was a time when possession of a microscope was a rare and novel thing. However rare it may be, it is certainly no longer novel. The same might have once been said of the photographic camera, but in the present day it might be more rare to encounter one who does not possess a camera. With the proliferation of cellular smart-phones it may seem that everyone is in constant possession of camera. This situation is apt to elicit a particular behavior; the placement of a camera at the eyepiece of a microscope.

Initially existing photographic apparatus was adapted to the microscope by the operator to meet their individual tastes and needs. Before long, manufacturers began putting out specialty photographic apparatus intended to be placed at the eyepiece of the microscope. Sometime later specialty microscopic apparatus-possessed of a beam splitter and photo tube-that required even more specialized photographic apparatus became the standard, below are two 35mm cameras of that sort.

Two varieties of integrated photomicrographic apparatus.

Simple, and easy to use, versatile cameras like the above provide variable levels of tertiary magnification. Shutters are integrated and fittings for remote shutter release are provided, along with connections for automated exposure meters, and on some models other computerized accessories. Unfortunately, these sorts of camera are very non-standardized. One might notice that the lower optical connection on the above are of significantly different size, neither compatible with any of the standard eyepiece diameters. Apparatus of this type is only suited for use with microscopes equipped with a photographic head, and even then one can not be certain of physical-never mind optical-compatibility.

Modern stands equipped with photographic apparatus are frequently of questionable quality and universally expensive. Vintage stands tend to be incomplete and similarly expensive. Unless one is lucky or particularly knowledgeable this sort of apparatus is best pursued only cautiously. Instead, one might take a page from the microscopists of years ago and seek to adapt standard photographic apparatus to photomicrographic work.

The next few posts will treat with this topic with a goal of producing professional quality photomicrogaphs conveniently and usefully. If this goal may be reached economically in a manner that provides optically superior results it is only because of the countless microscopists who labored with cumbersome tools and complex arrangements  to provide our understanding of the required techniques.