Since the development of digital cameras, micro photography has become a lot easier. You can now take an unlimited number of pictures and view the results immediately. This gives much more room for optimising the photographic settings and illumination. A good picture starts with a well optimised illumination and this is something that cannot be said often enough. It is less relevant if a simple compact camera or an expensive DSLR camera is used. And it is also not necessary to equip the microscope with objectives that have the highest correction such as (plan) apochromats. A good illumination is most important for a good quality microscope image, more than anything else. This is also discussed in the article 'Getting the best out of a basic microscope'.
Micro photography is quite different from macro photography. With micro photography we go a big step further and to make good microscope pictures, some knowledge about microscopy is necessary. When doing micro photography, the microscope is the most important tool, not the camera. Micro photography is not only about taking beautiful photos, it is also about the content and about gathering knowledge and information. A less beautiful microscope photo therefore does not have to be less valuable. In this section I will discuss some possibilities for photography through a microscope and what is required for this.
Smartphone and tablet
Many cameras are nowadays suitable for micro photography. Let's start with the camera that almost everyone carries around: the smartphone. The quality of smartphone cameras has improved greatly in recent years and they have even become better than some compact camera's. With a smartphone it is possible to take good microscope pictures. Even with some tablets reasonable microscope photos can be made, I will show a few examples in this section. Smartphone and tablets have a small lens and that is an advantage because a wider range of different microscope eyepieces can be used for photography. With such a small lens you can get very close to the front lens of an eyepiece. Many Huygens eyepieces have a low exit pupil, which makes it almost impossible to get near the front lens with a DSLR camera and have the circular field of view in focus. Even with some compact cameras this is difficult. If you want to take pictures with such cameras, a wide-field (WF) eyepiece with a high exit pupil is needed. Using a smartphone, the edge of the circular field of view can be brought into focus with all eyepieces.
The easiest way to photograph through a microscope with a smartphone is to hold the smartphone camera above the eyepiece until you see the field of view as a sharply outlined circle. This takes some practice in the beginning, especially to keep the phone stable. The trick is to hold the middle finger of the left hand (closest to the camera lens) against the microscope tube to provide support. It is easier to photograph through a Huygens eyepiece with a low exit pupil because you can get closer to the front lens of the eyepiece. With WF eyepieces, you have to hold the smartphone a few cm from the eyepiece which makes it more difficult to support with one hand. There are special smartphone adapters that you can attach to the microscope tube and in which you can clamp the phone. This way it is possible to watch everything live on the screen of the smartphone instead of looking through the eyepiece. Because a smartphone has a small sensor, the photo will not have a very large resolution, at least, compared to a system camera. The quality of the image can be further improved by using an eyepiece with a higher magnification, like for example a 15x eyepiece instead of the usual 10x eyepiece. The idea behind this is that the sensor records a smaller part of the image, so that the entire sensor surface is used for gathering information from that part. So, any details will be better recorded. Photos taken this way can be enlarged a bit more before they become "grainy". It is important to realize that fewer pixels are needed as the objective magnification increases. An image taken with a 40/0.65 objective contains less information than when taken with a 10/0.25 lens. Therefore, using higher power objectives with a smartphone will give better results. It is also possible to take acceptable pictures with a tablet, although this is a bit more inconvenient.
Shown below are some images I took with a smartphone, the Samsung Galaxy A8 (SM-A530F, 16 MP), Samsung Galaxy A3 (SM-A300FU, 8 MP) and a tablet (Huawei Mediapad T3, 5 MP).
Euglena with red eyespot and cyanobacteria (Anabaena), photographed with the Samsung Galaxy A8. Carl Zeiss Neofluar 40/0.75 objective with Kpl12.5x W eyepiece.
Cells from Elodea with numerous chloroplasts, photographed with the Samsung Galaxy A3. Zeiss-Winkel 40/0.65 achromat with Olympus P15x eyepiece.
Filamentous algae (Spirogyra en Mougeotia), photographed with the Samsung Galaxy A3 using 2x digital zoom. Zeiss Neofluar 63/1.25 objective with Zeiss Kpl12.5x eyepiece.
Histological slides from human (left) and plant (right), photographed with the Huawei tablet. Zeiss-Winkel 100/1.3 achromat (left) and Zeiss-Winkel 40/0.65 achromat (right).
With a compact camera, excellent microscope photos can be taken. The most useful are cameras with an internal zoom because there are no external moving parts. These cameras can be held close to or even against the eyepiece. I have used an Olympus Stylus 725 SW for a long time. In the beginning, I used this camera on a tripod that I placed in front of the eyepiece and that worked fine. With eyepieces that have a lower exit pupil, such a camera can be held against the eyepiece and rest on it, which also provides support.
If you want to capture the finest details in a microscopic image, a system camera is the way to go. With both micro 4/3 and DSLR crop sensor cameras, excellent micro photos can be taken. A full-frame camera is not really needed for micro photography, unless you want to make high-resolution videos. There is an important aspect in choosing a system camera for micro photography: vibrations. Cameras with a mechanical shutter mechanism can cause vibrations which results in blurry images. In micro photography, vibrations have a major impact, much more than with normal photography because of the high magnifications. I use a Olympus PEN E-PL1 mirrorless micro 4/3 camera and a Canon EOS 600D DSLR, both somewhat older models that still work fine. The Olympus PEN causes significant vibrations when using short exposure times as this camera does not have an electronic shutter. The vibrations caused by the shutter mechanism can be greatly reduced by using longer exposure times like 2 seconds or more. When I use the Olympus PEN, I only make pictures of more or less static objects because I use an exposure time of 2.5 seconds. The Canon 600D contains an electronic shutter and has the so called mirror lock-up feature. With this camera, the picture can be taken in LiveView and no vibrations are generated. The mirror is flipped up and the photo is taken before the mirror returns to it's default position. To avoid manual vibrations with both cameras, I use the self timer with a 2 second delay after pressing the button. Canon cameras that are very well suited for micro photography include the 500D, 550D, 600D, 650D, 700D and 750D. But there also more models suitable.
After choosing a particular camera, the camera has to be connected in some way to the microscope. Micro photography with a system camera can be operated with or without a camera lens. The setup without a camera lens is simpler and here a compensating eyepiece is used to project the image onto the sensor. With the Olympus PEN, I have used a Sigma 30 mm lens for a long time and that works quite well. This lens has the advantage that there are no externally moving parts when focusing, everything happens inside. This makes it possible to use the camera without the need for mechanically connecting it. The following image shows a setup with the Sigma lens that I have used a lot with horseshoe stands. The Olympus PEN camera with Sigma 30 mm lens is simply positioned on a silicon gasket that is placed on a Ihagee microscope adapter. The combined weight of camera and lens is sufficient to ensure a stable position. It is a convenient set-up for a horseshoe stand; after the photo has been taken, the camera is removed and visual observation can be continued. A second advantage of the Sigma 30 mm lens is that the entrance pupil is lying forward. This makes it possible to use any microscope eyepiece, regardless if it is high eyepoint or not. Even the most simple Huygens eyepieces can be used without any difficulties
Olympus PEN E-PL1 with Sigma 30 mm objective placed on a Olympus horseshoe stand.
It goes without saying that better quality photos can be taken with a system camera due to the higher resolution. The limitations of cameras with small sensors only become noticeable when an image is enlarged. The following images show a comparison between the Olympus PEN and the Huawei tablet with a slide of Cymbella.
Comparison between the Olympus PEN (upper image) and Huawei tablet (lower image) with a slide of Cymbella. Leitz achromat 40/0.65 with Leitz Periplan GF 10x eyepiece.
Crops from the previous images. Olympus PEN (left) versus Huawei tablet (right). Leitz achromat 40/0.65.
Nowadays I only use the Olympus PEN and Canon 600D without a camera lens, so no relais optics. The image is projected onto the sensor by the eyepiece. The better-corrected compensating eyepieces are particularly suitable for this and for the Leitz Dialux II, I use a Periplan GF 10x for projection. The set-up with the Canon 600D on the Leitz Dialux II is shown below. I have noticed that images taken without a camera lens have a bit more contrast. That makes sense as there is less glass in the optical pathway.
Adapting the Canon 600D to the Leitz Dialux II without camera objective. A Periplan GF 10x is used as projection eyepiece.
To evaluate whether the camera adaptation was done correctly, I use a object micrometer and some wet slides. If the optical alignment is not correct, all kinds of aberrations will become visible. I also often use a slide with pollen because chromatic aberration is quite easily noticeable as blue colored edges around the pollen grains. The following images show that there are no significant aberrations with the Canon setup.
Object micrometer photographed with a Leitz NPL Fluotar 25/0.55. With a Periplan GF 10x eyepiece, the image is projected onto the sensor of the Canon 600D. A little bit of blur is visible at the very edges but in a microscopic slide that has some depth this will be hardly relevant.
Pollen grains of different plant species photographed with the Canon 600D and a Leitz NPL Fluotar 25/0.55. A Leitz Periplan GF 10x is again used as a projection eyepiece. This is the entire image as recorded by the Canon's sensor.
A fresh plant slide can also be used for detecting optical aberrations. The epidermis of Dracaena marginata is well suited for this as chromatic aberration will easily become visible around the fine calcium oxalate crystals and the walls of the cells.
Epidermis of Dracaena marginata photographed with the Canon 600D and a Leitz NPL Fluotar 25/0.55. The quality of the image is good over the entire length. Blurred areas are caused by differences in thickness of the specimen and therefore not all details can be simultaneously in focus. With these types of small crystals, any chromatic aberration will become visible quickly.
I use a similar camera setup with the Zeiss Standard. Here a Zeiss Kpl10x eyepiece is used to project the image onto the sensor of the Canon 600D.
Water from a ditch in which mainly resting stages of Euglena can be seen. Photo taken with a Carl Zeiss apochromat 40/1.0 and oblique illumination. Without any camera optics, the image is projected onto the sensor of the Canon 600D with a Zeiss Kpl10x eyepiece.
In most laboratories, special c-mount microscope cameras are used to capture all images, because of the speed and convenience of such cameras. They are expensive cameras, but they do not provide better images than an older DSLR camera that you can buy fairly cheap at the second hand market. The quality of photos taken with a DSLR camera will usually be higher.
Microscope objectives, eyepieces and condenser
The influence of the type of objective and condenser on the photographic quality is often overestimated. The quality of the slide and illumination has a much bigger impact on the final results. Moreover, it depends on the type of slide which objective makes sense to use. A larger working distance and better depth of field is often more important than a higher aperture.
Objectives with or without plan-correction?
Plan objectives correct for field curvature and ensure that the field of view is flat so that everything from the center to the edge is in focus. These objectives are preferably used with very thin slides and where there are no significant differences in thickness within the specimen, for example thin tissue sections or stained blood smears. It makes little sense to use a plan objective with a thick specimen that is not flat. In such a slide you will have blurry parts due to differences in thickness, so some parts in the field of view will be sharp up to the edge while other parts are not in focus. Plan objectives also don't have much added value for observing pond life. The price of plan objectives is considerably higher than objectives without plan correction; extra lenses are needed to correct for field curvature. But the image quality in the center of the field (this is where you usually focus on) can never be better with plan objectives than with non-plan objectives. With older plan objectives that have a less efficient anti-reflex coating, the presence of the extra lenses may become noticeable. Due to the the extra lenses, more reflections and thus stray light may be generated resulting in an image with poor contrast.
I don't use plan objectives that often. The idea that there is so much extra glass in these objectives, just to correct for some field flatness, does not really appeal to me. I have always the following thought in my mind: what is being observed and photographed is usually lying in the center of the field and not on the edge. Furthermore, for photography I don't find it necessary to capture as much of the field of view as possible.
Achromats, fluorites or apochromats?
It is a myth to think that you need expensive and highly corrected objectives for micro photography. In principle, you don't need anything else than good quality achromats. With normal achromats, excellent images can be obtained. See also 'Praise for the humble achromat'. Anyone who cannot achieve satisfactory results with ordinary achromats is doing something wrong. There is always a compromise between depth of field and resolving power and it is important to consider which objective is useful for a particular type of specimen. A high resolution apochromat places high demands on the quality of the slide. The higher the resolution on the objective, the shorter the working distance and the lower the depth of field will be. It can happen that the image of an apochromat turns out to be extremely disappointing because the quality of the slide leaves much to be desired. And some apochromats contain many lenses which can result in poor contrast, especially with older objectives.
If you want to use better corrected objectives, then in my opinion the fluorites (such as the Zeiss Neofluar and Leitz Fluotar objectives) are a very good choice. In general they give images with more contrast than apochromats and less chromatic aberration than achromats.
In my opinion, fluorites and apochromats will never be a complete substitute for achromats because the latter have a greater depth of field and working distance.
Many different types of eyepieces exist. For photography it is important that a distinction is made between compensating eyepieces and eyepieces with less or no correction. It is recommended to use a compensating eyepiece for high power achromats (>20x), all planachromats, (plan) fluorite and (plan) apochromates. With most low power achromats (10x and less), it is best to use an eyepiece that compensates less or not at all. Compensating eyepieces are often marked with a 'C', K, 'P' or other letter combinations that indicate the word 'photo' or 'compensation'. Sometimes however, there are no markings that indicate if the eyepiece is a compensating one. An example of that are the Olympus WF eyepieces, used for objectives with 37 mm parfocal lenght. These eyepieces are indeed compensating and recommended for photography. When in doubt, it is wise to look up the optical specifications given by the manufacturer. It is always important to judge the microscope image critically. How does the image look on the edge: is there any chromatic aberration or distortion visible? In case of a mismatch between objective and eyepiece, chromatic aberration and distortion occur mainly at the edge of the field of view. In general, best results are achieved with objectives and eyepieces from the same manufacturer. But there are many exceptions to this. What's most important is to use a compensating eyepiece for objectives that need correction. Most of the time, a 40/0.65 achromat for example, will give a better image with a compensating eyepiece from another manufacturer than with a non-compensating eyepiece from the same manufacturer. And no-name microscopes often come with eyepieces that barely compensate. See also: 'The right combination of objective and eyepiece'.
Sometimes it is said that an achromatic condenser is recommended for color photography. Perhaps there was some truth in this in the pre-digital era, but nowadays the impact of software to improve the colours in the image is many times greater than the achromatic correction of a condenser. Most microscopes are equipped with a two-lens Abbe condenser that has no correction. This condenser is well suited for nearly all purposes. The influence of an achromatic and aplanatic condenser on the quality of the image is being overestimated, it is important to realise that a condenser does not actually contribute to the creation of the image. Highly corrected condensers are expensive and in many cases they do little to improve the image. These types of condensers will only be useful in very critical and high-resolution microscopy where the maximum resolving power must be achieved.
Structures and details in microscope pictures can be greatly enhanced with image processing software. Especially contrast enhancement does a lot. For processing pictures I use the program RawTherapee but I am quite careful with image editing. Mainly white balance, contrast and brightness are adjusted in moderation. I believe that an image should look pretty good from the start, without any editing. It is fine to remove any annoying spots in the image caused by dust particles that have settled on a lens or the camera sensor. Personally, I'm not in favor of erasing certain details in a photo just to make the subject in question look nicer. For example, if a picture shows algae that are surrounded by bacteria, then I think those bacteria simply belong there. These are the algae in their natural habitat that they share with other organisms. In my opinion, it is the different sizes and shapes of different organisms in the same image that gives much more meaning to that image.