by Howard Webb, Missouri, USA
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I have always loved darkfield images; there is something almost magical about the glow against a black background. However, while I could enjoy viewing such images by eye, my attempts to photograph these images were disappointing. After thinking the problem through, and playing with the controls of my digital camera, I have found that good results can be obtained consistently and with little trouble. You need to understand a bit of how cameras are different from the human eye (and brain), then just do a bit of playing. I am not going to go into the 'hows' of setting up the microscope for darkfield illumination, there are already good articles on the topic (Making Darkfield Illumination Easy by Mike Samworth and Wim van Egmond). What I want to describe are the issues involved with photographing these images with a digital camera.
When normally using the microscope, light enters from below the subject and refracts through it, then enters the lenses and finally your eye. With darkfield illumination, you block the light that directly enters the subject. A small ring of light is permitted around the subject, which reflects off of it and into the lens. The blocking of the direct light gives the black background (darkfield), on which the brightly illuminated subject is superimposed. The difference of reflected light from refracted light, and the extreme contrast between subject and background, are what give these images their richness.
It should be noted that darkfield images are not better or worse that normal illumination, they are just different. While the high contasts may produce a more artistic image, this is not necessarly a better image for scientific use. For scientific purposes, different lighting conditions serve different needs. Do you want to see internal structures or external surfaces? Is the subject transparent or opaque? Darkfield illumination is another 'tool' in the 'toolbox'. The more tools you know, the more you can do.
The Eye and the Camera
The human eye and brain are able to process a wide range of light. A camera, under the same circumstances, can handle only a limited range. If the camera adjusts to the bright subject areas, the dark areas are black. Inversely, if the camera can distinguish the dark images, the light areas are 'burned out' (image right). While not scientifically accurate, for illustration's sake it is like saying the eye can detect images in a light range from 1 to 100, but a camera can 'see' only a 20 point range (i.e. 1 to 20, 40 to 60 or 80 to 100). The eye is much better than the camera; you have to recognize these limitations, and work with them (or let them work for you).
Most cameras with 'auto-exposure' try to adjust the light level to give the overall image a 50% neutral grey tone. Given a subject against a black background, the camera will try to lighten the image (to make the background more grey), which tends to 'burn out' the central subject. This is a normal photographic problem, and most cameras have a way to fake the camera out by setting the 'stop' up or down to adjust for these high contrast situations. This is the heart of the solution. With a high contrast between subject and background, you have to adjust the camera to properly read the subject, and lose the background to the extreme (in this case, to blackness). This is where the narrower range of the camera works for you; even if your darkfield illumination is not perfect, the narrower range of the camera will push the dark areas to a uniform rich black.
In a previous article I outlined how to use the Intel PC Camera for microphotography. I am using the same setup for these images. If you are using a different digital camera, read its help file; there should be similar controls, as what I am using are standard features. The Intel camera provides settings to control many of the features, though under normal conditions they can be ignored (and are often forgotten). The default setting is for 'auto-exposure'. With one click I turned the default off, and started playing with the 'brightness' control (a slider bar). By moving the settings window to the side, it is possible to see the live image in the microscope at the same time you are playing with the controls. Moving the brightness control is similar to adjusting the aperture of the microscopes light source. So far, I have found it best to adjust the microscope to normal darkfield illumination, then play with the brightness control. Another option is to make a coarse adjustment with the camera, then continue making fine adjustments with the aperture (there is some interchange between the two light controls).
As noted in the other article, darkfield illumination gets tricky at high magnification. When I have tried to push it to 400x, I can still get a good image, but the background coloring is not as reliable. My 'masks' are pieces of plastic with black dots painted on. The accuracy of the dots (or lack there of), and the difficulty of positioning the mask likely contributes to this problem.
There is one problem that I have not been able to get around, and that is depth of focus. With a regular camera, it is possible to stop down the aperture and increase the length of exposure; gaining more depth of field. Digital cameras have no such exposure control. Thus, while I can get some nice images, they all have a relative narrow range of focus. With fairly thick specimens (such as daphnia), it is not possible to have the whole specimen in focus at the same time. If anybody has any suggestions, please let me know. I have read of CCDs used in astronomy for long exposures (minutes to hours), but these require special cooling (defeating my goal of being easy and inexpensive).
About the images:
All daphnia were collected around St. Louis, MO, USA
1) Daphnia (pulex?) from Grasso Spring, 100x, 27kB: note the detail of the antenna.
2) Small Ceriodaphnia from Forest Park, 100x, 8kB.
3, 4, 5) Larger Ceriodaphnia from Forest Park (St. Louis, MO) 100x, 37kB, 37kB & 41kB
6) Small Ceriodaphnia from Forest Park 400x, 23kB: note the ciliates attached to the back.
P.S. In the last article I did not have a good close-up of the camera and its relationship to the microscope. Since then I have taken such a picture, and enclose it here (63kB).
Contact: Comments to the author sent via our contacts page quoting page url plus : ('hwebb','')">Howard Webb.
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