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Seeing Three Dimensions in the Microscope

Wayne

Microscope images often look flat and lack detail. There are two reasons for this: First, most microscope lenses have little depth of field, so only a thin plane is in focus; Second, most microscopic critters are transparent and their organelles are transparent, too.

Cyanobacterial Mat Hoffman Modulation Contrast

This is a photomicrograph of a Cyanobacterial mat sample, taken from the bottom of the small pond in Heron’s Head Park salt marsh we call “Old Site 1”. In this photomicrograph, taken at 3x optical zoom with my Nikon Coolpix 885, I used a large laboratory Olympus microscope at 200x magnification, equipped with Hoffman Modulation Contrast optics.

Invented by Robert Hoffman, this optical system adds slits, polarizers, and a wave-amplitude filter to the usual brightfield optical path. These elements combine to create artificial shadows where the refractive index changes. In addition, the objective lenses have a wider focal range. The resulting artificial enhancement of tiny changes in focus and refractive index around organelles and cell walls gives a distinctive and useful three-dimensional appearance to the photomicrograph. Look at the photomicrograph above for awhile and you will notice that sometimes the filament walls appear convex in three dimensions and some time they appear to be concave.

The next photomicrograph was taken of a sample from a thin, shallow stream in another salt marsh at the south end of the Bay.

Cylindrothecia diatoms from Drawbridges, the weep stream.

These diatoms, species of Cylindrothecia, were taken from a sample of the central stream at the site we call “The Weep”, near the Drawbridges in the Don Edward San Francisco Bay National Wildlife Reserve. Notice the Cyanobacterial filament on the right.

The bottom of this small, slowly moving stream, was appeared covered with yellow fluff. Like many diatoms, Cylindrothecia is motile. Here we see a videomicrograph with the Modulation Contrast optics removed to enable enough light to get through for the camera. Magnification was 200x.

Modulation contrast is a real light hog. My Olympus microscope has only the standard 30W filament bulb for illumination. To get enough light through for the Hoffman Modulation Contrast optics to really work in a videomicrograph, I had to remove the diffusing filter which gives a uniform blue-gray background. I also had to shoot at a lower magnification. In the first videomicrograph, I used ordinary birghtfield optics with the diffusing filter in. In the second, I removed the filter and used Modulation Contrast.

Compare the videomicrograph above with the one below, taken at 100x magnification with the diffusing filter removed. The color is off – but the three dimensional contrast is good.

The ciliates swimming among the diatoms are not only three dimensional, but show their internal organelles well.

My next step is to buy or build a more powerful illuminator. I think a 150W fiber optic illuminator, kept outside the microscope to prevent overheating, but with the fiber optic component inserted in the illumination port, will work.

The Weep site stream also yielded a mystery. When we first sampled it, in September, the Cylindrothecia were a minority. The dominant organism was a diatom, but a much larger diatom. We have not identified it, but this is what it looked like:

Orange area.

Here the magnification is 200x and the Modulation Contrast is on. In December, when we sampled the stream and found the Cylindrothecia, we also found that the large “orange” diatoms, like this one, had been pushed back to one small patch of orange mud at one corner of the Weep site.

This is a substantial change in population. We do not know what caused it.

CONTINUED NOTES – DECEMBER 25, 2006 …

I was able to borrow a fiber-optic light from the EXPLORATORIUM, courtesy of Peter Richards. Over the weekend of the Christmas holidays, I tested the use of this light. This is my “Done-Found Report” […what I done and what I found].

The fiber-optic light was manufactured by Dolan-Jenner Industries [OEM to Technical Instrument, San Francisco]. It was designated Model MI-150 Fiber-Lite. This light uses a 150W EKE Projection Lamp with a Dichroic Reflector. It is equipped with two flexible 0.5-in fiber optic light guides.

Initially, I simply removed the 30W bulb from my Olympus Microscope and aimed the end of one light guide into the convex lens at the beginning of the microscope light path. This actually resulted in considerable improvement, the light being sufficient for good 400x Hoffman Modulation Contrast optics photomicrography. It was still insufficient, however, for 1000x oil immersion photomicrography under Hoffman Modulation Contrast optics.

I then removed the convex lens and ran the fiber optic light guide into the microscope light port and all the way to the prism that reflects the light up into the microscope optical path. This made a much greater improvement, permitting adequate photomicrography at 1000x oil immersion. Keeping the flexible light guide centered along the light path was a little difficult.

My final Experimental Setup consisted of removing the light guide from the Dolan-Jenner Fiber-Lite source and using a 0.75-in lucite rod with the ends polished very smooth. This created a large rigid light guide from the light source along the base of the microscope to the prism. The result was a very great increase in available light.

Fiber Optic Experimental SetUp

Experimental SetUp to test enhanced light.

This photograph was taken in normal daylight, so you can see how bright the light passing down the lucite rod is. This placed a 0.75-in diameter light right at the prism, centered along the axis of the light path.

Here is what happened: At 400x it was possible to use full polarization on the Hoffman Modulation system and still have enough light to turn the light down from maximum. Shown below is a photomicrograph taken with the Experimental SetUp, using a slide of a sample from the Heron’s Head Park salt marsh, sample site OS-1, Cyanobacteria and Melosira. The magnification was at 400x and the Sony camera was set for 3x zoom.

400x Results CyanoMelosira

Both the detail and the contrast effect are very good. This is probably at the limit of improvement with Hoffman Modulation contrast optics.

Next, I tried a photomicrograph at 1,000x oil immersion with the Hoffman Modulation optics on. I used the Nikon camera. This is the result:

1000x Cyano Results

I have been able to print this image on 8.5 x 11 glossy premium archival photo paper with very good results. This image is reduced in size for downloading to the server, so it might not print as well as the native image, but it can be easily printed to 4×6 or 5×8 photo paper.

I have ordered a used Dolan-Jenner Fiber-Lite [OEM Cole-Parmer Fiber-Optic Microscopy Illuminator] from Aunt Beas’s Specialty Items. The unit ordered comes without the flexible light guides, which I do not need. I may increase the size of the lucite rod to 1-inch. This would just fit down the light-path tunnel in the Olympus Microscope, and would carry maximum light. I will turn the end of the rod on a lathe to 9/16-in so it will fit snugly into the light port of the light source. I will also create a mount to position the light source box at exactly the right height for the microscope light-path tunnel.

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