Giorgio Carboni, September 2002, updated December 2003
Translated by John G. Davies
Figure 1 - Spirogyra in 3D. Use bi-colored spectacles to view.
Use of Polarized Filters
Use of the Occluded Objective
Use of Displaced Slides
Use of Oblique Illumination
Use of Colored Filters
Use of Liquid Crystals
Limit of High Power Stereoscopy
Bibliography and Internet Resources
Our eyesight normally allows us to see things in three dimensions: width, height and depth. This is known as stereoscopic vision or commonly as 3D. The ability to perceive depth, volume and the distance between objects is important. When we look at a photo, television or a film we can only see things in only two dimensions. In the same way, high-powered microscopes lack any depth of field, so objects appear flat. In this article I describe how to convert these instruments to produce stereoscopic images.
In order to perceive depth it is necessary for one eye to make the observation at an angle to the other. When this happens the brain fuses the two different images into a single stereoscopic one. It is the slight difference in angle that creates the impression of volume and depth. Normal microscopes can magnify between 50 and 1200X using a single objective at a time. The majority of these instruments are used in Biology, whilst others are used in metallurgy, mineralogy and other areas. The simplest models are equipped with a single eyepiece, whilst more complex one have binocular eyepieces and may be equipped to use a camera. Even though they have two eyepieces each provides the eye with identical images because a prism in fact splits a single image, thus the image appears completely flat.
Many people would like to have the advantages that three-dimensional images produced by stereoscopic microscopes offer. In the observation of protists it enables one to follow their movements as they change depth and to appreciate the internal disposition of organelles. This article describes how it is possible to produce these effects with a normal microscope using a single objective.
Before describing the methods of high-power stereoscopic observation it is necessary to say something about Köhler Illumination which is the more diffuse illumination system in the microscopes. Figure 2 shows that the lamp filament and the specimen come into focus at different levels along the ray path, so when the specimen is in focus the filament is not and vice versa. This avoids superimposing the two images and the specimen is evenly illuminated. In the diagram the two paths are separated for clarity. Note that in the illustration the image of the specimen is focused near to the eyepiece, however during the observations the light that comes out of the eyepiece is parallel and the image is focused at infinity.
The image of filters used must never come into focus at the same level as that of the specimen, but must always be displaced. From the theoretical viewpoint the best position for these filters is at the aperture diaphragm. However, this position is in practice inconvenient, so they have to be positioned between aperture diaphragm and the field diaphragm. Be aware that if the filters are placed too close the field diaphragm their image will come into focus on the specimen. The most convenient position tends to be just above the illuminator, though this can be rather too close to the field diaphragm. During this article this is the position that is referred to because it is convenient and usually works well. Adjusting the position of the condenser can create a situation where the filters are out of focus and the specimen is clearly visible. However, if there is insufficient separation between the filter images and the specimen mount the filters on a tubular support to distance them from the field diaphragm.
USE OF POLARIZED FILTERS
The first technique that produced three-dimensional images required the use of two polarizing filters arranged on the illuminator so that horizontally polarized light attains an half of the objective and vertically polarized light attains the other half of the objective. Then polarizing filters were placed in the eyepieces of a binocular head so that each eyepiece only allowed the rays polarized in one direction to pass. Each eyes thus received two different images seen from a slightly different angle making stereoscopic vision possible. Due to absorption by the polarizing filters, it is also necessary to have an illumination system of a certain power.
Sheets of polarizing plastic can readily be obtained from photographic shops or in this website: http://www.3dlens.com/ (Product No: #P210, Polarizing efficiency: 99.98%). Cut two strips that are polarized at 90° to each other and mount them above the illuminator as shown in Figures 3 and 5 without superimposing them. The line of contact between the two filters must be orientated vertically (figures 3 and 6). The two filters can be mounted using screws or glued with silicone. Cut away the excess parts.
Place a polarizing filter on each eyepiece (figure 4). For the initial trials these can be cut from the same sheet as the other two and can be held on the eyepiece by adhesive tape. Later optical quality filters can replace those on the eyepieces, but the others suffice because of their position close to the lamp. Ones made from polarizing plastic are more than adequate here.
You may be able to obtain the correct-sized filters to be placed on the
eyepieces from well-stocked photographic supplier or from the USA - Edmund Optics -
http://www.edmundoptics.com/ . Once you obtained these filters, mount them on the eyepieces. The filters placed on the eyepieces must be orientated in such a way as to allow light polarized in one sense to pass through one of them and light polarized in the opposite sense to pass through the other. We will now see how to do this.
Now, having fixed the filters to the illuminator and eyepieces, switch on the illuminator and put a slide containing freshly-collected protists on the stage. Using the 20X objective lens adjust the height of the condenser so the contact line between the two filters is in focus and central in both eyepieces' field of view. No unpolarized light must pass between the two lower filters, they can be overlapped by a fraction of a millimeter.
Rotate the polarizing filter of the right eyepiece until the left-hand side of the field is obscured as much as possible. Do the same thing with the filter on the left eyepiece, this time obscuring the right-hand side of the field.
Now adjust the condenser again to obtain optimum illumination and to move the contact line out of focus. You will see that the field becomes bright again. At this point, you should be able to see in three exciting dimensions. If the specimen appears in an opposite relief (pseudoscopy), just rotate the filters on the illuminator through 180° to obtain the correct image.
Figure 5 - Polarizing filters
Figure 6 - Polarizing filters
Adjust the illumination level and the diaphragm, after which you can begin your observations. As required change magnification, then adjust the diaphragm, illumination and the height of the condenser. Increasing the magnification also increases the stereoscopic effect and between 200 and 400X provides the best conditions.
This method uses one half of the objective to observe the specimen from one direction and the other half for the other direction. In order to achieve this, the image of the polarizing filters must be in focus at the plane of rear focal point of the objective (figure 2). This will occur precisely when the filters are arranged at the aperture diaphragm. In reality, such precision is not required. What is important is that when the specimen is in focus, the filters are well out of focus. If the dust on the illuminator comes into focus along with the sample, use a tube to raise the half-moon filters a couple of centimetres. Once the initial trials are complete the filters may be cut into semicircles to fit a ring constructed to hold them snugly on top of the illuminator.
I have obtained excellent results with this method and the smear of water under a coverslip, normally only a few tenths of a millimeter thick appears to be a pool several meters deep. Unfortunately, for different reasons observation in 3D using the polarizing filter technique doesn’t always work. If you fail to obtain noticeable results check that there are no other polarizing filters left on the condenser or elsewhere along the optical path. Sometimes one optical component of the binocular box may interfere with the polarization of the light. Some prisms and non-metallic mirrors have this effect. Observation in 3D of permanent preparations often doesn’t yield good results.
If you are about to buy a microscope and you want to know if you can adapt it for stereoscopy, do as follows: take with you two cuttings of polarizing plastic. Switch on the microscope and place one of the pieces of plastic at the exit of the illuminator, so as to cover it completely. Place the other piece on top of the right eyepiece and by rotating it verify that there is a point in which there is a discrete extinction of the light. Do the same thing with the other eyepiece.
I have only used this technique for observing protistans and have to say that I'm not tired of observing them yet. To finally see the form of an Amoeba as it extends it pseudopodia upwards is inspiring. What can I say about Ciliates that rise and descend through a forest of cyanobacteria? It is a real joy to see Euplotes as it explores a strand of Spirogyra, passing under then over it like a gigantic reed. Now I actually see it rise and sink, instead of just moving in and out of focus! The interior of a Vorticella, with the movements of its food vacuoles and the contractile vacuoles, is wonderful. A friend, who is passionate about Diatoms, told us that our method allowed him finally to appreciate their three-dimensional structure and it had revolutionized his observation methods. This technique also increases the depth of field because the eyes can accommodate more comfortably having the impression of depth.
It is impossible to list every case where there might be an aesthetic or
practical advantage in having the third dimension at high magnification. In
order to see the advantages that this technique offers, try comparing it with
standard observations in different cases. Stereoscopy particularly offers
advantages when there is a lot of detritus present because they do not appear as
a single clump as they normally do, but actually lie at different levels and out
of focus. It is so spectacular to observe protists in three dimensions that once
you have tried this technique you will not be able to do without it.
This technique is useful for direct observation, but unfortunately it does not lend itself to photography or filming in 3D.
USE OF THE OCCLUDED OBJECTIVE
This method involves first closing one half of the objective and then the other whilst taking the two photos. It is useful for photographing stationary objects. Actually it is not essential to close the objective, only to intercept half of the beam by placing a piece of card on the diaphragm or more conveniently, on the illuminator. Figure 2 shows how this corresponds with the partial occlusion of the objective. The stereograms have to be observed through a stereoscope or a 3D slide viewer. Alternatively they can be used to obtain an anaglyph to be observed with red-cyan spectacles. Obviously this technique does not lend itself to direct observation.
USE OF DISPLACED SLIDES
Like the previous technique, this method allows photography of stationary objects and does not permit direct observation. It uses horizontal displacement of the slide between two photographs, so that one becomes the left image and the other is right partner. It has the advantage of maintaining optimum definition and the colors of the image.
To observe the adjacent picture of Spirogyra try to superimpose the two photos whilst keeping the eyes parallel. In order to do this, gradually move closer to the screen until the two pictures fuse into a single, out-of-focus stereogram. Move away slowly, trying to keep the images superimposed until the image becomes clear. If you can cross your eyes easily, look at the second and third stereograms whilst trying to look at the left picture with the right eye and vice versa.
USE OF OBLIQUE ILLUMINATION
Some condensers can be de-centralized, that is they can be displaced from the principal optical axis. This results in angled rays illuminating the specimen. In order to obtain a stereoscopic image it is necessary to take two photos, one with the condenser displaced leftwards and the second with is moved to the right. In a similar way, the mirror can be slightly rightwards and then to the left on microscopes with a moveable mirror, so the specimen is illuminated from different angles for each photo.
USING COLORED FILTERS
The following procedure allows the direct observation, photography or filming of moving objects to be done relatively easily. I describe a method whilst not devoid of problems offers certain clear advantages. The technique is similar to that based on polarizing filters except that it uses colored filters instead. A red and a cyan filter are placed adjacently on the illuminator (figure 11), cyan lies halfway between green and blue. The images obtained by this method are called "anaglyphs" (superimposed stereograms) and the images show characteristic red-cyan colors. The photos must be observed through red-cyan spectacles. By convention the red filter is on the left which results in the left eye receiving a slightly different image from the right.
Obtain two pairs of bi-colored spectacles from photographic suppliers. Remove the filters from one pair and place them on the illuminator. Put on the second pair and look through the binocular microscope. When this is done correctly it is possible to see the specimen in three dimensions. If the specimen appears in opposite relief, change over the filters on the illuminator.
Some people are rather prejudiced against this technique because of the image's modified colors and the inconvenience of wearing the red-cyan spectacles. I have to say that it isn't really that bad because one becomes used to wearing the glasses and the colors don't appear so garish after a short time, rather they become greyish. Surprisingly, the colors often appear quite normal, not quite as true as with normal observation or using polarizing filters, nevertheless it is nowhere as bad as you might imagine.
As I have said, this method has the great advantage of allowing observation of moving specimens to be made easily. They can be filmed in 3D and easily projected, the impression of three dimensions being contained in a single image. This means that a single shot with one camera is sufficient, whereas when polarizing filters are used two synchronized cameras are needed. If you are limited to direct observation then the polarizing filters method is certainly the best.
The colored filters method requires a binocular microscope, but to take photos a simple student's monocular microscope is adequate. For microscopes equipped with a mirror, it is necessary to obtain colored plastic films, as near to the color of the spectacles as possible, and place them in front of an illuminator with an opalescent bulb. Figure 12 shows how to do this with a sheet of card.
Unlike the polarizing method, a video camera can be mounted on the microscope and moving 3D images can be examined on a monitor or TV. This enables the image to be observed by an entire class as long as red-cyan spectacles are available.
http://www.microscopy-uk.org.uk/mag/art98/diat.html Some magnificent anaglyphs of microorganisms.
USE OF LIQUID CRYSTALS
A weak current can convert a transparent liquid crystal screen
from transparent to black. Using this principle it is possible to make 3D films
with just one video camera. A pair of liquid crystal screens placed on the
illuminator, in the same way as the polarizing or colored filters, can
alternately allow light to pass from one side of the illuminator or the other.
As we have seen, this corresponds to using one half of the objective and then
the other. By synchronizing the liquid crystal screens with the frames of the
video camera, alternate frames correspond with left and right halves of the
objective. In order to see these images in 3D it is necessary to use special
liquid crystal spectacles synchronized with the projection system.
Several research laboratories are beginning to offer TV screens that allow 3D recordings to be shown without the need to wear spectacles.
THE LIMITS OF HIGH POWER STEREOSCOPY
As is well known, any increase in magnification causes a corresponding decrease in depth of field. This limitation is particularly obvious when making photographs, but is less so in direct observation when it is possible to change focus to look at different levels. As a remedy for this poor depth of field it is possible to superimpose several photographs, but the images produced in this way are not sharp. Recently, programs have been produced that recover the clear part of each photograph.
These techniques of making stereoscopic observations and photographs at high
magnifications will open up new horizons in your use of microscopes. I am sure
that once you have tried them you will find the methods hard to give up,
especially for the observation of protists. These techniques are within the
scope of any amateur microscopist. However, these
methods need a lot of trials and adjustments to produce satisfactory results.
The optics of microscopes, stereoscopy, polarization and anaglyphs are vast fields and cannot really be fully described by the schematic information that I have supplied. An understanding of optics will significantly improve your ability to obtain good results from our techniques. Much supporting information is readily available on the Internet.
The sight of microorganisms in pond water swimming up and down seen in 3D at high magnifications is incredibly fascinating. You will not see anymore Spirogyra as a mass of matted strands, but as separate fibers lying at different levels sloping up or downwards with their elegant helical chloroplasts inside each cell. At last, you will see if that strands have left-handed or right-handed spirals. You will notice that the quality of observation is noticeably improved because these techniques not only offer an aesthetic improvement, but the addition of the third dimension provides further information about the shape, size and position of the objects and organisms that you are observing. All this helps you to understand them better.
BIBLIOGRAPHY AND INTERNET RESOURCES
1 - Rudolf Kingslake, Applied Optics and Optical Engineering, Academic
Press, 1967, vol 4, p 60.
2 - http://www.microscopy-uk.org.uk/mag/art97b/wim2.html 3D Photography through the microscope, by Wim van Egmond
3 - http://www.physicsclassroom.com/Class/light/U12L1e.html Some basic information on polarization
4 - http://www.microscopy-uk.org.uk/mag/articles/poldw.html Technicolor Nature. By David Walker. Useful information to buy polarizing filters.
5 - http://www.microscopyu.com/ Nikon site dedicated to microscopy.
6 - http://micro.magnet.fsu.edu/primer/microscopy.pdf Optical Microscopy.
7 - http://www.stereoscopy.com Excellent information about stereoscopy.
8 - http://www.3dmetro.com/new/links.html Links on stereoscopy.
Internet keywords: stereoscopy links, 3d links, stereoscopy books, 3d books