XX. Documentation

This section would have been called "photography" a few years ago. Today there are a number of things that can reproduce the image from a microscope. These include scanners, tv. and computer enhanced tv.

Still 35mm and large format Polaroid are the mainstays of documentation. Polaroid is used mostly in industry were instant prints are used for Q.C. documents. Slides are the main stay of bio-med photography since they can be used for talks or publication.

Modern photomicrographic systems are so easy to use that there is no reason why you can't take excellent pictures. All modern photo systems are completely modular so that you can switch film sizes, backs and formats with ease. The best reason to own a modern photomicrographic system is the automatic exposure controls. These really work!

Fully manual systems are still made that can do both Polaroid and 35mm. For 35mm they are a complete pain and for Polaroid they are almost as bad. The only reason to get one is that you are going to take pictures of only one specimen type on Polaroid all the time. Then you can zero in on the correct exposure time and just keep using it otherwise buy an automatic system.

All manufacturers make automatic camera systems to attach to the trinocular tube of their heads. The systems vary based on the overall complexity of the electronics in the system. All manufacturers make modular camera systems. These modules include the control unit, the shutter unit, photo eyepieces and the camera back. The cameras built into some research microscopes resemble the add on systems to a great extent.

The control unit usually has the electronics to allow you to input the film information. Then the control unit determines the exposure time and shows it to you. Control units will have a method for doing a manual exposure, although this is a very rare procedure, and the ability to over and under expose the picture.

Some control units allow you to lock in an exposure time. This is a good feature to have. If you have to make a montage (a series of photos at high power that makes up the whole structure) then you will want to take them at one exposure time so they all look the same.

The shutter assembly attaches to the trinocular head. Each manufacturer does it slightly differently. This is the one weakness of an add on system. An add on system will tend to be more susceptible to vibration than the built in camera of a research microscope. However with a little care an add on camera will work just fine. Make sure that the shutter assembly is properly attached to the trinocular. Usually the shutter assembly will fit over the trinocular tube that has a photo eyepiece installed. A photo eyepiece servers the same purpose as the eyepiece you use to see with. It provides the final magnification for the user, in this case the camera.

Photo eyepieces used with 35mm camera backs will usually be of low magnification. This is necessary to reduce empty magnification. Usually a photo eyepiece will be around 2.5X to 4X. There will be additional magnification from the trinocular. Each manufacturer provides a magnification chart for each back and photo eyepiece. For visual observation we know that the ratio of NA to magnification shouldn't exceed 1 to 1000. In photography that ratio shouldn't exceed 1 to 500. Later on when you show the slide or reproduce it for publication you will invariably add magnification to it. After all when you project a slide it will wind up wall size.

There are photo eyepieces made that are very high magnification. These are usually used for automated measuring applications were high magnification is necessary and empty magnification is acceptable. These should not be used for routine picture taking. The shutter system contains the light sensing, metering, equipment. This can range from a simple solid state device to a very sensitive photo multiplier tube. The more sensitive the device the more expensive the system. These devices can be used to detect light over the whole field, called averaging, or in a very small area, called spot metering. Both meter types have their uses. Most high end units let you use both. Very recent systems meter the field with an array or grid of detectors. The information from this grid is then processed by a computer in the control box. This is neat new technology but the old stuff still works.

Averaging metering works very well if the specimen covers the whole field. Spot metering works at its best were the field has very few specimens and they are very different than the background. A classic use of spot metering would be in fluorescence with a few scattered cells in the field. You would place a cell under the spot area and lock in that exposure. This would be more accurate than averaging a lot of background that you aren't interested in with a very little amount of fore ground.

However in a lot, if not most circumstances, averaging metering works just fine. While a spot and averaging meter system is the most versatile and an array system even better if you can't afford them an automatic averaging metering system works just fine.

Most shutter systems have a place on them for a focusing eyepiece. This allows you to focus the camera and the specimen. These are really not necessary on most microscopes. The manufacturers design their cameras to be parfocal to their eyepieces. If you are using a modern camera on an older microscope you may very well need a focusing eyepiece otherwise use a photo reticule equipped eyepiece in the trinocular.

The camera back contains the film and determines the format. All modern automatic camera systems can do a wide range of formats. The usual ones are 35mm, 4 X 5, sheet film Polaroid, pack Polaroid and automatic eject Polaroid and 8 X 10. Each one of these formats has its use. Polaroid, 4 X 5 and 8 X 10 are usually used by industry since they need large prints immediately. Most biological scientists and geologists use 35mm since they want slides for talks and papers.

Some systems can use roll film formats such as 2 1/4 by 3 1/4. This should have been a commercial success since this is a widely used format for professional photography. But commercially it never made it probably because medium format slide projectors are not very available. If you shoot a lot for publication this could be a very good way to improve your quality. Ask the manufacturer if they have a roll film back.

There are some systems that can use 16mm motion and time lapse systems. These systems have been replaced by tv. and time lapse tv. Plus they were never all that popular any way. If you need one they actually exist and work very well.

The core of a time lapse film system is an intervalometer. This connects to the control unit and at programmed in times tells the camera to take a picture. Some of them can turn the microscope light on and off in sync with the photo system. These can also work with a 35mm motor drive back. If you need one of these you need one badly. How else can you take an exact series of photos seperated by a known time? But not many people need one.

35 mm back come in motor drive and non-motor drive. Motor drive backs are a whole lot easier to use. You never have to remember if you wound the film. Most makers have built in, automatic dark slides. These allow you to remove the camera back while it still has film in it. Older backs had dark slides but you had to pull them out of the way. I once shot thirty six pictures on the dark slide. I really like the automatic ones. The other advantage is if your department has only one photo equipped microscope you can shoot a few pictures using your back and then remove it and some one else can use the microscope with their back. If bucks are a real issue and you are willing to be a very careful worker you can attach a conventional 35mm single lens reflex camera to the trinocular tube. You will need a photo eyepiece and an adapter tube. Usually you will have to focus through the camera back although some adapters are par-focal.

This gives you an inexpensive, automatic camera system however there are draw backs. Vibration can be a real problem with these systems, they tend to be way up in the air. Use a camera back that lets you lock in an exposure time and then lock up the mirror in the camera. This reduces the amount of vibration when you make the exposure.

The worst problem with these systems is the lack of meter sophistication and sensitivity. They are designed for far different duty than photo-micrography. You will have to work to get bright field exposures right. They will not really do automated exposure fluorescence. If you are doing work that doesn't involve fluoresence then this approach can save you money and cost you time. Most manufacturers make an adapter to attach their brand of 35mm single lens reflex to the microscope.

Diagnostics, a very good company, makes adapters to put just about any 35mm camera or photo system on just about any microscope. Their stuff really works and is quite well built.

So every ones pictures come out perfectly right? Well actually no. The problems are in a few easy to correct areas. First you must set the microscope up correctly. Reread the section on Kohler illumination and the condenser diaphragm. Set the microscope up each time you change the objective and be very careful about the condenser diaphragm.

Next is focus, you must focus the microscope correctly to get a good looking photograph. Each manufacturer provides a photo reticule for you to focus. This is located in the right eyepiece on most mid-range systems, in a separate focusing eyepiece attached to the camera or both eyepieces may focus. This last system is used by large research microscopes.

Most modern microscopes have the reticule in the eyepiece. The head and eyepieces are parfocal with the camera. This is much more convenient than having to look through a camera eyepiece. Some users distrust parfocal heads but I have always found that they work very well and are a lot easier to use.

The photo reticule has two components, a focusing target and a format outline. A focusing target is usually a crosshair made up of two closely set pairs of lines in the center of the field. It can also be a circle of two closely spaced lines. The focusing target must be in focus for you to get acceptable results. To focus it turn the eyepiece counter clockwise (away from the microscope) until the target is blurred.

Now focus clockwise (towards the microscope) quickly until the reticule is in focus. If the reticule is a cross hair try to focus only on the horizontal or vertical line pairs, any residual astigmatism you have will make it impossible to focus on both. If you wear glasses use them when you are taking pictures.

Always focus quickly. Our eyes are controlled by our brain and the brain wants in focus images. If you try to focus slowly the brain will over ride the eyes and just say "Enough is enough". Focusing quickly whether it is the reticule or the image will produce the correct focus every time.

Now that the microscope is set up, the reticule is focused and the image is in focus we can look at the other part of the reticule, the format outline. This shows what will appear on the film. Some manufacturers have both 35mm and Polaroid formats in the same reticule while others have only one format. Make sure you have the right one in.

All modern photo systems have a way of varying the magnification to the film. This is usually the photo eyepiece. For the best results use a low power photoeyepiece to avoid empty magnification. High power eyepieces are used for industrial automatic inspection systems and other unusual applications.

If the photo reticule is in the eyepiece the manufacturer will include lines to show you the picture using each of the different eyepieces. Large research microscopes have ways of showing you the photograph before you take it. This is called "framing the picture".

When you are using 35mm film always frame loose. Don't put important part of the picture out at the edge. Slide mounts cut of a portion or the picture and slide projectors will do the same. Put the important parts of the picture in the inner two thirds of the photographic field.

If you do a lot of photography you will appreciate the convenience of a rotating stage. If you really do a lot of photography you will need a centerable, rotatable stage with a mechanical stage. These are not easy to use in a high volume bio-med application but for photography they make framing the shot so easy to do.

Focusing low power shots can be hard. Since low power objectives have low NA they also have lots or depth of focus. It can be confusing to properly focus these objectives. The best way to do it is to use a focusing telescope.

Focusing telescopes provide a very magnified look at the reticule and the image. These are temporarily put on the eyepiece with the photographic reticule. The focusing telescope is then focused on the focusing target. After the target is in good focus the image is brought into focus.

Some research microscopes have low power automatic focus. These actually work quite well. These built in systems use a variety of optical trick to focus the image. While they do sometimes fail they will tell you when they have a problem. If you do a lot of low power photography you need to look into one.

Automatic exposure systems on modern photo systems are extremely accurate. There is no real reason to use manual exposure any more except for the most extreme circumstances. However all systems have some way of modifying the exposure, why is this?

The answer has to do with film speed ratings. The film speed is the rating of the films sensitivity to light. This is the DIN or ASA film speed rating. Film speed ratings have to assume a set of contrast and color ranges in the slide. Since most film is used to make pictures of people the contrast range is very different than for photomicrographs.

The contrast range of a photomicrograph is at most 1 to 16. Most films are designed to reproduce at least 1 to 100. The low contrast and color range of microscopic specimens is very different from the usual image that most films were designed for. To get the best exposure for your type of specimen you need to bracket the exposures.

Bracketing is over and under exposing the picture from the point that the camera picks as ideal. All manufacturers have ways to do this. Usually there will be a dial to adjust a bias control. You can also change the ASA speed on the camera control box.

When you start out taking pictures take one picture at a plus one bias adjustment, one at zero bias and one at one half bias adjustment. If you have to adjust the ASA then have one shot at the next lowest ASA, one at the correct ASA and one at the next highest ASA. This give an over and under exposure range to choose from.

When the slides are developed see which one has the best exposure for your purposes. After doing this once or twice you should know what bias adjustment is best for you. As long as the specimen is approximately the same you can use the same bias adjustment.

While correct exposure is necessary for correct color the color temperature of the light must also be correct. Color temperature, rated in degrees Kelvin, is a red to blue measurement of the color of the light. The degrees Kelvin rating of a light source is called it color temperature. The lower the color temperature the redder the light.

Films and other documentation systems are rated in the color temperature they are designed for. To get the best color the film and the light source must be matched. If the source and the film don't match then the back ground and the specimen colors will be off. When discussing color film there are two type, tungsten and outdoor.

Tungsten has a color temperature of 3200 deg. Kelvin. This matches the color temperature of a microscope lamp at optimum voltage quite well. The other type of film is outdoor film. This has a color temperature of 6400 degrees Kelvin. This is much bluer than the tungsten color temperature. To use it with a microscope you must use a blue filter designed to convert tungsten light to outdoor light. Not every blue filter will do the job. Make sure it is designed to be a conversion filter. These can be bought from the manufacturer, third party filter makers and really good camera stores.

I use medium speed, tungsten, Kodak Ektachrome for my shooting and find that this is better than using outdoor film and a conversion filter. If you get a consistent orange-red background on your slides it probably means that you are using outdoor film without a conversion filter. If you get a consistent blue tint it means that you are using tungsten film with a blue filter.

There are films with higher ASA speeds but as ASA speeds increase the films become "grainier". This means that as ASA speeds increase the actual silver grains that make up the film have to become larger. This makes the image appear broken up rather than smooth. In the bad old days this was a real problem but modern films have made great strides in reducing grain size.

Outdoor film is considered to be 5800 deg. Kelvin. Since microscopes don't use sunlight I think it is easier and better to use tungsten films. There are filters to convert tungsten color temperature to outdoor color temperature but they significantly reduce intensity and I don't like the color rendition.

As the voltage to a lamp increases the color temperature increases. A lamp will produce from 2800 deg. Kelvin to 3200 deg. Kelvin at its optimum voltage. If a lamp is rated at 12 volts you need to run it at 12 volts when you are taking a picture. If there is to much light for the exposure system then use a neutral density filter.

Some manufacturers make filters that correct their light sources for a perfect 3200 deg. Kelvin. These are a good idea and usually inexpensive. However the difference between 2800 and 3200 deg. Kelvin is not all that important.

For years there has been the myth that there is a "right" exposure for a micrograph. The best exposure for a micrograph is the shortest one the system will do. Faster shutter speeds help suppress vibration and vibration is one of the great destroyers of quality photos. If your photos look blurry and you are focusing correctly, suspect vibration.

Vibration is a real problem with fluorescence since fluorescent exposure times are longer than bright field. One way to reduce exposure times is to increase ASA times. High speed Ektachrome is a good film for fluorescence since the trade of grain size for faster exposures is worth it.

When you are setting up a photo system check the area for vibration. A good way to do this is to take a shallow pan and fill it with water. Place it near the photo system and leave it for an hour or so and come back and look at it. If you can see ripples on the water there is to much vibration.

A good place is set up a photo system is on a sturdy table well away from the wall. Walls tend to carry vibration from the building machinery. If you can't get away from vibration there are air suspended tables that do an excellent job. However they are expensive, bulky and difficult to operate.

Filters are a necessary part of photography. The most used filter is the Didymium. This filter enhances contrast on H and E stained specimens. A green filter can increase contrast for black and white film. Neutral density filters can help reduce light when necessary.

Black and white 35mm film is still used by a lot of disciplines. Cyto-genetics folks use it a lot and any one doing black and white prints for publication should use it. Unfortunately there is not the support for black and white there once was.

Now if you want good prints the odds are you will have to do it your self. In my area there isn't a really good black and white lab. Don't believe the story that you will get good results running it through a color negative machine. The results are poor if it comes out at all.

Black and white film can be correctly processed quite easily. Get good quality stainless steel tanks and reels. Practice loading the reels, once you learn it is easy. Make up the chemistry precisely as the package tells you and keep the temperatures of the developer and stop bath precisely at the recommended temperature.

Temperature control is one of the great secrets of good black and white film developing. Bad temperature control leads to grainy, fuzzy negatives. The other secrets are consistency and timing.

When developing black and white negatives always be consistent. If there is a problem it is much easier to identify if you are consistent. Time all process by a good lab timer. Times in the development process are critically important.

Were do you find out all this time and temperature information? First of all each package of chemicals has a very good instruction sheet. Kodak has wonderful information sheet on all their films and chemicals. Call them and get the information you will need.

A good place to start with black and white is to get the Kodak information bulletin on their remarkable film Technical Pan. This is what you should use for all black and white except, maybe, fluorescence. For fluorescence you may need the faster Tri-X film.

For most uses, however, Technical Pan is great. Its resolution and contrast range is superb, far beyond what the optics can provide. This film can do just about anything, you just have to get it to do it. The data sheets tell you everything you need to know. If you follow the data sheet and are consistent you will get world class prints.

When you get to the print phase make sure your enlarger is set up correctly. Check the manufacturers manual for instructions. Make sure that the lens is square to the bottom board. Like any documentation system vibration will destroy print quality so isolate your enlarger from vibration.

Let me be frank here. I have recommended Kodak films for all applications that they make film for. The reason is simple, they make excellent, consistent film and they offer support to the scientific community. They have specialists in the needs of scientists of all disciplines. Since they support our community I think we should support them. Polaroid makes several films that are great for instant documentation that is frequently needed in industry. Their black and white print films are very fast with good contrast and resolution. They make a black and white print film that gives both a print and negative. This can be great if you need copies of a print later.

Color film is where Polaroid suffers. While there new films designed for the automatic ejecting cameras is better there is still problems with color and consistency. If you need instant color prints Polaroid is the best, better than tv. printers. However the cost per print is high and the inconsistency of the prints can make the hassle factor to high. The more you need color prints the more you should take a look at tv. printers.

A tv. printer uses a tv camera to send the image from the microscope to the printer box. The box the grabs that image and prints it on a special paper. The print sizes range from 8 X 10 to wallet size, different manufacturers have machines that make different size prints. Taking the picture is simple, just look at a tv monitor and move the specimen and adjust the microscope until the picture looks right. Push a button on the printer and out your picture comes. The trick is to adjust the monitor to look just like the print.

This is real easy to do then what you see is what you get. This technology only works for prints right now but stay tuned. Photography is far from dead but for the first time we are seeing digital technologies that are giving it a run for its money in some areas.
What this means to you
To make good pictures you must set up the microscope correctly. Focus the photo eyepiece. Always focus rapidly.