There are a variety of different factors that determine the achievable resolution. Some of these factors can not be actively influenced by the microscopist, others can. Some of the factors play a larger role, others a smaller one. In the following post, I want to summarize some of these factors.

Objective-related factors

• Correction of lens errors: In contrast to achromatic objectives, apochromatic objectives focus more colors of the spectrum to one point. This results in a sharper image.
• The numerical aperture of the objective: This value is printed on the objective. The higher the value, the higher the resolution. The numerical aperture is a dimension less value which represents the cone of light that can be caught by the objective.

Lighting system

• General color of light: The shorter the wavelength, the higher the resolution. If your microscope uses halogen or tungsten lamps (instead of LEDs), then the color of the light will shift towards the red end of the spectrum with increasing age. This will reduce the resolution. The color of the light also changes with its intensity. If you turn up the light to maximum intensity, then the color of the light will be more towards the blue end of the spectrum (shorter wavelength and higher resolution). LEDs do not change their color with age or brightness.
• Light spectrum (color range): The color range may also impact on resolution. In the case of monochromatic light, chromatic aberration does not play a role and the light can be focused on one point.

Specimen-related factors

• The correct thickness of the cover glass: The correct cover glass thickness is extremely important for high numerical-aperture objectives. For other objectives, the effect may not be noticeable.
• The correct refractive index of the cover glass: This is something that you do not have to worry about, this is the task of the cover glass manufacturer.
• The correct refractive index of the mounting medium: This one should be as close to the refractive index of glass as possible.
• Thickness of the mounting medium: the thinner the better.
• The presence of immersion oil: Objectives that carry the label “OIL” need the correct immersion oil for best resolution.

Adjustments of the microscope

• The correct condenser diaphragm setting: This setting must match the numerical aperture of the microscope in use.
• The correct setting of the correction collar: Some objectives have a correction collar (a turnable ring) to adjust to the cover glass thickness. Most objectives do not have one, however.

Maintenance-related factors

• The cleanness of the optical parts: Dust and dirt generally decrease image quality and are a big annoyance, especially if one uses dark-field microscopy.

Stability of the photomicrographic system

• Moving objects: Moving cells naturally cause a blurring when long exposure times are used. This decreases resolution of the moving object.
• Stability: A shaky photographic system generally decreases resolution of the image.

The checlkist: how to obtain the best image quality

• Use new light bulbs and turn up the light. This will reduce the wavelength of the light. Alternatively, use a blue filter.
• Use cover glasses of the correct thickness and make sure that the mounting medium has a refractive index which is close to the refractive index of glass.
• Adjust the condenser aperture diaphragm to the numerical aperture of the objective
• If you use oil immersion, make sure that the oil has the correct refractive index
• Use fresh light bulbs (low in red light, high in blue light)
• Keep the microscope free of dust
• Make sure that the objectives, eye pieces are clean

Aperture: f/19.9, Exposure time: 10sec.

Aperture:f/22.6, Exposure time: 20sec.

In order to obtain good looking macro images one has to take several measures:

Technical set-up

• A solid, stable tripod: A heavy and stable tripod is absolutely necessary. Even the slightest vibrations (people walking, wind, shutter vibrations, etc.) will translate into a blurry image. I used a tripod designed for heavier video cameras.
• High f-stop values: Macro images generally have a low depth of field. In order to increase the depth of field it is necessary to increase the f-stop values. This again results in longer exposure times.
• Reducing shutter vibrations: Shutter vibrations can be minimized by using the mirror-lock up function of the camera. When the live-view feature of the camera is enabled, the mirror is in the “up” position. During release, the mirror does not have to swing up (because it is already up) and this significantly reduces vibrations. Not every SLR camera has a mirror lock-up feature, however. In this case, one can use a trick to completely eliminate shutter vibrations. Adjust the camera to have a long exposure time (let’s say 20 seconds). Cover the objective with a black cardboard, without touching the objective. Then release the shutter and wait 1 second for the system to stop vibrating. Then remove the black cardboard, this starts the exposure. One second before closing of the shutter cover the objective again. The opening and closing of the shutter (and mirror swing) will then take place while you have the black cardboard in front of the objective. Amateur astronomers use the same technique for taking vibration-free images of the night sky. They cover the telescope aperture with a dark cloth (without touching the telescope, of course).
• Long exposure times: This may come as a surprise for some. Long exposure times can result in more steady images because the duration of the vibrating camera/tripod system (after release) are much shorter than the total exposure time during which a steady image reaches the sensor of the camera.

Composition, artistic aspects etc.

• Sufficient ambient light: To prevent hard shadows, I decided to use the natural, indirect light of the room to take the picture of the rose. This results in longer exposure times, which makes a very stable tripod a necessity.
• Contrast: Make sure that the main object is set off from its background. This can be achieved either by blurring the background, or by making sure that the background has a distinctly different color.
• Exposure time: If you use a black background, then the camera may expose longer than necessary. The black background “fools” the camera into thinking that the image is too dark. This may result in the object being overexposed. Underexpose by 1-2 stops if the main object is too bright.
• Post processing: Crop the image and adjust the colors so that the background is completely black (if you use a black background). This will increase the impact of the picture.

Dandelion macro image.

Lens: Sigma 20-300mm APO DG MACRO (macro up to 1:2)
Camera: Canon EOS 450D
Exposure Time: 2 sec. (long time to minimize vibrations)
Aperture Value: 6.62 EV (f/9.9)
ISO Speed Rating: 100
Focal Length: 300.0 mm

The male pine cone (or flower) is responsible for forming pollen. These pollen grains are also visible in the image above. For a zoom-able image of a female pine cone, visit the following link: Virtual microscope: female pine cone (Pinus)

Field diaphragm is wide open. Reflections from the side of the tube are very strong.

Field diaphragm is half open. The reflections are less.

Field diaphragm is closed. Only the direct light is able to reach the camera.

Taking a picture of the tube with a webcam. Any camera with a small lens would also have done the job.

Advantages of Köhler illumination for photography

Köhler illumination increases the contrast of a photomicrograph because it reduces stray light and glare caused by reflections inside the microscope. On the right side you can see images taken through a trinocular head with a web cam. The more that the field diaphragm is closed, the less the reflections coming from the side of the tube. The bright spot in the center is the light which comes directly (unreflected) from the light source. In order to see a picture, it would be necessary to remove the lens from the webcam and project the image directly on the sensor of the webcam. In this case, the lens was left on to be able to see the side of the tube.

For more background info on Köhler illumination, you may be interested in the following two posts:

• Advantages of Koehler Illumination
• Adjusting Koehler Illumination

Stacking

Microscopic images generally have a low depth of field. It is possible to take several images of different depth of fields and to combine them in such a way that the final image is sharp throughout. By carefully turning the fine-focus knob a specified amount, it is possible to section through a complete specimen. Care should be taken, however: If too many parts of a transparent specimen are in focus in the final image, then these parts may cover each other, thus reducing the information content of the final image. Two cell organelles which are located behind each other, both being in focus, will cover up each other, and it is not possible to say which part belongs to what organelle.

Stitching

In this method, different overlapping images are assembled together into a larger final picture. While stacking combines the images “vertically”, stitching produces a larger final image by “horizontally” combining them. By stitching, it is possible to overcome the limited field of view. Stitching can be accomplished by using a panorama software. When choosing the software, one should take care that it allows for the combining of images both horizontally and vertically (some only permit for horizontal combination). Microscopic images often do not offer much image complexity. For this reason, the software may have problems assembling the images automatically. It pays off to do a little planning beforehand.

• How large will the final image be? The processing requirements increase significantly with increased image size.
• What camera resolution should be used? The choice of camera resolution has a significant impact on the final image size and required processing power. One should first test, if a high camera resolution is indeed necessary of if it is not simply results in empty magnification. Read: Required camera resolution for photography through the microscope
• How much image overlap should be used? More overlap may make it easier for the software to automatically assemble the pictures, but at the same time more pictures are needed to cover the whole specimen (which again increases work time).

All of the images of the category Virtual Microscope were stitched together.

Background clean up

The optical surfaces (especially the lighting system and condenser optics) are rarely completely free of dust. These disturbances will be present in the image, whether or not a specimen slide is present. It is now possibly to mathematically subtract these disturbances from the image. A picture with and without a specimen has to be taken at the same magnification and using the same exposure time. The empty image (without specimen) is then subtracted from the image containing the specimen.

Alternatively, it is possible to clean the background by selecting the specimen without background and copying it to a new clean background. This system was employed when taking a photograph of the tick (See: Virtual microscope: The Tick). An automatic selection only works well if the specimen’s color or brightness is significantly different from the background.

Increasing contrast

Contrast enhancement is one of the methods which, when done correctly, does not result in any loss of image information content, provided that the image does not use the full brightness spectrum from white to black in the first place. Nearly all photo editing programs contain a “levels” or “histogram” function, with which one can adjust the contrast.

Sharpening

Sharpening the image may subjectively increase image quality, but it will not result in a higher information content. Excessive sharpening introduces artifacts, it may enhance image noise and may enhance irrelevant image components, such as dust and dirt. Before the image is sharpened, it is probably better to increase the contrast. This will sometimes also give an impression of a sharper and more pleasing image.

White balance adjustment

This is a critical adjustment if one wants to obtain reproducible results. Microscopic light will show a different color temperature, based on the intensity level. Turning up the light to a high intensity will also shift the color temperature towards the blue end of the spectrum. A lower intensity setting will increase the red components. The age of the light bulb also shifts the color temperature towards the red. Digital cameras can adjust the white balance automatically, but this may not be a reliable setting, as the camera uses a predefined standard. A specimen which contains many red components, for example, may fool the camera into thinking that the light source is too red. The camera will then shift the color balance toward the blue, which does not reflect the real nature of the specimen. This is a particular problem of colorful images of crystals and specimens which cover the full field of view, without a visible background from the lamp. Some cameras also have a custom white balance function. In this case an empty reference image without specimen is taken. The camera will then use this image as a basis for correcting the white balance of all subsequent images.

Photo editing software also permits users to automatically or manually adjust the white balance. An automatic setting will also take the specimen itself into consideration (just like in the automatic camera white balance setting described above), and the results may not be pleasing. I generally make white balance adjustments manually. In this case, one has to click on those parts of the image that should be considered white, usually the background.

Comparison of resolution. There is practically no visible difference between 3MP and 12 MP. The limiting factor is therefore the microscope or specimen, and not the camera resolution.

The method of determining the optimum resolution is quite simple:

• Take a low-resolution and a high-resolution picture of the same specimen. I took two pictures each, one with a setting of about 3MP and one with the maximum resolution of 12MP. In both cases, the file compression was low (and JPG image quality high).
• Enlarge the low-resolution image to the size of the high-resolution image. In my case the low resolution (3MP) image had 2256 by 1504 pixels. It was enlarged to 4272 by 2848 pixels (12MP).
• Compare the two images. If the high-resolution image shows details that are not present in the low-resolution image, then this indicates a loss of information when taking a low-res photograph. Otherwise one can safely use the low-res setting of the camera. In this latter case the limiting factor for image quality is not the resolution of the camera, but rather the optics or the specimen.

The result? There was barely any discernible difference between the images. With the specimens that I used, it is perfectly OK to use the smallest camera resolution of 3MP. The limiting factor, so to say, is the optical system of the microscope and/or the specimen itself. For this reason, it is not necessary to choose a high resolution camera setting. Other specimens may deliver finer images, so the differences may become evident then, but from what I found, a small image resolution of 3MP is sufficient.

There are several possibilities to reduce the vibrations:

Long exposure time: The microscope-camera system vibrates for the fraction of a second after shutter release. One should therefore use a long exposure time (2-5 sec.). The camera will therefore collect most of the light when the system is steady. Of course, this does not work for moving objects.

Very short exposure time: Alternatively the exposure time can be significantly reduced (about 1/250sec). This is so fast that the vibration will hardly be recorded. This requires much light, however.

Micro flashing: One can also use a flash system mounted above the light source. With experimentation, it is also possible to make a system like this oneself.

The cardboard technique: A similar technique is used in astronomy to make steady images. The camera is set to “bulb” (B) and the shutter is opened. The light intensity has already been adjusted, but the light source is covered by a dark piece of cardboard. The cardboard is then removed for exposure. This method is indeed free of vibrations but requires long exposure times to be practical.

Mirror lock up: Some digital SLR cameras have a mirror-lock-up function. The integrated mirror of the camera can be moved into an “up” position. This reduces the vibrations significantly because the mirror of the camera does not have to swing up during exposure.

Flexible camera-microscope connection: Here, the microscope does not carry the weight of the camera. Rather, the camera is mounted either on a tripod or on another separate system. A vibration of the camera is therefore not passed on to the microscope.

So what methods do I use? I use a combination of several measures: I use a mirror lock-up function, a self-timer (2 sec) and a “long” exposure time of about 2 sec. During the mirror lock-up, it is not possible to look through the viewfinder to focus. Focusing has to be done already beforehand, or one could use the live-view feature to focus and evaluate the picture on the LCD screen before exposure.

This is a darkfield image of a tick. Ticks are blood-sucking arthropods. They possess 8 legs and are not insects, but rather are related to the spiders. Ticks are known to transmit various diseases, such as Lyme’s disease and encephalitis.

For more information on the tick, read the following post: The Tick (Ixodidae).

For more information on the pine cone, have a look at the following post: Female Pine Cone (Pinus) The specimen size is approximately 20mm from left to right.

Slide duplicator attachment: The left duplicator is mounted instead of the camera objective (via T2 adapter ring). The right one is attached to the existing objective (via filter threading)

Both systems compared.

The slide/film holder is the same in both cases.

Digitized slide showing vitamin C.

Digitized slide showing vitamin C.

There are several ways to digitize the slides:

• Using a slide or film scanner: This is the method of choice if you want to retain image quality. These devices are connected over USB to a computer. On the down side, scanning takes a long time and a film scanner is also not cheap. Some better slide scanners have a dust removal system.
• Use a flat-bed scanner: This is possible, if the resolution of the scanner is high and if there is a background lighting. Some flat bed scanners come with an appropriate slide holder. I found this system too time consuming, however.
• Get the slides scanned by a company: I did this once, it was expensive, but the quality was good. This is probably suitable for a smaller number of slides
• Photographing slides with a dedicated slide duplicator: This duplicator is directly mounted on the camera, instead of the existing objective. There is a slide/film holder attached. The slide duplicator that I initially tried was designed to reproduce 36mm slides again on 36mm analog systems (or digital cameras with a large sensor – the “full-format” systems). My digital camera’s sensor is smaller than film size. As a consequence it was not possible to fit the whole slide on the image and I always had added magnification. The objective allowed me to zoom in, but not zoom out (what I would have needed.) There are objectives like this that are specifically made for digital SLR cameras with a smaller sensor. So watch out if you get one of these devices.
• Photographing with a duplicator in front of the objective: This system is mounted in front of the camera’s existing objective. It contains extra lens elements to magnify the slide. This is the system that I used, and it worked well. The adapter is screwed into the filter threading of the camera’s original objective, so be careful that they are compatible (or use an extra adapter ring). One possible problem may be, that there are now many lens elements between the slide and the camera’s sensor. The image quality may suffer because of this. For my purposes, this was perfectly fine. Considering the generally low resolution of microscopic images, the quality loss was negligible. This duplicator also allows me to zoom in. This way I can take overlapping pictures of the slide and assemble them (“stitch” them) using panorama software. This way it is possible to reproduce the slide with an extremely high total resolution – but it’s time consuming (and it’s questionable if the slide / microscopic image has the necessary resolution in the first place.)

About exposure time

It’s very important to rest the camera body as well as the objective (whatever system is used) solidly on a stable surface. The objective should not be able to vibrate in relation to the camera body. If both are stable, then the optimum exposure time (to minimize vibrations) should not be too critical. Because I am in no hurry, I set the exposure to about 2 sec. The whole system will have vibrated out (and be steady) for the most part of the exposure. Long exposure times are more important when the camera is mounted on a microscope. In this case the effects of vibrations are much more evident. To minimize vibrations even more, I use the mirror-lock up feature of my camera.

About white balance

My camera allows me to adjust a custom white balance. I first take a blank picture of the white screen (the adapter system has a white screen) of the and use this as a reference image. The camera will then automatically adjust the white balance of all images that are taken. If your camera does not allow for the use of a reference image, then you should set the white balance manually based on the actual light source used. It’s not a good idea to use auto-white balance, as there is a color drift. Depending on the algorithm used, the camera may assume that the brightest spot on the image represents white (or a shade of grey, if darker), which may not be the case.

Why talk about drawing microscopic images, if it is now possible to record the images using digital cameras? Drawing is not an old-fashioned or outdated method, rather it complements the possibilities of photographic documentation.

Advantages of Drawing Microscopic Images over Photography

• Combining different focus levels into one picture: Especially high-magnification images suffer from a low depth of field. A drawing is able to combine the different focus levels. It is now also possible to use image stacking software to combine different (digital) photographs from different focus levels into one final image.
• Removing artifacts: Dust and dirt do not have to be included in a drawing, but they are automatically part of a photograph.
• It is possible to draw a “typical” structure: The artist is able to look at several different specimens and then produce a final, typical drawing of the specimen.
• Emphasizing: The artist is able to emphasize different structures of the specimen, and to ignore others. This becomes useful if the drawing is to be used for identification purposes. This way a drawing can aid an inexperienced viewer. A photograph is often more complex with unnecessary details.
• Training of observation: Drawing takes practice and requires careful observation. These two aspects are trained.
• Same style: For publication purposes, it may be an advantage to show different microscopic specimens in the same style and size. Artists can use the same drawing style even for vastly different specimens. It is then possible to arrange the drawings on the same page next to each other without causing too much visual confusion.

Drawing Techniques

• Drawing without technical aid: For right-handed people, look with the left eye through the eyepiece of the microscope and with the right eye at a white drawing surface. You may need to adjust the angle of the drawing surface (placed right of the microscope) appropriately. With a bit of practice, your brain will combine the microscopic image and the white sheet of paper into one single image. You can then trace the image onto the paper.
• Drawing tubes: These devices can be installed beneath the microscope head. It will direct the image into a tube and project it directly on the table to be traced.
• Using a small mirror: A small mirror is mounted in front of the eye piece to project the image onto the drawing surface. The image can then be traced.

Kiwi Fruit: stacked with the software Combine ZP.

Materials: microscopic slides, cover glass, a soft kiwi, tissue paper.

Method:

1. Take a small piece of the soft part of a kiwi (not the seed and not the white center) and place it between the slide and the cover glass.
2. Carefully tap against the cover glass with a hard object, such as a pen. This is to test if the kiwi is actually compressible (or if it is not ripe enough). A hard kiwi may result in a broken cover glass.
3. Place a small piece of tissue paper on top of the cover glass and carefully and gently press down on the cover glass using your fingers (provided that the fruit is soft enough). Excess kiwi juice will be absorbed by the tissue paper. Be careful: do not move the cover glass horizontally. A thin, green kiwi film should have formed between slide and cover glass.
4. Observe under the microscope as normal.

Note: The image on the right shows some (unidentified) structures found in a kiwi fruit. The final picture is a stack of 12 images, processed with the program Combine ZP. Stacking of the images increases its depth of field. Without stacking, some of the green bubbles would not be in focus.

An opened field iris diaphragm increases the width of the light beam. This setting is used for low magnifications (large field of view)

An closed field diaphragm decreases the width of the light beam.

A closed field diaphragm.

• Uniform specimen illumination: Before the advent of Koehler illumination, a diffusing glass was placed over the light bulb. This had the disadvantage of reducing the light spectrum.
• Reduction in specimen heating: A heated specimen increases evaporation of the water beneath the cover slip and also reduces the dissolved oxygen, a potential problem when viewing live organisms.
• Reduction of light reflections in photographic work: Excessive light is eliminated reducing reflections inside the optical system. As a consequence the contrast of the photographic image increases.

The Koehler field diaphragm is designed to restrict the light beam only on this part of the specimen which is actually observed. Especially at high magnifications only a very small part of the specimen needs to be illuminated.