USE of the LOGITECH QUICKCAM PRO 9000
WALTER DIONI CANCÚN, MÉXICO
In the previous article I described the behavior of my new camera using normal lighting in bright field. I think it should be clear that in this configuration, I find the Logitech extremely useful, although it has behaviors that a professional microscopist (and some senior amateurs) might object to.
Like many digital cameras it is sensitive to deficiencies in the illumination such as slight unevenness of intensity across the field which the eye can often tolerate. Setting the microscope's lighting, whether Köhler or critical (for my stand critical) to the optimum before undertaking photography is essential. Despite the limitations of my own microscope's lighting, it still gives me very acceptable documents from an amateur’s point of view.I'm used to using a number of techniques of image enhancement which are beneficial, and usually help me to better understand the structure of the studied subjects.
These include Oblique Illumination, Darkfield Illumination, Rheinberg Illumination, Polarized illumination, and the Reflected Illumination at low magnification (also called Incident Light).
In consultations I made with some colleagues before installing the camera, I was told that some of their Microcular Cameras (ultimately webcams installed with relay sensor lenses replacing the eyepiece of the microscope, so that the image covers the total field sensor) showed some difficulty to accept the application of some of these techniques, or definitely did not accept them. So my first task for adapting the camera for use in afocal technique, was to test its capabilities. First results were disappointing. But a few tricks got some acceptable results.
An introduction to the topic may be
In what follows, I will try to confine myself to the special features which I found necessary to obtain the best results with the Logitech. Also I will try to limit the images to the description of the result obtained with this camera.
1) Before anything else, Köhler or Critical lighting must be installed, as usual.
2) I had not previously insisted on an important detail: a diaphragm field should be used in the lamp of the microscope. If, as with most budget microscopes, the lamp does not have a field iris diaphragm, the enthusiast who wants to use the Logitech, if did it not already, must read now this MICSCAPE articles and apply its recommendations
Rose Marie Arbur : http://www.microscopy-uk.org.uk/mag/artjan01/whitelite.html
Not to use a field diaphragm will inevitably lead to an excess of diffuse light reaching the sensor, producing glare, contrast and colors.
3) Install the camera (if not already done so) and verify that you are working in the default configuration. Not to do so will result in major defects (Irregular backgrounds, Central spots of bright light, Aberrant contrast or color, etc.). Once the white balance is set, disable changes to this option. Immediately disable the auto-focus. The image will most likely be too light, but this will be fixed easily by slightly decreasing lighting and contrast. Install filters, diaphragms, or whatever you wish to use, and properly adjust light, contrast and color intensity. Carefully manipulate the camera and the microscope commands to optimize the image. Remember that, unlike the traditional technique, and in contrast to the field diaphragm, the aperture diaphragm must be correctly adjusted to give the optimal image. Later, if deemed necessary, a small adjustment of the aperture iris to improve resolution can be made.
The basic tools: there are multiple devices used to produce this oblique effect. They are the simple "Mathias wedge (or arrow)", the "Gerlach Fork (1976)", and the "black stops with eccentric hole " known from the late 19th century, which are usually placed in the microscope condenser filter holder.
Dieter Gerlach: Das Lichtmikroskop, Thieme (Stuttgart), 1976
Once the mechanics of the process are understood, the amateur microscopist can develop many variations for this really useful and visually effective lighting effect. The DC-3 enables an efficient use of all these devices with very good results. In almost all of my work, there are examples of this.
Fig. 1 - Logitech - Brightfield – X10 – zoom to 1400 px - “flat” illumination
Fig. 2 - Logitech – Brightfield – x 10 – idem –Gerlach Fork
Fig. 3 - Logitech – Brightfield – x 10 – idem – Mathias Arrow
(also see the image in the polarized light seccion)
Fig.4 – The fifth pair of legs of a female copepod. Mounted on GAF (see http://www.microscopy-uk.org.uk/mag/artmay03/wdpart3c.html)
In the upper right corner is the full egg sac. Reduced clipping of 800x600 px from the original picture. The stop used was a 15 mm diameter dark disk, decentred tilting the filter holder. x40 objective.
Figure 5 - This is the "flat" version in Brigthfield
Not all subjects respond favorably to this technique. Obviously subjects with moderate relief and well-outlined structure are ideal. Diatoms, micro-crustaceans, micro-arthropods in general, spicules, fibers, or hair, provide opportunities to experiment with this technique. Dry mounts are a good observation medium. Any other media will interact with the subject according to their refraction and only experimentation will show their real behavior.
Fig. 6 - articulation, fly wing, brightfield, obj. 4x
Fig. 7 - Idem. Neutral background. Mathias arrow . x 4 – one pass through NetImage
Fig. 8 Idem, Mathias arrow - 4 x - Dark background. One pass through NetImage, gamma control
Fig. 9 Fly wing - 12 mm stop - x 10 - without post-processing, except a moderate background cleanup
Fig. 10. A classic subject x 40, 15 mm stop. One pass through NetImage. The focused nucleus shows its 3 nucleoles.
Figure 11 - Diatoms with the 40x objective - A group at the edge of a preparation kindly donated by Dominique Voisin - 15 mm very dense stop. Without any postprocessing but size reduction.
Fig. 12 - Body of a mosquito larva mounted in PVA-G. "Polarized Darkfield"
well as allowing "Brightfield",
almost similar to that obtained removing any filters, and stops, a good variety
of intermediate densities and even "dark field" are produced by
the crossed polarizers. For some subjects, with a slight displacement of the
condenser filter holder, interesting highlight effects can be achieved. Of course, polarization affects only the
background. The subject is only illuminated by unpolarized light, as in
the other dark field techniques.
His article: “On a new differential Couloured Substage illuminator” was published in the page 364 of Volume 5 (series 2), of the Journal of the Quekett Microscopical Club published in 1896. It is interesting to note that, although there is on-line a virtual edition of the magazine, in the Internet Archives, I could not find this volume. But the vol. 6 (1894-1897) in series 2, with an entry in December 1896, page 346, where there is a "Notes on colour illumination" and other of October 15, 1897 in whose 438 page you can read a relatively short article about "Note on a new modification of double colour illuminator", both are published on-line.
Figure 13 - The picture above shows the two hind legs of a female copepod, the point of attachment of the bag of eggs, and the first part of this bag. Eggs are deformed due to dehydration suffered when mounting the piece. The subject is greenish, as was shown before in bright field images and oblique illumination, but with the use of a Rheinberg filter with blue center and red ring, it appears to have been colored with carmine or eosin. The center was not sufficiently dense, but the effect is still good.
Fig 14 - the same previous filter, slightly eccentric because of a slight displacement of the filter holder, highlights the relief features of the exoskeleton and muscles in the fifth leg and the beginning of the queue of a male copepod. In this case the price of the relief is the strong red dominant on the background
There are many articles in MICSCAPE library that can serve as an introduction, or efficient guide, to this so appealing technique. Look under
Techniques - lighting and light sources
French readers can also find information more than enough in the Forum Mikroscopia and its Microscopies magazine.
Popular Science: Oct. 1934 pg 69.
One Way With Simple Polarizing Units and Imitate Feats Performed
Popular Science Octubre 1938: pag 200.
To prepare crystals to observe or photograph in polarized light, unpretentious scientific nor mineralogical, this article has enough information: Pavlis:
Fig. 15 - aqueous solution, dried on the slide of potassium dichromate. Completely crossed polars plus Oblique illumination (Mathias wedge). Reduced from 1000x1000px. This is my first crystallization. The capricious forms adopted by crystals often suggest landscapes. My imagination dictates me "Tropical forest" as a name for this image. The suggestion and description of this mixed lighting (polarized and oblique) technique can be read in the article of Paul James:
and also in the more complex Ian Walker article
Fig. 16. The same field of view, but without the oblique illumination stop
Fig. 17. The same field of view: A rotation of only a few millimeters
INCIDENT illumination – REFLECTED Light
This technique scans with the low powers of the microscope (mostly 4x in my micro) the surface of opaque subjects. One technique, used by amateurs, is a derivation of the Lieberkühn Speculum. Although the instrument was designed to be attached to the objective and have a paraboloid design, to assure the focusing of the light over the object, the truth is that in many cases one can get good results with reflectors recovered from simple hand torches with appropriate diameter.
I used this method with the Logitech, but soon I discovered that for some subjects when working near the window of my lab, the daylight was enough for the Logitech to capture the subject without having to employ any reflector.
Fig. 20 - A drop of water, encompassing a drop of air, over the ovary of a flower of "Allium sp." Lighting: a handheld flashlight, obj. 4 x. Direct light without diffusers
Fig. 21: Ovary superior, flower of "Allium sp", x 4, Lighting: a cylinder of architect paper, handheld flashlight with two1.5 V batteries. Single picture. Of course as the subject was appropriate, a 'Stack' for CombineZ could have been attempted.
There are several works on the Internet which propose small homemade instruments equipped with LED's, fed with batteries and controlled with potentiometers, that can work with efficiency for reflected light.
Fig 22 - The above image is a shot with the 4x objective, of the surface of a Mexican coin in common use. The coin has a diameter of two centimeters. The area covered is about 4 mm in diameter and shows the head of an eagle seizing a snake.
LIVE SUBJECT PHOTOMICROGRAPHY
AND VIDEO RECORDING
One of the problems to overcome in both cases is the depth of focus of the camera. It is imperative to careful obtain a very thin preparation of the subject to be photographed. In the following video a proper recording of the displayed Philodina clearly would have required a much thinner preparation. The rotifers move vertically, often out of focus, and this prevents a consistent study of their anatomy.
Fig. 23. Click on the photo to view the video
As always before start working, all the routine of initiation as explained at the beginning should be applied, preparing good, clean and thin wet mounts, or using any extra-thin wet cameras as described in
PICTURES OF MOVING SUBJECTS
If for still images it s surmountable, as we saw earlier, it is really limiting when it comes to video.
The first logical format, starting with the largest, is the “native” 2 Mpx. However a single 1600 x 1200 image is partly displayed out of my 15-inch screen, set to 1440 x 900 px, even if, as in this case, just use the sensor partially. Therefore, while it is a most useful picture format for still subjects, it’s not suitable for videos.
In my microscope, with my current lighting capacity, speeds that I really get using the objectives 4x and 10x are of 10 fps capturing at 2.0 and 1.3 Mpx configurations, 15 fps at 960x720, and 30 fps for VGA or 320x240 videos. Incidentally, the Cavalue image that prompted me to try the Logitech, was recorded in 1600 x 1200 format at 15 fps.
With 40x objectives and 100xOI, rates
vary only between 12 and 18 fps. However in my working conditions 12, 15 or 18 fps
videos have shown an acceptable continuity of the movement and a sufficient
definition of photographed subjects.
With 40x objectives and 100xOI, rates vary only between 12 and 18 fps. However in my working conditions 12, 15 or 18 fps videos have shown an acceptable continuity of the movement and a sufficient definition of photographed subjects.
Sequence of 3 pictures 200x200 (fig 24-25-26)
Sequence of 2 pictures Fig. 27 – 28
In the first short clip, after a relatively long stay under the coverslip protozoa are moving close to the flocs of microalgae, where they find food and higher oxygen content,. Several minutes later, protozoa accumulate in a crowded band near the edge of the coverslip where they still have a good ability to exchange gases. The two videos were recorded in separate sessions. Both times the speed was recorded at 30fps.
where you can select the desired parameters. With the "Recording duration" command off, videos can reach a very long duration. If enabled, you can set a specified time, after which the recording will stop automatically.
My intention, buying the Logitech,
was to have a 2 Mpx camera, to achieve a good electronic resolution of my
microscope field of view with all my objectives, including in particular the
Not wanting to do that at the moment, as I explained in the first part, I was limited to accept the conditions of the direct afocal system, and as a result, to accept a camera of only 0.8 working Mpx.
Fig. 30 Gyrosygma sp. - 100xOI Objective. From a preparation given to the author by Dominique Voisin.
Comments to the author,
, are welcomed.
Published in the April 2010 edition of Micscape.
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