USE of the LOGITECH QUICKCAM PRO 9000

for photomicrography
History of a near-failure, or a semi-success

PART  III

see here Part I, and Part II

WALTER  DIONI                                                                                                            CANCÚN, MÉXICO

INTRODUCTION

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. 

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.

 All are standard lighting techniques as use with amateur equipment, and have been used for a long time, so, if some beginners falter on these pages, they will find a wealth of information by entering the corresponding terms in their browser, and, especially, reading the articles in the MICSCAPE library.

An introduction to the topic may be

http://www.microscopy-uk.org.uk/mag/artdec03/wdonion2.html

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. 

 Because it is important for the best performance of recommended configurations, I must stress the preparation steps before starting the photomicrographic work as I described them in the 2nd. Part:

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

Ian  Walker : http://www.microscopy-uk.org.uk/mag/artsep02/toptips11.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.

OBLIQUE  ILLUMINATION

 A technique that generally gives good results, is lighting with transmitted light laterally displaced. With subjects of a certain thickness and more or less complex structure, it not only helps to better solve optically the structural and spatial relationships, but also produces pictures with a strong additional interest. With certain subjects and in special conditions it even produces images that mimic those produced by DIC microscopes. It is therefore logical to explore the possibilities of the Logitech from this point of view. Remember that by the very nature of the adopted optical process, images produced with this technique suffer some distortion and are not appropriate to carry out measurements.

 (Search "Oblique illumination" in MICSCAPE library, and please read the articles about "Circular Oblique Illumination (COL)" by Paul James). It is essential for you to practice it. But it must be remembered, as the author says, that not all subjects and not all optical systems respond optimally. Amateur microscopists must know that it is difficult to duplicate exactly the original conditions described by James, but that this does not exempt him from taking the test. (You can be sure that you will be rewarded.)

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.

http://www.schwaben.de/home/mathias/emikro/kontrast1.html

Dieter Gerlach: Das Lichtmikroskop, Thieme (Stuttgart), 1976

http://www.microscopy-uk.org.uk/mag/artoct03/wdoblique.html

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.

 The oblique illumination, and even the dark field illumination to be described below, were already well known to microscopists even since the 19th century. A review of the microscopy books published on-line in INTERNET ARCHIVES easily verifies this. Visit:

http://www.archive.org/index.php

 Withthe 4x and 10x objectives it is possible to install the dark field effect with simple devices as such as opaque circular stops placed on the condenser filter holder, or even with the Mathias arrow. This is why it is one of the most often used methods to highlight appropriate subjects. It is generally said it is difficult to obtain the same effect with the 40x. However, with the Logitech I achieved it using stops from 15 to 20 mm diameter, depending on the subject, and by carefully adjusting lighting and contrast.

 Some colleagues using microculars, whom I consulted when I was unable to install the effect, informed me that some of these cameras do not allow not Rheinberg nor Dark Field.

But with the Logitech I can achieve a good approximation, provided that the central opaque disk used has enough density. To get the proper effect I needed to superimpose up to 4 and sometimes 5 central black stops, printed with my inkjet printer. But this, in addition to being tedious, it also increases the opacity of the transparent peripheral ring. So, now, I'm preparing my stops by cutting the central disc from a fully opaque material, and pasting it with nail polish over a relatively rigid disk of the most transparent plastic available. All my stops are made today that way.

 The stop can be properly centred if you print a pattern, corresponding to the two concentric circles (any decent drawing software allows this), and sticking the disk in the center position using, for example, nail polish.

But even using a suitable stop, with some subjects the gamma value of the photomicrograph should be decreased a little, for the black background to look uniform.

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.

 Don Thompson, in his article (http://www.nhm.ac.uk/hosted_sites/quekett/Technical/DonThom-DGI.html) drew attention to a special method of achieving darkfield using polarizing filters. He prepares stops similar in geometry and diameter to dark field stops, but instead of using opaque disks he used Polaroid disks. One of them put in the filter holder of the condenser. Over the front lens of the microscope lamp he puts one bigger one. With subjects focused, spinning the disk on the lamp produces the extinction, with a deep blue background and the subject brightly lit.

Fig. 12 - Body of a mosquito larva mounted in PVA-G.  "Polarized Darkfield"

As 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.

RHEINBERG  LIGHTING

 In 1896, Julius Rheinberg intelligently understood that the very essence of the dark field technique allowed him to design a system of optical coloration for the  microscopic objects.

   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.

 You know that disks are used whose center has a color, while the ring around it is in a contrasting color. As virtually all articles on the subject insist, centres have to be of a very dense color. This is true in any case, but it is critical for the Logitech. A very transparent color center (or grayish if one chooses black) will not produce the desired results, and the image will have an excess of "noise".

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.

 Since the simple eccentric displacement of a dark field stop can produce oblique lighting (remember figure 4) it is logical to verify the behavior of an a similarly displaced Rheinberg filter.

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

POLARIZED  LIGHT

 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. 

 But for who have greater possibilities and a more serious interest in chemistry, or mineralogy, the work of Ian Walker and others, in MICSCAPE, may offer a guide. 

 Of course not always have amateur microscopists been able to enjoy the attractive aesthetics of polarization so easily.  This item from Popular Science from the years 1934-1938 can serve as examples:

 Using a Microscope for Polarized Light Experiments

Popular Science: Oct. 1934 pg 69.

http://blog.modernmechanix.com/2007/05/15/polarized-light-experiments/

One Way With Simple Polarizing Units and Imitate Feats Performed

Popular Science Octubre 1938: pag 200.

http://www.popsci.com/archive-viewer?id=hiYDAAAAMBAJ&pg=200&query=polirized+light

To prepare crystals to observe or photograph in polarized light, unpretentious scientific nor mineralogical, this article has enough information: Pavlis: http://www.microscopy-uk.org.uk/mag/indexmag.html

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:

 http://www.microscopy-uk.org.uk/mag/artfeb01/polarise.html

and also in the more complex Ian Walker article

http://www.microscopy-uk.org.uk/mag/artjan05/iwsworlds.html

Fig. 16. The same field of view, but without the oblique illumination stop

 The defects of these images depend on my equipment. My filters are plastic sheets, cut from low-quality polarized lenses used to see three dimensional films and cheap plastic polarized lenses sold to protect the eyes from the sun. Surely gelatin, or better glass filters, as may be purchased for classic photographic cameras, could offer sharper images. But, while the amateur micrograph does not aspire to match Brian Johnson’s work (see his magnificent crystallizations accompanied by abundant and interesting information in almost every issue of MICSCAPE for several years to date) a Logitech and a pair of cheap polarized glasses will assure countless hours of aesthetic pleasure. The minimum rotation of the "analyzer" will cause major changes in the color of the images.

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.

 In cases where this is not enough, one or two hand flashlights generally allow a good lighting of the subject. However, because the flashlights tend to produce unpredictable reflections on some subjects, I usually illuminate through diffuser housing, such as simple screens or translucent plastic cylinder (see "Topical Tips – Updates about Well Known elementary contrast controls)

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.

 As the metal and the relief image reflected light in inconvenient ways, producing hot spots, I used a a diffusing screen built with a small cup of translucent plastic, making a hole to allow the use of the 4 x objective.  Lighting was also made with the same hand flashlamp, and therefore is unilateral. Two light bulbs on opposite sides have given better control of the reflected light.

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

 http://www.microscopy-uk.org.uk/mag/artoct09/wd-microaquaria2.html

STILL PICTURES OF MOVING SUBJECTS

With the larger sizes (from 1.3 Mpx onwards) still images of mobile subjects take on average a second to be captured, so notstill images of the majority of moving living things may be taken. 

 The capture rate with useful formats is typically only 1/10 to 1/15 of a second. All those accustomed to photography (and more those that knew, as I do,  the old "box cameras" of Kodak) know that the minimum speed to freeze a slow movement is 1/25 of a second, and that a somewhat more rapid movement just stops more or less adequately with 1/100 of a second (which of course is not enough for an athlete, a race horse, or a car) No webcam, even those which are proposed as very quick (2 Mpx, CMOS, 60fps - Philips or HP) can arrive at that speed.

 Still images from 320x240 to 960x720 can be taken at tenths of a second intervals (the smaller the format, more rapid the shot speed) provided that on the TAKE PICTURE menu, the command parameter "take picture after:" is disabled. If this command is active, the interval between clicking the mouse on and taking the image is a minimum of 1 second, and none of the possible mobile subjects to register will produce an acceptable image, except by a very unlikely chance. The DC-3 shares this limitation. Only the flash, or high intensity lighting equipment, can use very short exposure times, which can allow a faithful record of beings in active movement.

 Whoever wants to use faster speeds should use consumer digital cameras. Michel Verolet, using a Canon DS 50, handheld over his microscope eyepiece has photographed rotifers with only 1/1000 second exposure. It is clear that this requires a microscope with adequate optics and lighting... and the development of an intelligent and precise maneuver.

 VIDEO

 It is when faced with this task, of course, that the limitation imposed to the image size by the afocal technique becomes clear and more restrictive.

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.

 The first viable format is 1.3 Mpx. But I prefer, for the best quality of images, the format of HD960x720, which is what I normally use for my videos. Even with the field of view reduced to 40% or 50% the displayed circle is sufficient. But although short videos of up to 30 seconds may be viable, with increasing recording time the net file size in MB becomes quickly excessive for publication, even with this format.

 If the entire field sensor were covered, the 640x480 format which recorded 20 fps, would be of choice, and for certain subject and magnifications, 320x240 could be useful. With the Field of View I have now, both formats are very unsatisfactory, specially for videos of small, mobile subjects such as protozoa for example. Keep in mind that the quality and the recording speed depend very much on capture size and lighting.

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.

 I include three AVI files approx. 20 seconds each, from the same material, recorded with three different sizes, through the 40x objective.
They do not claim technical qualities of any kind. I simply use them to show, with a fast-moving material, the capture sizes and speeds collected.

Clicking the icon will be show the short video. I think the best video quality was recorded at 960x720 px

Sequence of 3 pictures 200x200 (fig 24-25-26)

 The following two videos that reach 30 fps were recorded with the 10x objective

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.

 If you click the arrow to the right of the VIDEO record command, a configuration menu will be displayed.

Fig 29

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.

IN  SUMMARY

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 4x.

From this point of view, my adventure was a failure. When I started it I did not realize that, to achieve this result in afocal geometry, I had to change or remove the camera lens, and change the eyepiece of the microscope for a good brand Super Wide Field (SWF) eyepiece, with 20 to 23 mm diameter of its Field of View, and, preferably, with high eyerelief.

 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.

 Nor am I satisfied with the behavior, in afocal geometry, for the recording of videos in large formats (2.0 and 1.3 Mpx), because although I can get discrete recordings for personal use, I find it difficult to adapt them to share on the network. Although that perhaps may be solved with appropriate software. 

 It is easy to understand why all the specialists in diatoms, protozoa, desmideaceans, etc. when using webcams have adopted what we call astronomical techniques, covering the entire field of its sensors.

 But I was successful at other things also important to me, and probably to many beginners or amateurs with few resources, whose limitations I share.

 I have a supplementary camera which is exactly 10 times more powerful than the one integrated in my microscope. Still limiting me to use the maximum rectangle cutout of the actual view field, my publishable images are of real 800x600px, i.e. 6.25 times larger than the original image provided by my current microscope camera, and 1.6 times greater than the resized output. This size is usually the best suited for vision online on the web, and also the most used in forums and galleries in the network.

 I also like more the Logitech images, because of its performance of color, structure, and texture. And find its wider field of view more manageable and usable than the previous I have. I can, of course, also use mosaics, for larger images. This is a modest 2 image Mosaic:

Fig. 30 Gyrosygma sp. - 100xOI Objective. From a preparation given to the author by Dominique Voisin.

 The use of a modest digital zoom, effective with thin preparations with good natural color and contrast, which only require, apart from logical and normal processing in photomicrographs of any size, a minimum post process (essentially contrast adjustment, noise and moiré elimination) appreciably extends the range of sizes useful for reproduction on the Internet.

 Comparing the cost (90 dlls. camera, cents the adapter) against the lower price microculars (usually only producing VGA size images), and whose quality, as some revisions and my own experience show, is at least questionable, I found the  balance sheet very favourable.

 Those who want to equip a modest amateur microscope with a cheap and efficient camera, at the cost of a bit of post-production, they can use with confidence a 2 Mpx webcam with CMOS sensor. Not all of them, however, reach the quality necessary to be useful in microscopy. The 2 Mpx, of a good brand, which I use as a Webcam on my computer was my first choice, but could not withstand the test.

 You may get nice stills and even videos of good quality and acceptable size, to enjoy them on your screen or share with your colleagues. If you use a Logitech 9000 these pages can serve you as an introduction; if you are looking for other brands, you must know that you start a risky but interesting adventure, similar to mine. No webcam operates as a dedicated photomicrographic camera. To obtain an acceptable performance involves devoting to each picture the time it requires.

 Moreover, accepting the cost in money and labor that this represents, you may think of adding, at some point, a "relay" to really get full coverage of the sensor. You will always have a better camera than an "economic" VGA microcular, and practically at the same price.

 Those who decide to try removing the lens of his camera can also use the old but effective method recommended in MICSCAPE by Wan Yu (http://www.microscopy-uk.org.uk/mag/artaug01/wyuphoto.html) Although the technique alters the camera by removing its lens, (and is a good Zeiss Tessar lens), does not require "relays" (ie requires no extra investment) and use the very eyepiece of the microscope to project the image onto the sensor. One of my correspondents is thinking in doing that, and, according to their results, this could be my next project.