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.
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