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**Ali Abbass** · 04-24-2023, 02:03 AM

Advanced microscopy equipment and methods

We touched on microscopic photography previously and on two successive episodes, the emergence and even the adoption of the most advanced microscope, but now we show in detail the methods with the necessary equipment for more advanced microscopic imaging.

Today, most of the works that are done with a microscope include photography,
Either for educational or archival purposes, for the photographer who deals with jungle life, even if he is an amateur, the disparity and different stimuli and shapes on the microscopic level achieve interesting possibilities.

Camera selection:
In microscopic photography, the microscope alone has to form the image, and it is better equipped to do so than any part of a camera. It is necessary not to underestimate the value of an image that has been carefully and carefully enlarged by the use of an additional camera lens. This means that for good quality microscopic photographs, it is sufficient Working with a single-lens reflex camera is much better
From a live viewfinder camera.
The body of the 35 mm camera is light and compact enough to be installed directly on the eyepiece of the microscope with a special connecting tube, and there are studio microscopes that feature a vertical tube for the camera.
In terms of ( Trinocular Microscopes )
It can convert the image either into trinocular microscopes, viewing tubes, or photographic tubes, by means of a prismatic lens.
As for the third category of cameras, they are made for microscopic photography upon request, and these can often be obtained as part of the microscopic apparatus. With adjustable blowers and a wide speed range.

Choosing a microscope:
It is not necessary for the appropriate microscope optics for viewing to be of high quality for photography, especially when working with color film, and it is not surprising that the best microscope is expensive, and often the final choice is a compromise between optical quality and price. For microscopic photography, we find that some of the microscopic components are of special importance.

Subject lens (*):
The main lens in the microscope is called the subject lens. Its magnification energy is inscribed on its holder and is usually between five and a hundred times. The most important quality in the subject lens is its ability to analyze details. The usual scale for these is known as the numerical aperture (*) (NA), which is A number calculated from the angle of optical suppression needed to fill the lens aperture. The higher the aperture number, the better the resolution. The maximum numerical aperture is 1.0 in air, but some subject lenses are designed to be dipped in a thin oil film which allows a numerical aperture of up to 1.40.
The most common of the subject lenses are those known as ACHROMAT, which are the least expensive and work only partially to correct the most common optical aberrations. Here, we find colored fingers accustomed to the edges of the image.
As for the objective lens of the best quality, it is the apochromat, which is highly corrected.

- eye lens ( * ) :
The secondary lens in the microscope is the eyepiece (*) and it magnifies the first image and displays it either on the eye or on the film in the camera. As for microscopic photography, a compensatory eye lens is needed to correct some distortions in the subject lens. It should be noted here that the pair of the subject lens and the equivalent integrated eye lens gives the image of the best quality.

capacitor
This lens located under the subject's table gathers the light in the lamp and focuses it on the sample. There are three types of condensers: abbée, aplanatic, and achromatic. The latter is the most corrective for optical aberrations, which makes it the best for microscopic photography. It is necessary to adopt a method to focus the condenser in order to give equal illumination.

mechanical platform
In order to achieve accurate adjustment of the sample location and image formation, there is a mechanical platform with very small distances that allow movement that exceeds its accuracy and shortness of what can be done manually, and this is an actual good, and if the platform is able to rotate, it becomes better and better.

Basic operation:
The simplest method for illuminating a sample is bright field illumination, which exposes the sample against a background.
Bright enlightenment, as the name suggests, in this case, light is passed through the sample in
The truth, where the sample is thin and transparent. The standard microscopes are characterized by the presence of a mirror under the subject, whose angle can be changed to expose the light upwards from a separate lamp, although some advanced models are equipped with implicit light sources. As for the principle of illumination of the bright field, it is based on concentrating an image of the lamp in the surface of the sample in a way that makes it fall on the center of the viewing field, covering the whole field evenly. This means that the lamp is an essential part of the optical system.

Light sources:
The standard light source in microscopy photography is a tungsten filament lamp, which is mounted with an adjustable condenser lens and diaphragm. A tungsten-halogen lamp is best for color microscopy photography, as long as the glass cover does not darken or change color with time.
As for a bad lamp of this kind, it arises with active living beings that perform certain movements
It requires more enlightenment to avoid blurring, and one of the solutions in this case is the use of a flash unit.
Electronic pocket. However, an auxiliary tungsten lamp is required to be placed in the same location for viewing and focusing before photographing.

- the movie:
The typical film for microscopic photography is that characterized by soft stippling and varying light contrast - from moderate to high - speed is not usually important, as long as still samples can be photographed with long exposures if necessary and it is better to illuminate moving subjects with an electronic flash and this means that most soft-stack films suitable, but the following are the best:

- Black and white (Kodak Ektapan 4163) in the form of slides for use with a viewing camera, then film - photomic rography Monochrome, So (410 in 34 mm format,
Also from Kodak and in 35mm format
(Kodak Panatomic-X)
Measures 6 x 6 cm, 6 x 7 cm.
- Colored
Color film
( Kodak Photomicrography 2484 )
Slides measure 4" x 5" and have a 35mm structure, then a 35mm structure in the Kodachrome 25.

- exposure;
Since the optics of the microscope do not allow the use of an aperture diaphragm, the exposure must be adjusted by other methods. Changing the shutter speed in the camera gives one-stop adjustments, but to achieve finer adjustment, the bulb can either be relocated or zero density filters can be used. Exposures usually mean using test film on a series of locations and as a rough guide, one can say about the shutter speeds involved in a typical bright field setup with the use of aza 32 film. It may range from 1/4 second to 1/125 second.

**Ali Abbass** · 04-24-2023, 02:07 AM

- smudging
Since it is necessary to work on thin transparent sections of bright field based microscopic photography, many of the samples will not be visible if they are not prepared in a special way. Here, staining comes to play a complementary role in many microscopic works. There are biological staining materials of all colors, as the choice depends partly on the sample and the type of film used. Typically, on tissue papers, for example, two opposing staining agents are used for different parts of the sample. There are two most common staining materials used together on sections of animal tissue paper, eosin, which is red in color, and hemotoxylin, which is blue, although more staining materials can be used together. The colored films differ from each other. In how it records certain smudges, eosin, for example, gives a good result on Kodak Photomicrography 2483 color film, but it gives a weak result on Kodachrome film. The trial and error method is necessary to know the quality of smudges on the sensitive layer in a particular film.

- color balance:
Microscope lamps and color films are generally balanced at about 3200 Kelvin for tungsten light and 5500 Kelvin (K) for daylight work. Any difference in color temperature between the film and the lamp can be compensated for by using an appropriate color correction filter. These are available in different strengths, either to lower the color temperature - that is, to make the light more yellow - or to raise it - to make it more blue. This is despite the existence of a table that guides us to work in this field, but we will need to conduct individual tests in order to bring the color balance to a typical level.
Far from correcting the basic color balance, it may require some additional filtering to absorb the unwanted ultraviolet radiation from the zirconium arc, the carbon arc, or the electronic flash. To achieve this purpose, the (2B) filter is usually sufficient.
To compensate for minor color differences between spots on the film or chromatic haze in the condenser, we use filters (CC05) or (CC10) in red, blue, green, or blue.
Dark (cyan), yellow, or fuchsia.

This, since most microscopic specimens are so thin that they are very transparent, the biggest problem with microscopy photography becomes achieving enough light contrast to record an image. The solution is smearing in bright field microscopy photography, but this may not always be possible. With live samples or enough with some microorganisms and cellular structures - in this case, it is necessary to find another method to increase the light contrast that includes different lighting methods, including what is quite complex, in terms of theory at least.

Dark field:
When the lighting is set up in such a way that the background is dark instead of light, then transparent samples can be seen with a much greater amount of clarity, with improved light contrast. To achieve this effect, a dark field stop is placed in the middle of the light beam under the condenser, thus turning the light into a hollow funnel. When a sample is placed on the platform, the light is scattered upward towards the subject's lens. But since this method leads to losing a lot
From the light of the lamp, it is necessary to use a high-speed film or an electronic flash during photography, although this method remains typical with its low energy for working on aquatic organisms such as plankton (*).

Partial contrast:
The partial light contrast method is one of the most retouching methods, and it is possible to reveal internal details of completely transparent targets, and it is based on that effect, as the edges and fine details in the sample deflect the light that passes through them, and the result is that the sample passes part of the light directly without modifying it, while the rest is scattered. . In the partial optical contrast microscope, a special layer behind the lens of the subject slows down the direct light ray by a quarter length. The sample has improved.

Interferometric contrast:
In the optical contrast system, there is a wave. Then, when the direct and scattered beams meet, again, the details become visible, because the two rays in the light emit equilibrium interference systems with differences in the sample. There are other interference optical contrast methods that give a dependent result, but the light is split not by the sample, but by the optical system. For the microscope, the Normasky system, for example, is based on the use of prismatic lenses to slit the light, where one ray is directed to pass through the sample and the other to avoid it. The first ray is called the target ray, and the second is called the reference ray.

Polarization:
One of the other lighting methods that can achieve bright results that attract attention is that which works with two polarizing filters. If another polarizing filter is placed in the path of the ray that was previously polarized, it will not allow light to pass through it unless it is set at the same angle as the first filter, and if it is rotated by 90 degrees. The light turns off as well. There are some samples that also polarize the light, so that if they are placed between two polarizing filters - they are called the polarizer and the analyzer - they turn the light partially so that it becomes visible again. In varying amounts, the resulting overlapping colors can be very interesting.
In microscopic photography, there are many dilemmas, the causes of which are due to the low efficiency of optical waves at high magnifications, and one of the solutions to that is to search for Shorter wavelengths as a way to form an image. This measure should allow for high resolution and magnification, provided that the ability to control and direct the radiation in the same way as light can be refracted. This is the principle of the electron microscope, which is a device that can analyze details down to two billionths of a centimeter, which makes it Capable of making magnifications more than a thousand times greater than what the best optical microscope can do, and although such equipment is found only in specialized laboratories, the electron microscope succeeds in extending photography to new frontiers and is able to produce pictures that are astonishing in their beauty.
Magnets are used instead of lenses to focus a beam of electrons on the sample. This beam pushes other electrons off the surface. And these, in turn, are attracted to a sensor that converts the data into an image to be seen on a television screen. This simplified explanation ignores many technical complexities, as the complete equipment concerned will occupy the largest part of a small room.
An additional improvement made to the electron microscope only in the 1960s is a means of transmitting the electron beam through the sample so that each point on the surface of the subject can dissolve individually, which allows for a greater depth of field. At small magnifications in particular, the scanning electron microscope can produce images with depth A field whose scale the eye finds difficult to believe, for we are so accustomed to seeing shallow focus from short distances that the facial image of an ant with such fine detail is all rather unnerving to us.

But when the scanning electron microscope displays its image on a cathode ray tube. Photography is limited to taking pictures from this screen, which is similar to a television screen.
The work is usually done with a normal black and white film with a moderately fine dotting, as long as the colors, which are a function of the visible wavelengths, are absent from the image.

التصوير المجهري .. معدات وأساليب متطورة .. مجلة فن التصوير اللبنانية _ ع٣٥

التصوير المجهري .. معدات وأساليب متطورة .. مجلة فن التصوير اللبنانية _ ع٣٥

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