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Combining Circular Oblique and Oblique Illumination to Enhance Detail and Contrast in Light Microscopy, Part I

William Mark

Abstract

Enhancing detail and contrast in transparent specimens has always been a primary interest of microscopists. To such ends, several optical techniques have been developed, such as oblique illumination, circular oblique lighting (COL), phase contrast, differential interference contrast (DIC), and various similar techniques. What is presented in this article is a unique combination and application of COL, oblique illumination, and polarized light. In addition, stop shape and the addition of wave plates are also discussed.

Introduction

Although COL and oblique illumination are some of the oldest variations on brightfield microscopy, they are still powerful tools for improved contrast and enhancing detail. As a focus of research, both COL and oblique illumination, and variants, have been extensively studied and written about by many authors over the years. The purpose of this article is to discuss variations and combinations of these techniques that may not have been evaluated in the past with advantages and disadvantages of such.

The interest in COL and oblique illumination, with respect to this paper, stems from a desire to reproduce DIC-like images at a lower cost. It is well known that COL and oblique illumination can enhance contrast, and therefore the ability to highlight structures not easily visible under brightfield. A discussion and comparison of these techniques is given by Ted Clarke (2004). One of his key observations is that while both COL and oblique enhance contrast, oblique appears to give only “directional” resolution (dependent on orientation of sample and oblique mask). This is in comparison to COL in which resolution is not directional. The observed resolution enhancement of both techniques is consistent with Abbe’s theory with respect to increasing the order of diffraction maxima entering the objective and thus improving resolution (Anon., 2005).

In summary, both COL and oblique have advantages and disadvantages. The advantages of COL, in comparison to oblique illumination, are that the enhancement of resolution is slightly better, non directional, and has little impact on light intensity. On the negative side, there is no good way to control illumination with COL especially if high wattage light sources are used. This is because the COL setup requires that the condenser and field iris remain fully open or resolution enhancement is lost. An advantage of oblique illumination is directional contrast which results in subjects taking on a 3D appearance. Unfortunately this 3D effect is gained at the loss of light intensity.

The use of polarized light in combination with COL termed “Polar Annular Lighting” has been studied by Oku (2009) and, as illustrated, is capable of excellent resolution with respect to diatoms.

A study was undertaken to design a stop and filter system containing elements of the above mentioned systems which could overcome some of the drawbacks of each while preserving the optical benefits of both.

Materials

Experiments were conducted using a Zeiss GFL microscope equipped with a Zeiss 1.4 NA Achr/Apl condenser and intermediate tube beam splitter. Light was supplied by a 6v 15 watt bulb with tungsten filament. Objectives were either a Zeiss 40X 0.65 NA Plan Achromat (0.17), or Nikon CF 60X 0.85 NA (0.11-0.23) dry objective with cover slip correction collar. The camera utilized was a Moticam 2300 equipped with a Zeiss KPL 10X eyepiece and a Moticam 16 mm intermediate lens. One photo utilized a Nikon CF 10X Photo eyepiece which is noted in the caption. The specimens are from an 8 form diatom test slide by Klaus Kemp. Opaque masks were cut from black electrical tape and mounted on 1¼ inch (31.75 mm) standard glass filters. The circular polarizing film, ¼ wave plate film, and polyester film (used for the repair of automobile windshields) were all cut into 32 mm diameter round filters using a commercial gasket cutter. Circular polarizers were cut from commercially available film, and arranged so that they produce a black background when crossed (crossed Nicols).

Method

The crescent mask for prototype 1 was cut with a 40 mm diameter punch and the ellipse cut with a 42 mm diameter punch. The width of the ellipse is approximately 7 mm at the center, and the length is 24 mm. The ellipse is placed in the absolute center of the 1¼ inch (31.75 mm) inch glass filter slightly overlapping the crescent oblique mask (see Figure 3). The COL stop for prototypes 2 and 3 is a 10 mm in diameter circle, centered in the plain glass 1¼ inch (31.75 mm) filter. The crescent mask for prototype 2 was cut with a 32 mm diameter punch. The polyester film filter and plastic wave plates are 32 mm in diameter. One polarizing filter is placed in the microscope nosepiece (just under the head and above the objective). The other portions of the filter assembly are placed over the field diaphragm on the base of the GFL microscope, i.e., the light exit port. Images were converted to black-and-white via desaturation based on luminescence using the shareware program GIMPshop.

Optical setup sequence:

1. Focus on specimen

2. Open field diaphragm and condenser iris fully

3. Cross polarizers so that the background is black; this can be adjusted to off cross if desired. In most cases the polarized filters have been crossed unless otherwise noted.

4. Insert special filter

5. Focus condenser up or down until the observed image of the specimen is just free from a quasi “phase” effect. There may be some slight darkness on the edge of the field of view. A phase telescope can be used to center and focus the special filter in the objective back focal plane.

6. Rotate the special filter with the polyester film sheet until a portion of the background is bright green.

7. Insert ¼ wave plate and rotate it until background becomes bright. This can also be used to adjust brightness since the field and condenser irises must remain in the fully open position.

Development and Results

A simplified schematic of the general setup utilized is illustrated in Figure 1. Absent from the image is the condenser, field iris, and field lens. In order to simplify the image, these items have been intentionally left out.

An eight-form diatom test slide by Klaus Kemp was selected as the test subject. Nitzschia sigma and Stauroneis phoenocenteron were utilized for assessment of resolution based on the fact that these diatoms have known striae spacing of approximately 435 nm and 714 nm respectively (University of Texas, 2004). Numerous “special filter” designs were constructed and tested: these are illustrated in Figure 2. These early designs incorporated both an opaque mask for the center stop, and a polarized portion for additional contrast. These initial masks, while improving contrast, did not drastically improve the image. It was also found that the best image was obtained then the filter was rotated such that the polarized portion became opaque, completely blocking the light. It was therefore decided that certain components of these early designs would be retained (such as the elliptical center stop), but the materials changed and other shapes were introduced.

It also became evident that in order to produce the desired 3D effect there would need to be included a crescent-shape mask. These are classically used to produce oblique illumination, but often they are placed so that the majority of the direct light is blocked. It was decided to move the mask back, more toward the edge, and include an elliptical center stop. These changes led to the development of the mask seen in Figure 3. Noting that many crystalline and semi-crystalline materials have very low retardation and birefringence, it was decided that an attempt would be made to augment the filter with a thin semi-circular piece of polyester film. It is believed that the polyester film semi-circle shaped filter serves the purpose of retarding a portion of the light by a certain wavelength thus resulting in the sample being illuminated by two light beams of differing retardation. This combined with the center stop and semi-circular oblique mask results in detail enhancement. This filter setup will be referred to as prototype 1. Image results are shown in Figures 4, 5, and 6. Figure 5 was taken with the same filter setup as Figure 4, except that the polarizer and analyzer were not crossed as noted in the photo caption.

Nitzschia sigma taken with the prototype 1 filter system. Zeiss GFL, 40X Zeiss Plan Achromat lens, Zeiss 1.4 NA Achr/Apl condenser, 15 watt tungsten bulb. Modified stop full setup polarizer, analyzer, polyester film, and 1/4 wave plate, polars crossed. Black-and-white conversion based on luminescence. Camera system is a Moticam 2300 with Zeiss KPL 10X eyepiece.

Nitzschia sigma taken with the prototype 1 filter system. Zeiss GFL, 40X Zeiss Plan Achromat lens, Zeiss 1.4 NA Achr/Apl condenser, 15 watt tungsten bulb. Modified stop full setup polarizer, analyzer, polyester film, and 1/4 wave plate, polars NOT crossed. The polarizer was rotated approximately 45 degrees from the crossed position. Black and white conversion based on luminescence. Camera system is a Moticam 2300 with Zeiss KPL 10X eyepiece.

While the resolution is good and 3D effect is present, the resolution appears to be directional and related to the orientation of the elliptical center stop. This is compared to a simple COL stop (shown in Figure 7).

Nitzschia sigma diatom taken utilizing simple COL. Zeiss GFL, Nikon 60X P Plan Achromat lens, Zeiss 1.4 NA Achr/Apl condenser, 15 watt tungsten bulb. Black-and-white conversion based on luminescence. Camera system is a Moticam 2300 with Zeiss KPL 10X eyepiece.

Based on the above results the stop was again modified, with removal of the center elliptical stop and substitution of that with a round centered stop. This will be referred to as prototype 2. A slight variant on this design without the polyester film sheet was also evaluated; this will be referred to as prototype 2a. A schematic of each design is shown in Figure 8, image results are shown in Figures 9, 10, and 11.

Nitzschia sigma diatom Modified filter with COL center stop crescent shaped oblique mask and semi-circle polyester film filter (prototype 2). Zeiss GFL, Nikon 60X P Plan Achromat lens, Zeiss 1.4 NA Achr/Apl condenser, 15 watt tungsten bulb. Black-and-white conversion based on luminescence. Camera system is a Moticam 2300 with Zeiss KPL 10X eyepiece.

Nitzschia sigma diatom. Modified filter with COL center stop crescent shaped oblique mask (prototype 2a). Zeiss GFL, Nikon 60X P Plan Achromat lens, Zeiss 1.4 NA Achr/Apl condenser, 15 watt tungsten bulb. Black-and-white conversion based on luminescence. Camera system is a Moticam 2300 with Zeiss KPL 10X eyepiece.

Stauroneis phoenocenteron diatom. Modified filter with COL center stop crescent shaped oblique mask and semi-circle polyester film filter (prototype 2). Zeiss GFL, Nikon 60X P Plan Achromat lens, Zeiss 1.4 NA Achr/Apl condenser, 15 watt tungsten bulb. Black-and-white conversion based on luminescence. Camera system is a Moticam 2300 with Zeiss KPL 10X eyepiece

The last modification that has been studied thus far is shown in Figure 12. This setup is slightly different than the general setup shown in Figure 1. The main points of difference between the general setup and this modification are the absence of a polyester film sheet and opaque mask. There is no true oblique mask with prototype 3: rather, the oblique effect is provided by the crossed polar filters and the plastic wave plate. The crescent shaped area remains dark (by the crossed polar filters), while the area under the wave plate is altered into a bright region. This filter arrangement is referred to as prototype 3. Diatom test images utilizing the prototype 3 setup are shown in Figures 13 and 14.

Nitzschia sigma illuminated by the prototype 3 optical setup. Zeiss GFL, Nikon 60 X P Plan Achromat lens, Zeiss 1.4 NA Achr/Apl condenser, 15 watt tungsten bulb. Black-and-white conversion based on luminescence. Camera system is a Moticam 2300 with Zeiss KPL 10X eyepiece.

Stauroneis phoenocenteron diatom illuminated by the prototype 3 optical setup. Zeiss GFL, Nikon 60X P Plan Achromat lens, Zeiss 1.4 NA Achr/Apl condenser, 15 watt tungsten bulb. Black-and-white conversion based on luminescence. Camera system is a Moticam 2300 with Zeiss KPL 10X eyepiece.

Discussion

Prototype 1

Although the prototype 1 setup provided enhanced resolution and thus better image quality over brightfield, the observed enhancement provided only directional resolution benefits. Depending on the orientation of the subject or elliptical mask, certain features of the test diatoms could be seen or not seen. This directional resolution is definitely not preferred and was inferior to the resolution observed with a simple COL setup. The shadowing is also directional. Prototype 1 did impart an acceptable 3D aspect to the image and with the inclusion of the ¼ wave plate filter it is possible to control specimen illumination. Although not shown in this paper, the inclusion of the polyester film filter did seem to improve the contrast and thus aid in image quality with prototype 1.

Prototype 2 and 2a

It is clear from examination of Figures 7 and 9 that prototype 2 provides resolution improvements on a par with the simple COL setup (if not slightly better). In addition, there is a distinctly 3D aspect to the images. As in the case of prototype 1, this 3D effect is a direct result of the elliptical oblique stop utilized in the design, is directional, and depends on the position of the crescent-shaped mask. It is also clear from an examination of Figures 9 and 10 that the inclusion of the semi-circular polyester film filter has improved contrast. It is believed that this may result from illuminating the sample with light of two different retardations, the differential retardation being a result of the added retardation provided by the polyester film filter. Again, as in the case of prototype 1 with the inclusion of the ¼ wave plate filter, it is possible to control sample illumination.

Prototype 3

Prototype 3 represents a change in both filter setup and optical train. Images produced via this prototype have very good resolution, comparable to that obtained via COL and via prototype 2. Resolution enhancement is also not directional, although the shadowing effect is. As in the design shown as prototype 2, prototype 3 also has the potential to illuminate the sample with light retarded to different degrees. By partially crossing the polar filters and rotating the clear plastic wave plate, it is possible to control the shadowing and sample illumination (although not quite as simple as with prototypes 1 or 2).

Conclusions

While all prototypes offer some improvement in contrast, resolution, and shadowing over brightfield, the resolution improvement provided by prototype 1 is directional, and thus, depends on sample and filter orientation. Prototypes utilizing a centered round stop give non-directional resolution improvement: this, combined with an oblique crescent-shape (either as an opaque mask or as a cross polar darkfield), adds the desired 3D effect to the images at a very small cost of light intensity. The optical train illustrated also allows for the adjustment of light intensity which is not possible with the normal COL or oblique setup. Finally the inclusion of a polyester film half circle appears to further enhance contrast as seen in comparing images taken with prototypes 2 and 2a. It is clear that a simple design, such as prototype 2 or prototype 3, can offer the microscopist on a budget image quality and an overall appearance similar to those obtained via DIC at a cost of around $20.00 (USD). With some additional experimentation, either of these systems (prototypes 2 or 3) could be adapted to any microscope, or, conversely incorporated into a condenser.

Cited References

Clarke, T. (2004) Evaluation of a Prototype BF-DF-Oblique-Circular Oblique Lighting (BF-DF-Obl-COL) Condenser. Retrieved September 28, 2011, from www.modernmicroscopy.com:
http://www.modernmicroscopy.com/main.asp?article=53

Anon. (2005) Innovation 15, Carl Zeiss AG, 2005 “From the History of Microscopy: Abbe’s Diffraction Trials”. Retrieved September 29, 2001, from http://www.zeiss.com/C125716F004E0776/0/D1B2BF1284180E37C125717C003EDF5A/$File/Innovation_15_18.pdf

Oku, O. (2009) Mic-UK. Retrieved September 29, 2011, from Microscopy UK: http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artjun09/oo-diatom.html

University of Texas (2004) Bio 205 lab PDFs Diatoms. Retrieved September 28, 2011, from University of Texas EDU: http://www.sbs.utexas.edu/bio205l/lab_pdfs/diatoms.pdf

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William Mark Ph.D.
Director Product Design
Pfizer Consumer Healthcare
William Mark

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Enhancing Detail and Contrast in Light Microscopy Using a Combination of Circular Oblique (COL) and Oblique Illumination, Part II

William Mark

PDF Version

Abstract

Work presented in this article is a continuation of a previously published document (Mark 2012). In the present article, enhancing detail and contrast in transparent specimens is accomplished using a deviation of the previously developed circular oblique lighting (COL) and oblique masks. The new filter arrangements incorporate a quarter-wave plate step-wedge, and an auxiliary field lens; in addition to a COL central stop, these modifications allow for the use of objectives ranging from 16X to 100X and provide an incremental enhancement to image contrast over the previously discussed designs. While the concept is still the same (combining COL and oblique illumination) the execution is slightly different, with enhanced flexibility. What is presented in this article is a unique combination and application of COL, oblique illumination, quarter-wave step-wedge, and polarized light for the enhancement of detail in transparent specimens such as diatoms.

Introduction

This article is a continuation of previously published work dealing with simple combinations of COL, oblique illumination, and polarized light to improve resolution in transparent specimens such as diatoms. While the previous work focused on traditional oblique masks, elliptical and round COL stops, and polarized light with simple wave plates, the goal of this work is threefold:

1. To simplify some aspects of the previously established technique (such as the oblique mask).

2. Develop a system that is amenable to a wide range of magnifications.

3. Improve flexibility and resolving power over the previous setup.

While the concept is similar to the previously published work (combining COL and oblique illumination) the execution is different in three key ways:

1. The inclusion of a step-wedge increases the diversity of retarded light striking the specimen; this has been found to enhance resolution and improve contrast.

2. The oblique illumination can be varied and is provided by crossed Nichols rather than a static mask.

3. The central COL stop is smaller in size (2.7 mm vs. 7 mm) allowing for greater light throughput.

4. Inclusion of an additional field lens makes it possible to use the setup with lower power objectives such as 16X.

This study was undertaken to further develop an optical system that would allow for maximal resolving power using a standard halogen light source based on the previously discussed COL and oblique combinations.

Materials

Experiments were conducted using a Zeiss Universal Photomicroscope II equipped with a Zeiss 1.4 NA Achr/Apl condenser. Two standard Zeiss biconvex field lenses having a focal distance of approximately 100 mm (as determined experimentally). Light was supplied by a 12V 100W halogen bulb and Zeiss illuminator. Objectives were a Zeiss 16X 0.35 NA Plan Achromat, Zeiss 40X 0.65 NA Plan Achromat, Zeiss Neofuar Pol 63X 0.90 NA dry, and Zeiss 100X 1.25 NA oil Plan Achromat.

Cameras used were one of the following: a Canon EOS 60D mated to a Zeiss intermediate basic unit 47 60 10 and Zeiss 4X projection eyepiece; Panasonic DMC GF1 mirrorless camera with Zeiss intermediate basic unit 47 60 10 and Leitz 4X projection lens; Moticam 2300 equipped with a Zeiss KPL 10X eyepiece and a Moticam 16 mm intermediate lens; or a Nikon Coolpix 4500 attached to a Leitz Periplan eyepiece. The specimens are from an eight- or ten-form diatom test slide.

Method

The circular polarizing films were all cut from commercially available film into 32 mm diameter filters using a commercial gasket cutter, and arranged so that they produce a black background when crossed (crossed Nicols). The COL stop was made from self-adhesive foil tape cut to a diameter of 7/64 inches (or 2.7mm). The additional lens is a standard Zeiss auxiliary biconvex lens, focal length approximately 100 mm, and was placed just above the step-wedge. The step-wedge was constructed using a 1” x 3” glass microscope slide, on which 7/8” x 1.25” sheets of polymer ¼ wave retardation film (WF-OG4-WE, from Aflash Photonics OPD 135 nm +/-10 nm Center wavelength 560 nm) was applied and held in place. The leading edge of the first sheet was placed in the center of the glass slide and each subsequent ¼ wave sheet was placed 1 mm from the previous leading edge. All sheets were arranged with the fast axis parallel with the length of the slide. Six sheets were stacked to form the step-wedge (Figure 1).

Figure 1: Schematic representation of step-wedge.

This type of wedge is known as a Fox Wedge (Clay 1914) and is usually constructed of mica sheets of a given retardation. Since difficulties were encountered when trying to source a mica step-wedge, a wedge was constructed using ¼ wave film instead. The ¼ wave film wedge gives ¼ wave, ½ wave, ¾ wave, 1 wave, 1-¼ wave, and 1-1/2 wave distinct bands of retardation in crossed polarized light.

Images converted to black-and-white were done via desaturation based on luminescence using the shareware program GIMPshop.

Optical setup sequence:

1. Focus on specimen.

2. Field and condenser iris fully opened.

3. Cross polarizers so that the background is black. Remove the analyzer from the light path.

4. Insert COL stop filter and center it in the objective back focal plane using a phase telescope or equivalent. Insert the analyzer into the light path. The background should be black.

5. Slide in the step-wedge until the desired objective back focal plane image is achieved. It has been my observation that the best image resolution is seen when the step wedge leading edge is near the outermost edge of the COL field stop as projected onto the objective back focal plane.

6. Focus substage condenser up or down until the observed image of the specimen is just free from the appearance of a central “dark” spot. The condenser can be kept at a lower position (slightly lower than the position where the dark spot is observed), or at a higher position (just above the position where the dark spot is observed). There may be some slight darkness at the edge of the field of view.

7. The step-wedge may be rotated to control light throughput, and slid in or out to enhance or reduce the oblique effect.

8. For a 16X objective, the lower auxiliary field lens is left in place, while the standard field lens is removed from the light path (Figure 2). For 40X the auxiliary field lens should be removed. For greater than 40X both the auxiliary and standard field lenses should be in place.

9. For images utilizing the 100X objective, both the objective and condenser should be oiled using a good quality immersion oil.

Development and Results

A photo of the actual setup is shown in Figure 2, and a simplified schematic of the general setup and optical elements utilized is illustrated in Figure 3.

Figure 2. Shown above is the general optical setup for experiments.

As mentioned earlier, the stop and wedge are adjusted by viewing the objective back focal plane, as shown in Figures 4 and 5. It is important to center the COL stop, but likewise important to center the condenser and light-source filament. A phase telescope or equivalent device can be used to facilitate.

Figure 4. Objective back focal plane image of COL stop and step wedge without analyzer in place.Figure 5. Objective back focal plane image of COL stop and step-wedge with analyzer in place.

Eight-form and ten-form diatom test slides were selected as the test subject. Striae spacing for test diatoms are given in the table below:

Table 1. Diatoms imaged with critical parameters.

Diatom

Length (mm)

Striae Spacing

Amphiplura pellucida

80

0.273 , 0.21 (0.22-0.25) 4

Amphipleura lindheimeri

100

0.365

Surirella gemma

60-100

0.33 (0.40-0.50) 4

Nitzschia sigma

150

0.4353

Stauroneis phoenocenteron

170

0.7143

Results and Discussion

Photographic results of the setup are shown in following figures. Figures 6 through 12 were taken of a professionally mounted diatom test slide. This represents a best case scenario. Figures 13 through 15 were images taken from fresh mounted samples. Figures 13 through 15 represent samples in less than ideal mounting. As can be seen in the photographs, good resolution is obtained with all the diatoms examined. In fact, even the most challenging striae, spacing is resolved (Amphiplura pellucida at ~210 nm), with moderate optics (Plan Achromat). In addition, overall color is kept natural. Good detail and image quality is also maintained in specimens that have not been prepared in an “ideal” mount, regardless of camera, as can be seen by an examination of Figures 13 through 17. Application of this technique with a professionally mounted stained sample is shown in Figure 18.

It is believed that the enhanced contrast seen in these samples is a result of the combined effect of annular lighting (provide by the COL stop), the polarization state of the light, and the interference colors; the latter effects being supplied by the crossed Nicols and Fox wedge. Each wave plate in the wedge not only changes the polarization state of the transmitted light, but also contributes an interference color. All of these combined factors result in the images as shown. Removal of any one of the three components results in an inferior image.

Conclusions

The system discussed in this article has shown the potential for very good resolution enhancement as well as contrast adjustment. These image enhancements can be achieved from a standard halogen light source and Plan Achromat objectives, and can be applied to a wide range of samples and sample preparations. Because a standard light source is used, the samples retain their natural color, which is of great advantage when examining live wet mounts.

Cited References

Mark, William, Ph.D. (2012) Combining Circular Oblique and Oblique Illumination to Enhance Detail and Contrast in Light Microscopy, Part I.

Clay, Reginald S. (1914) Practical Exercises in Light, Being a Laboratory Course for Schools of Science and Colleges. Page 171, Macmillan and Co., Limited, London, 1914

University of Texas (2004) Bio 205 lab PDFs Diatoms. Retrieved September 28, 2011, from University of Texas EDU: http://www.zo.utexas.edu/courses/bio206/diatoms.htm

Piper, Jörg and Chmela, Gunther. (2011) Advanced Techniques for Observation and Photomicrography of Subcellular Structures in Diatom Shells. Microscopy Today 19 (1) pg. 10

Journal of the Quekett Microscopical Club, Ser. 2, Vol. XII., No. 77, November 1915 pg 575 http://www.archive.org/stream/journalofquekett0212quek/journalofquekett0212quek_djvu.txt

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Table of Contents

- Combining Circular Oblique and Oblique Illumination to Enhance Detail and Contrast in Light Microscopy, Part I

- Enhancing Detail and Contrast in Light Microscopy Using a Combination of Circular Oblique (COL) and Oblique Illumination, Part II