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