Tuesday, July 16, 2013

Making things visible under a microscope requires the creation of contrast

Making pictures using a light microscope has been such great fun for decades.  I had my first serious experiences with a microscope in a histology class at Bradley University in 1976.   I was always struck by the organization, structure and shape of the subjects I was examining. I still have some primitive drawings from that class that I used to help learn to discriminate muscle cells from connective tissue.

Photographing at a microscope can be interesting and every subject has unique and interesting challenges, no matter how mundane. Certainly more complicated samples are more difficult to image but someone once told me, "it is just as much work to make a bad picture as it is to make a good one. Truer words have never been spoken.

The first thing that happens when you sit down to use a microscope is a basic orientation problem. A microscope produces circular images and the field of view is limited. It is almost like using blinders. Most of the field of view will be out of focus and it may also have illumination problems. A user must by adjust the focus and other instrument controls to form an image. Once the image and its structure has been created, subtle controls can then be adjusted to increase the quality of the image. This is required before any level serious investigation can begin. Quality often is described as contrast, visibility, uniformity of illumination. It also might include composition and effective subject isolation.
To locate specific structures often requires careful scrutiny of a subject to differentiate the important structures from the sample as a whole. When a sample is magnified 1000x, a 1" dimension becomes nearly 85 feet. Imagine scouring an area nearly 85 feet in length and looking for a "thing" that is only 1 inch in size. It is time consuming and requires significant concentration.  When photographing for science, the goals of the photography are very clear. The picture needs to contain the required information.

The following photomicrographs contain a fiber in a debris field of liquid and chemicals. There is no real purpose for the photographs except the object seemed interesting to me when evaluated using magnification.

The first photograph immediately below is the fiber at x25. It has been formed using Kohler illumination. The aperture diaphragm or the microscope's steering wheel of radiated energy was established to maximize image resolution. The aperture diaphragm in a microscope control's four attributes of an image, contrast, resolution, range of focus and intensity. It should never be used for brightness control.   The remaining photographs will be described beneath each.

An image Fiber is revealed in a liquid using brightfield illumination with an almost wide open aperture. Structure, contrast and image visibility is low. The image resolution surprisingly is high. In summary, the image visibility is low due to low contrast.

In this photograph, the fiber's image is much more revealed in a liquid using brightfield illumination and with a closed aperture. Structure, contrast and image visibility is very pronounced however the image's resolution has now been reduced. In summary, this image's visibility is high due to high image contrast. Fine structural detail has been lost been so visibility could been gained. Maximizing image detail is a balance of contrast, resolution and range of focus. An operator often can have two of the three, but not all.

To make more contrast, the fiber was photographed in a polarized light system and anything that is not birefringent is not visible and its transmission has been eliminated. The fiber is now easily visible but environment has been subtracted.

Adding a quartz wedge to polarizing light microscope created this result. Amazing.....

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