HIGH RESOLUTION PHOTOGRAPHS

WITH CIRCULAR OBLIQUE LIGHTING.

By Yannis Tsamouris, ATHENS GREECE

My passion with microscopy started when I was at the first grade of high school and I looked at a microscope preparation of living protozoa during a biology class. From that time on I became obsessed with microscopy and I bought my first toy microscope soon after. It was a Japanese microscope with glass lenses and from the first moment I experimented to achieve a better picture with higher magnification trying to achieve what I was seeing in the pictures of my biology books. Eventually my experiments ended with a broken microscope thrown into the garbage.

I started again many years after this wonderful experience when I had the spare time and the money to become involved with more professional equipment. eBay was a great help and for the last 15 years I've been gathering a lot of hardware and experience of manipulating this intricate machinery. I have to admit that this magazine was a great inspiration to me and gave το me access to many aspects of amateur microscopy and how to find practical solutions to problems that any amateur microscopist faces every day in handling these wonderful instruments. I experimented a lot with all the current techniques like dark field, Rheinberg light staining, Phase contrast and Differential Interference contrast (DIC). One of the first accessories I acquired with a Leitz Ortholux microscope was the Heine condenser. When I started taking photographs with a CCD camera I observed that I could take very clear pictures with high resolution. After many trials I ended up with the following results that show the hidden capabilities of circular oblique lighting that according to my opinion is capable of providing high resolution pictures without even immersion objectives. The microscope I used is a modified Zeiss Photomicroscope III with a Heine condenser adapted to it, a special illumination setup made by me which can provide quite parallel rays of light and a Zeiss Axiocam MRC5 CCD camera which gives superb results. The photographs that follow show that with this arrangement you can resolve even with a dry lens (Nikon Plan x 60 NA 0.85) the striae of Frustulia rhomboides which according to D.B.Murphy (Fundamentals of Light Microscopy pp 94) has a period of 0.29 μm/stria. According to theory this resolution can be achieved only through oil immersion lenses. The preparation I used is the 8 FORM TEST SLIDE and the 100 FORM SLIDE by K.D.KEMP

Frustulia rhomboides Nikon Plan X 60 NA 0.85 dry green filter

As you can see from this photograph, although the resolution is inferior compared to the microscope live image, not only the striae are resolved but also the pores. This is made more clear in the following picture taken with a 15MB photographic camera (OMAX) and processed with the Adobe Photoshop.

The most striking photograph I achieved was the resolution of the individual pores in the striae of the following diatom (Climacosphenia monligera) for which I calculated three (3) striae per μm and about four (4) pores per μm. The microscope image is very clear but the resolution of the MRC5 camera (pixel 3.4 μm)  is not enough to capture such small details. The sample is from Klaus Kemp 100 Type diatom slide.

    The SEM image which is hosted at Protist Central (photo credit Chris Lobban) shows a striae spacing of 0.40 microns and a pore spacing of  0.33 microns.

       This is further confirmed in the following photograph taken with the OMAX 15MB photographic camera

With the same technique and moderate optics the resolution and the depth of field is great.

Zeiss Plan x 40 NA 0.65 dry, green filter. Pleurosigma Angulatum 0.53 μm / stria

Pleurosigma angulatum Nikon Plan x 60 NA 0.85

With an oil immersion objective the results are more striking.

Nikon PlanApo X 100 NA 1.40 oil green filter. Amphipleura pellucida Period 0.25 μ/stria

Nikon PlanApo x100 NA 1.40 oil, green filter. Frustulia rhomboides Period 0.29 μm/stria

Nikon Plan Fluar X 40 NA 1.30 oil, green filter. Navicula hennedyi

Nikon PlanApo X 100 NA 1.40 oil green filter. Surirella gemma Period 0.50 μm / stria

Nikon PlanApo x 100 NA 1.40 oil, green filter. Pleurosigma angulatum Period 0.50 μm/stria

If we compare the above photographs with the results that we get with the customary techniques of Phase contrast and DIC the results speak for themselves.

Zeiss Phase PlanApo x 60 NA 1.40 oil green filter, Zeiss Phase condenser NA 1.40 oiled. Amphipleura pellucida.

Zeiss PlanApo x 60 NA 1.40 with DIC slide, blue filter. Frustulia rhomboides.

Some more photographs give us a glimpse to the beauty of the crystal palaces where the diatoms live. (Olympus x 100 NA 1.30 oil).

Pinnularia major Olympus A100 NA 1.30 oil green filter

Pinnularia alpina  Olympus A100 NA 1.30 oil green filter

Mastoglia splendida Olympus A100 NA 1.30 green filter

Eunotia clavata, Olympus A100 NA 1.30 oil, green filter v232g

Aulodiscus kitonii Olympus A100 NA 1.30 oil

If you study the photographs by zooming in you can appreciate the details they contain.

I dare to make the assumption that in the light that comes out of the objective lens after its interaction with the specimen there is a lot more information than we are used to suppose. It’s up to us to find the way to gather it. The COL provides an easy and cheap method to get the highest possible resolution with great depth of focus.

REFERENCE

Douglas B. Murphy “Fundamentals of light microscopy and electronic imaging”

A JOHN WILEY & SONS INC., PUBLICATION

All comments to the author are welcomed.

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