Visible Light Microscopy
Updated 15 February 2020
Diatom - Pleurosigma Angulatum, Leitz Dialux Polarising Microscope, 63X objective, dark-field illumination, Omax 1.3 MP camera
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My Leitz Dialux Polarising Microscope is a research grade microscope with 5 objectives. The microscope is now fitted with white light emitting diode illumination. It is in original condition with a good range of eyepieces, additional objectives and many accessories.
I fitted a 3.2 MP microscope camera from Omax. This is a very good low cost camera. The optical limitations of a microscope, and the computer display used, controls the final image resolution. With any microscope there are inconvenient physical limits to the image quality obtained, both for transmitted and reflected light.
A method is described for contrast improvement, when observing transparent samples. An additional plastic-film compensator is used along with normal polarised light microscopy. Dark-field and circular-oblique illumination techniques are discussed for improved contrast with difficult subjects, like diatoms.
There are brief descriptions of various LED lighting systems, macro-cameras, stereo-microscopes and a modified USB-microscope. On the right I have included a range of useful links, plus some images which can be enlarged by clicking on them.
These notes and links are for my use and also to help others. My limited experience with many areas of microscopy means there is a bias towards equipment, materials science and a few diatoms. Another bias is that I can't afford new research-grade equipment, so I try to achieve similar performance from selected low-priced or second-hand alternatives. I revise these notes periodically to correct mistakes. The photos shown here are scaled for the internet so some detail is lost.
Much of the performance of my Leitz microscope can be had quite inexpensively. Sources include the AmScope, Omax, Swift, Amazon and ebay websites. A search for the model numbers should locate the specified microscope. Use the web-site search box and enter the model text shown here in bold.
A monocular microscope should have a metal frame, three objectives, inclined viewing with a wide-field 10X eyepiece, transmitted and incident LED illumination with some control of lighting level and extent. This would suit family use where all sorts of items are viewed.
The AmScope M158-2L compound microscope is very good value at about US$91 A similar microscope is the Swift SW150 microscope for about US$80. Both microscopes have coarse and fine-focussing. The optics are all basically the same quality from similar origins. The 40-400X useful magnification range of these low cost microscopes is sufficient for most purposes. In addition to all the normal functions there is transmitted and oblique-incident LED lighting which can be battery powered and dimmed to suit. With this flexible lighting the microscopes can be used to look at small everyday objects. On my microscopes I find that, up to 400X, a fine-focus control is not essential, provided the coarse focus is smooth and the knobs are larger to compensate. Usually, on many microscopes, the feel of the focus controls can sometimes be adjusted by slightly turning the knobs in opposite directions.
Slide Preparation and Lighting
The quality of the slide preparation and lighting is much more important than the microscope optical quality or cost. The sample should not be too thick. For transmitted light work the sample should be immersed in water or some suitable mounting medium. A 0.17 mm thick cover-slip is added on top.
Good lighting passes through most of the glass surface area of the microscope objective. More information from the sample can reach the eye. This produces the best resolution. Light should not reflect or scatter off adjacent surfaces, which lowers contrast. Information from the sample is then diluted by optical noise. Fortunately this lighting is easier and cheaper with modern LEDs plus a condenser or aperture disk. A diffuse source of white light plus an aperture to suit the objective is all that is required for many applications.
Getting the Best Out of a Basic Microscope is about lighting techniques to obtain high quality results, using simple paper light-diffusers to improve resolution and contrast. Professional results can be obtained with just a little preparation. MicrobeHunter and some of the other references at right will be helpful for learning about microscopy.
My 42 year old Kyowa Polarising Microscope, shown above at left, now has a home-made lighting system which runs off any USB port. A diffuser made from a plastic container was added and three plastic aperture-caps, made from old paint test-pots, were drilled with different central hole sizes of 5, 10 and 18mm. This controlled the extent of the illumination. The contrast and resolution was improved. The images were nearly as good as those from a research-quality microscope.
The inexpensive Omax 1.3 megapixel digital camera is sufficient for most purposes, as discussed below. This camera would also work well with the basic AmScope and Swift monocular microscopes discussed above.
More Advanced Microscopes
Advanced microscopes can be obtained, for not too much money, from many suppliers. The range seems bewildering, but they do break down into few basic microscopes with a wide range of add-ons. A good microscope with standard DIN objectives, a single-lens condenser and a multiple-aperture disk to control the lighting, is the AmScope M200-LED 40-400X cordless monocular compound microscope. It has all the basics with nothing extra and is good value at US$116 on Amazon. The drift upwards in price to more features starts from here. As the price increases you get 3, 4 or 5 DIN objectives, a full Abbe condenser, binocular viewing, improved optics and a mechanical stage. For example the AmScope M230C 40-1000X LED monocular compound microscope with a double-layer mechanical stage would meet most needs for US$187.
The Omax M82E 40-1000X binocular compound microscope is sold for for US$209. The AmScope B120 40-1000X binocular compound microscope, also for $209, looks very nice.
Old microscopes are another option. Some good antique microscopes are available through dealers and auction sites. My 19th century French microscope is shown above at right. This microscope is still very usable with proper lighting. Alternatively, serviced educational microscopes are sometimes available.
Getting the Best Out of a Basic Microscope is about lighting techniques for basic microscopy. It shows how good lighting can produce very good images from the simplest microscopes.
An article on Teaching Microscopy to Young Children is worth looking at. This is essentially a very good microscopy lesson-plan for young children. Microscopes similar to the links above are recommended and their application is much wider than just biology.
Pippa's Progress shows amateur science with a microscope for young people with videos, information sheets and a very nice book.
What Price Optics shows there are only minor differences in the quality of the observed image when viewing a marine Cocceneis diatom, using a range of 100X objectives. The objectives have an age range from recent to more than 100 years old and a 100 to 1 price range.
Eyepieces and Objectives
A replacement AmScope 6 objective set and a modern AmScope wide-field 10X-18mm eyepiece can revive a typical old and well-used microscope. 10X is the magnification. If the objective is labelled 4X then the total magnification is 40X. 18mm is the field of view, or diameter, of the intermediate image located 10 mm below the top of the eyepiece-tube for a DIN standard microscope. The observed field of view, using a 4X objective, is 18/4 = 4.5 mm. This can be tested by observing a perspex ruler. It is not uncommon for old microscopes to have scratched or delaminated lenses so, in that case, new low-cost objectives and eyepieces will be a major improvement.
For many Leitz microscopes the intermediate image is 18 mm below the top of the eyepiece-tube, with Leitz standard optics fitted. This distance is a design property of the eyepiece. If you place a virtual image at this point it will be in focus, when viewed. This virtual image is usually inside the eyepiece and the top lens is used to observe it.
Field of view (mm) for a 10X-18mm eyepiece with 4X to 100X objectives and a 160 mm tube length
Note that a 25x eyepiece sold with some microscopes, to achieve the marketing gimmick of 1000x to 2500X, is not going to reveal any more detail. The image will simply look larger. It will be hard to use, particularly with glasses, because the eye needs to be very close to see the full field of view. The correct way to achieve high magnification is with a 60X objective or a 100X oil-immersion objective. An eyepiece magnification range from 5X to 15X is more comfortable to use. 10X is a good choice.
If glasses are worn an eyepiece with a high eye-point may be needed. This is the distance from the eye to the eyepiece where the full field of view is seen. If the eye is too far from or too close to the eyepiece this will not be the case. The minimum eye-point required is roughly equal to the distance from the eye to the front surface of the user's glasses. Lower magnification eyepieces often have higher eye-points.
One of the best eyepieces I have used is the Leitz Periplan 10X or 12.5X. They are not too expensive second-hand. With a 3.5X Leitz achromatic objective there is a little chromatic aberration at the image edge, although the field stop shows none. There is also a Periplan 8X eyepiece which has a higher eye-point and a 26 mm field of view. With a 4X objective the observed field of view is 26/4 = 6.5 mm. A second-hand Olympus WF10X eyepiece is also very good. With a 4X achromatic objective it shows a slightly flatter field of view with a little chromatic aberration at the edges.
On my Kyowa polarising microscope, with a 160 mm tube length, the AmScope objectives worked well. The stage micrometer was sharp almost to the edges using either an Olympus WF10X eyepiece or a US$19 AmScope wide-field 10X-18mm eyepiece . My Leitz Dialux polarising microscope has a 170 mm tube length. Leica states that 10 mm tube length differences will only matter for objective magnifications lower than 16X. However the AmScope 4X and 10X objectives do perform well for camera and eyepiece viewing. The built-in compensation of the Leitz Periplan eyepieces and the effect of 10 mm extra tube length may have combined to produce a good result with the AmScope objectives. The viewing is even better when the US$38 pair of AmScope wide-field 10X-18mm eyepieces are used. Some my other objective and eyepiece combinations do perform terribly, with strong chromatic aberration and curvature of field. In view of that, I have been fortunate with the lenses I am now using.
The image below was taken on my old Kyowa Polarising Microscope using 4X Plan, 20X, and 60X achromatic AmScope objectives and an Omax 1.3 MP camera. The field width is reduced to about 60% when compared with the table above. A USB LED light source was used and is shown in the lighting section below. The slide also looks very good using, either the AmScope WF10X-18mm eyepiece, or the Olympus WF10X eyepiece.
Bitterwood Stem - Kyowa Polarising Microscope - 4X, 20X, 60X Objectives, Brightfield
The AmScope wide-field 10X-18mm eyepieces are surprisingly good. The viewing is very sharp with nice contrast. Unlike the Olympus WF10X eyepiece, there is no chromatic aberration at the edge using either a Leitz 3.5X achromatic objective or an AmScope 4X achromatic objective. There is a low curvature of field and good sharpness near the edges with all the AmScope DIN achromatic objectives. This matching eyepiece and objective combination works very well with the 160 and 170 mm tube-length polarising microscopes I have here.
Vertically cropped images of a 2 mm long Leitz stage-micrometer with bar intervals of 0.01 mm are shown below. These images were taken with a 3.1 MP Omax camera on my Leitz Dialux polarising Microscope. The eyepiece views were sharper.
4X, 10X, 20X, 40X, 60X AmScope achromatic objectives - 0.01 mm Leitz stage-micrometer
Suggested Additional Objectives
A good compromise is to fit a 60X objective for US$42 in place of the 40X or 100X objective. Oil is not needed. For some applications, such as observing pond life, replacing the 10X objective with a 20X objective for US$24 allows more detail to be seen, while still allowing the tracking of subjects.
Note that the generic AmScope objectives and eyepieces are the same as on many other brands of microscope. I would fit 20X, and 60X, objectives rather than the supplied 40X and 100X objectives. The 20X and 60X objectives, as noted above, are not an expensive addition. I also have an AmScope 4X Plan Achromatic Objective for US$22, which is perfect for photography. This objective is par-focal with the AmScope DIN achromatic objectives.
I have converted all my microscopes to LED illumination. When converting a microscope to LED illumination excess brightness can be a problem. LEDs are not lasers, but they can be very bright. Coloured or diffusing filters may help. A means of dimming is helpful. If dimming is not possible then the white LED power rating should be less than 1 watt to replace a 15 watt incandescent lamp , and preferably much less when using modern LEDs.
For example I originally used a 2.8 volt, 20 mA, white LED with a 12 volt power supply. A (12 volt - 2.8 volt)/0.02 amp = 470 ohm resistor was needed in series to keep the current to less than 20 mA. The maximum power was less than 0.06 watts. I found that for general use this provided more than sufficient light for bright-field work. For dark-field and polarised light work more brightness will be needed. The LED voltage depends on the current and can be higher than 3.3 volts with a good heat-sink
I have also used a bare white LED chip recycled from an old light bulb. Tools for recycling included a small hacksaw, some tweezers, a knife and a small gas torch to gently heat the aluminium heat-sink from the back to melt the solder holding the LEDs.
Some light bulbs package 4 blue LEDs per chip. The blue LEDs are covered with a yellow fluorescent layer to make a white light source. The chip has a measured voltage rating of 4x2.8 = 11.2 volts at 50 mA. The illuminated surface area of 6 mm2 is much larger than a typical packaged white LED. The strong lens of typical 5mm packaged LEDs makes it hard to achieve even lighting. I mounted the LED in a forward position as the original and expensive Leitz tungsten lamp had an extended filament position.
The required series resistance is ((Maximum Power Supply Voltage)-(LED voltage))/I ohms where I is in the range from 0.02 to 0.2 amps. A white LED voltage is typically measured at 2.8 volts at 50 mA. An 8.4 volt recycled 3 LED chip from an old light bulb should be in series with about 68 ohms to run at about 50 mA with a 12 volt power supply. An 11.2 volt 4 LED chip would require about 15 ohms for 50 mA at 12 volts. It would help if the resistance could be varied to give a current in the range from 10 to 180 mA. The 4 LED chip can dissipate more than 0.5 watts on a suitable aluminium or copper heat-sink. Some cheap torches contain similar bare LEDs which could be used as microscope lights. The microscope light shown below runs from 8 to 12 volts, using a variable DC power supply.
Microscope light - a 3 LED chip on Veroboard supported by two 10 ohm resistors behind
USB light Sources
I have recently prepared some microscope lights powered by a USB hub or a USB power-bank. Many second-hand microscopes have mirrors or illumination based on obsolete lightbulbs and heavy power supplies. Converting to LED illumination removes a source of heat and often improves the viewing experience. USB illuminated microscopes are self-contained, portable and useful tools. Everything fits inside the microscope case. It is also greener, since older microscopes can now have an extended working life.
The USB 2.0 specified power output is about 2.5 watts, or five volts at usually more than 500 mA. For my Kyowa polarising microscope I converted a $5 camp-light which originally ran directly off 3 AAA cells at 4.5 volts. The flat 16 mm diameter 2.8 volt LED, mounted on an aluminium heat-sink, was ideal for this purpose. A switch wired in series with a 10 ohm resistor and a 200 ohm wire-wound potentiometer produced LED currents from 12 to 168 mA using a 5.1 volt USB power-bank. I have now updated to the mosfet circuit, below-left, to control this LED source.
I used some ground-glass as a light diffuser. Alternatively a square of white translucent plastic or thin white paper or tissue would do. Above this I added the bottom half of an old condenser comprising a diaphragm and a field lens. The illumination was bright and even. I added this light in place of the original mirror. The inside of this simple light is shown at right.
Microscope light sources derived from an inexpensive LED camp light
An earlier and even simpler USB light is described in the Beginner's section above. This light is now used to provide transmitted illumination for an old Cook, Troughton and Simms M6100 Greenough stereoscopic microscope. This microscope is shown at right, along with a view of the sub-stage lighting. For incident lighting there are two LED lights costing $7 each, so the total cost of lighting is under $20. A steel plate, added to the mirror cradle, engages with the magnetic base of the lamp to hold it steady. The 18 mm diameter aperture-cap now has a polariser added. Two of the three objective pairs have small polarisers in front. This aperture-cap can be rotated for polarised or normal illumination.
I am now testing the 16 mm diameter camp light LED, described above, because the white balance is better and it is very easy to align. So far it is working well and it is probably the best light source I have tested at a very reasonable price. The white LED consists of a 16 mm diameter light-yellow phospher disc dotted with 16 small blue LEDs. The light source position and the condenser height needs to be adjusted so these two sources are properly mixed to produce white light. This is no different from defocussing a filament image.
Because the light source is 16 mm in diameter a less severe field lens can be used in front of the light source. This will increase the light intensity through the microscope. I used a plano-convex lens with a focal length of about 40 mm. The convex side faced the light source. The lens, or the LED, was positioned until the the base of the field-diaphragm was slightly more than fully illuminated.
A simple N-Channel Mosfet circuit, shown at left, allows a 100 kohm potentiometer to control the LED brightness when connected to a 5 volt USB power supply. The LED current can be varied from 0 to 100 mA. In the camp light the dropping resistor is 1.8 ohms so this light source can operate at over 700 mA or approximately 2 watts, with some heat sinking. If the lamp is not bright enough, reduce the 22 ohm LED resistor value to 10 ohms. At 10 ohms the current draw should be about 150 mA, allowing for the approximatey 5 ohms on-resistance of the 2N7000 mosfet. For my purposes the light level produced is very good.
Choosing a Microscope Camera
A digital camera is useful as a recording device, but it almost never displays the additional detail seen by the eye. The eye can, to a degree, focus through the image to reveal more layers of detail at the same time.
For the usual range of microscope objectives the observed horizontal resolution, expressed as pixels, ranges from about 1800 pixels at 3.5x to 1100 pixels at 40X. In oil the observed resolution is doubled to a maximum of about 2000 pixels at 40X, falling to 800 pixels at 100X. For more see Numerical aperture and resolution.
A good camera covering much of the field of view should therefore have a horizontal resolution of 1000 to 2000 pixels. A 5 MP camera would exceed this specification and a 2 MP camera would be sufficient for work in air. A 3 MP camera meets this specification and would perform well as the sample is moved into position, and for focussing. A USB-3.0 interface would allow for faster image updates, although USB-2.0 is usually adequate - provided the camera resolution is not too high. A 21 inch computer monitor is typically 2.1 MP and not all of that 1920x1080 pixel area would be used in practice.
Matching Camera to Microscope Resolution is a useful tutorial which assumes perfect optics. A 3 MP camera would be about optimum using this guide. Required camera resolution for photography through the microscope can be simply demonstrated by taking an image at two resolutions, namely 3 MP and 12 MP. Anything above 3 MP shows only minimal improvements in detail with most microscopes. This argument only applies to microscopes, not telescopes.
Optimum microscope resolution assumes:
In reality few of these statements are true.
Field Curvature and Resolution
Although the image is sharp to the eye, there is almost always field curvature. There is a region towards the edge of the image circle which is not so sharp. This effectively reduces resolution when the objective image is projected onto the flat sensor surface.
The eyes can adjust focus a bit, when using the eyepieces. Fortunately only the sharper middle-part of the objective image is sampled by the camera. Expensive plan objectives do produce nearly flat fields, so more camera resolution might be justified, particularly at low magnifications.
Resolution of the Sampled Image
Because the sampled rectangle is well inside the full microscope image circle, the total number of recorded pixels required for best resolution will be reduced in proportion. So we are back where we started. Personally I would choose a camera that could almost fill a display that I could afford.
Resolution and LCD screens
A 1.3 MP camera has 1280 pixels horizontally and 1024 pixels vertically. If just the vertical resolution of a typical LCD display, at 1080 pixels, is considered then a 1.3 MP camera should be satisfactory. In general, the lower resolution cameras are less noisy and can cope with a wider range of illumination levels. Omax offers 0.75x, 0.5x and 0.37x C-mount reduction lenses which can be chosen to optimise the camera viewing area.
Not all the computer LCD screen area is used. The Touplite imaging program has a display window of 1500x900 pixels, which is smaller than the usable area on a 21.5 inch 1920x1080 pixel LCD display. This expands to 1500x942 pixels if the Mac toolbar is not displayed. So the maximum number of pixels that can be displayed using Touplite is 1.4 MP.
Resolution and Publishing
For some users, publishing requirements may dictate the required camera resolution. An image printed at 300 dots per inch on half an A4 sized page, with generous margins, would need to have a resolution of about 2 MP. A 3 to 5 MP camera would meet most needs. But typically many papers have montages of much smaller images. Each small image focuses on a particular detail. So again, 1.3 MP will do.
Sensors and Resolution
Bayer digital camera sensors have an overlay of coloured dots dominated by green. This halves the resolution of the sensor for a given colour. Some of that lost resolution is regained by interpolation, by using just the intensity data. It could be argued that a slightly higher resolution camera is needed to display all the image information.
Cameras and Trinocular Adaptors
Some microscopes have inconvenient trinocular adaptors which do not fit standard eyepieces. This makes many microscope cameras difficult to fit. There are a few options:
Omax 3.2 MP Microscope Camera
Four Omax Microscope Cameras
For my purposes there were four Omax cameras of interest.
The Touplite application has fixed magnifications of 10%, 20%, 25%, 33%, 50%, 67%, 75%, 100%, 150%, 200%, 300% and 400%. These magnifications can combine with the camera pixel settings to vertically fill the software viewing window at 1500x900 pixels on a 1920x1080 screen. The 1.3 MP camera matches reasonably well at 100% magnification with 1280x1024 pixels in the recorded image. The 2.0 MP camera matches almost exactly at 75% magnification to give a 1200x900 pixel on-screen picture and 1600x1200 pixels in the recorded image.
The aspect ratio of the 1.3 MP camera is 4 by 5. The other cameras all have a 3 by 4 aspect ratio.
The other cameras could use medium resolution for viewing and high resolution for photography. Touplite has this option. At medium resolution the 5 MP camera is a good match for the software viewing area with a reasonable frame rate. An Apple 21.5 inch Retina screen shows 2304 vertical pixels so the Touplite viewing height is about 1912 pixels. This matches the 5 MP camera at full resolution but the low 5 fps frame rate may be annoying. The optics of a high quality microscope actually provides a somewhat lower resolution, particularly in air, for the reasons described above. Note that the higher resolution cameras have a lower dynamic range, they have lower frame rates and they are noisier. As computers evolve, a USB-3.0 or USB-C camera might be a better option in the future. In my case cost was an issue.
The imaging software blurs slide movements so the higher frame rates, specified with the lower resolution settings, are not realised in practice. A small object moving within a stationary field of view would probably be rendered better. The USB-2.0 interface limits the data rate. A coloured image at 1200x1024 pixels has 3x256 colour levels making for a total data size of 943,718,300 M bits or 118 Megabytes of 8 bit information. Compression and buffering reduces this. The typical 30 MB/s data rate of a good USB-2.0 connection is stressed if too much image content is changing at the same time. The frame rates are higher if auto-exposure is disabled in Touplite. USB-3.0 is better for good video but it is more expensive.
Omax 1.3 MP Microscope Camera
I ended up purchasing an Omax 1.3 MP camera SKU:A3513U at US$94.99 plus shipping. The camera performs well and delivers very nice images, some of which are shown below and at right. In retrospect, the 2 MP camera may be a good choice as it can better fill the software viewing window, using the fixed Touplite magnification selections. The image is 7% wider, the frame rate is faster and the dynamic range is slightly less than the 1.3 MP camera. The video frame rate is higher, but probably not fully realised with USB-2.0.
I also purchased a 0.37x reducing lens for my original 2 MP JEPCAM which was made from an adapted web camera. Some images from this camera are shown at right. Modern microscope cameras are sufficiently inexpensive, and of such high quality, that it is no longer worthwhile modifying web cameras to suit.
I use the supplied Touplite software on my iMac and ImageJ for cropping and adding scale-bars. The Windows version of the supplied software is ToupView. It is more comprehensive and has more editing features, including scale bars. Touplite on the iMac is fine for my purposes. The latest 2019 Mac version of Touplite now has most of the useful features of ToupView for Windows. I can now do image stacking for an improved depth of field and image stitching for a larger field of view.
1.3 MP camera viewing area. Microscope with 4X to 100X objectives and 160 mm tube length.
Omax 3.2 MP Microscope Camera
I now have an Omax 3.2 MP camera SKU.A3530U at US$139 plus shipping. The performance is very good and there is a marginal improvement in the image quality at some magnifications. For most purposes there is no difference, but I now have a camera to use with one of my other microscopes. I can use both cameras with one computer and I can easily switch between them in Touplite. As a first purchase, this would definitely be my camera choice.
3.2 MP camera viewing area. Microscope with 4X to 100X objectives and 160 mm tube length.
Polarised Light Microscopy
Polarised light microscopy can produce some spectacular coloured images from ordinary subjects, such as fibres, sand, dust or even sugar crystals. With low cost microscopes some Polaroid can be added to one port in the stage aperture disk and a Polaroid cap can be added to the top of the eyepiece, as mentioned in the lesson-plan above. Alternatively two caps could be made, one for the eyepiece and another which fits on top of the light source. If there are prisms or mirrors in the light path just orient the top Polaroid for the maximum light transmission. This cancels any stray polarisation effects above the sample slide. Insert the lower Polaroid below the sample slide and rotate until most of the background light is extinguished.
Polaroid can be recycled from old LCD screens or old sunglasses. Clear thin plastic sheeting can also be added under the upper Polaroid cap or on top of the lower Polaroid to replicate sensitive-tint viewing. A clearer image will be obtained if the plastic sheets are in the lower position. Choose enough sheets to produce a magenta colour when inserted and rotated between crossed Polaroids. This allows some of the functions of a Leitz polarising microscope to be duplicated.
Vacuum cleaner dust art - Leitz Polarising Microscope - modified sensitive tint
Circular polarised filters for digital cameras can also be used. They are plain polarisers combined with a 1/4 wave plate. The effect is similar to adding plastic sheets between the polarisers. Use circular polarisers, one above and one below the sample with the threaded sides towards the sample. They won't need to be rotated for good viewing. There is only a slight change in the background when rotated, which is due to the 1/4 wave plate being fully effective at only one wavelength. The sample colours will be rich and often appearing less sensitive to slide orientation if two orders of the same colour are seen.
Leitz Dialux Polarising Microscope
My Leitz Dialux Polarising Microscope is in good condition, with a performance matching that of a modern research microscope. The microscope can carry 5 objectives on the turret. It has a trinocular head which allows a camera to be fitted. The rotating stage is supplemented with a precision x-y adjustable slide holder. Two condensers are available with full adjustments. Correct condenser adjustment delivers excellent viewing results with this microscope. The adjustments, which are described in many textbooks, confine the light to nearly fill the lens apertures without touching adjacent surfaces. This gives the best resolution and good contrast.
I use 12.5x Leitz Periplan eyepieces. My normal Leitz objectives are 3.5X, 10X, 20X, 40X, and 50X. The magnification range is therefore 44X to 625X. I also occasionally use a superb 63X objective, but it is not par-focal with the others. My current AmScope objectives are 4X, 10X, 20X, 40X and 60X and the magnification range is 40X to 750X
Polarised light is used in geology to help to identify minerals in thin section. It is also used widely for the identification of particles and fibres. A sample may exhibit birefringence between crossed polars. There is an additional rotation in polarisation direction which allows the crystal to become visible. If the refractive index of the crystal varies with direction then two polarised light streams are emitted with different strengths and retardations. This will produce colours because there is destructive interference of the two streams of polarised white light at particular wavelengths Some colours are removed from the white light spectrum. The colours are repeated, but become muted, as the retardation increases. The colours observed are summarised on a Michel Levy Interference Chart.
The McCrone Research Institute is an Illinois not-for-profit educational and scientific research organisation located in Chicago. Polarised light microscopy is used extensively by this organisation. It offers courses in microscopy and does research in support of its educational activities. Many of the links at right also provide more information about polarised light microscopy.
Compensating Plates and Imaging
A full-wave compensating plate rotates polarised light by 360 degrees. It is inserted at 45 degrees to both the polariser and analyser. This is also the position of maximum brightness for many birefringent crystals. The compensator shifts the interference colour wavelengths, from samples, by about 550 nm. This wavelength of 550 nm is called the retardation. The colours seen can be found from the Michel Levy chart after the compensating plate retardation is added. This means that low birefringent samples with almost monochrome interference colours are now brightly coloured. Yellow and blue are often seen, but many colours can be produced. The colours and extinction angles can be used to help identify some samples, or at least help to determine the optical sign.
A 1/2 wave compensating plate rotates polarised light by 180 degrees. The retardation is about 275 nm. The effect of this retardation is that the system of two polarisers and a 1/2 wave plate between are always in extinction at all polariser orientations. Light, without any sample present, is always blocked.
A 1/4 wave compensating plate rotates polarised light by 90 degrees. The retardation is typically about 137 nm. This generates circularly polarised light. Two polarisers with a single 1/4 wave plate between will always transmit light at all polariser orientations.
Two circular polarisers, with their built-in 1/4 wave plates facing each other, behave like the 1/2 wave case above. The polarisers are always in extinction, blocking any light. There will be a slight variation in colour as one circular polariser is turned.
Lastly, two polarisers, without any retardation, block light twice per revolution when one polariser is rotated.
Commercial full-wave plate retardations vary a bit, typically over a range of 530 to 560 nm. 1/4 wave plates vary over a smaller range.
Sensitive Tint Imaging Improvements
I have experimented occasionally with polarised light microscopy since 1974 when I worked in the DSIR. I found that modern plastic materials can behave like compensators. Some plastics used for packaging are typically biaxial and offer a maximum retardation of about 100 nm. This makes them ideal for trimming the response of the standard 530 nm full wave compensating plates. They can be used to improve viewing and photography through the microscope.
The following method gives well coloured images for samples which are transparent, with low contrast and low birefringence. This procedure is equivalent to trimming the thickness of the standard full-wave gypsum "sensitive tint" compensator. This produces the best possible contrast. For example an additional 1/4 wave compensator plate can be placed in the condenser, above the polariser, and rotated until the colours are suitable.
If a Berek or Quartz-Wedge compensator is inserted, a fairly sharp boundary is seen between first order yellow and second order blue. My procedure trims all the lighting exactly onto this boundary. Some low birefringent samples are seen as distinctive combinations of yellow and blue. The whole field of view now has the same magenta background colour.
For some gypsum "sensitive tint" plates this trimming may not be needed, but for the ones I have here from Leitz, Olympus and Swift, it is. This procedure is mainly an aid for photography. For quantitative work use just the gypsum "sensitive tint" plate.
The following image shows first-order yellow bordering second-order blue, using a Berek compensator which tilts a gypsum or magnesium fluoride plate. This creates a range of thicknesses equivalent to several orders of retardation. The corresponding interference colours are produced when the Berek compensator is inserted between crossed polars. Sample details are often seen more clearly at the magenta border between first-order yellow and second-order blue. The image is a little unsharp because of the tilted compensator plate above the objective.
Oamaru diatoms, Berek compensator, 10X objective, Omax 1.3 MP camera
For the photo below of the Diatom - Nitzschia circumsuta BW-113, prepared by Stuart Stidolph in 1982, I inserted a strain-free perspex plate, covered with a clear 0.006 mm thick mylar plastic film, above the condenser polariser. The mylar film was originally used in XRF sample preparation. It sticks to Perspex electrostatically. It has a maximum retardation of about 200 nm which is high, given the thickness. This is possibly due to the thin mylar being more severely extruded than packaging plastics. My Perspex sheet came from Bunnings and I was pleased to see no sign of birefringence between crossed polars. As far as I can tell it plays no role in the improved image properties observed. I inserted a full wave gypsum "sensitive tint" compensator into the standard slot above the objective.
The mylar film was oriented on the perspex until the background colour observed, bordered between first-order yellow and second-order blue. The background colour is set a bit more towards yellow than a normal full wave plate. Compensators made from some thicker plastics produced the same colours. In the past, I have also used mylar film from an oven bag, as a near full-wave compensator, for similar applications. Secure semi-rigid plastic packaging also works. Any slightly birefringent sample either shifts the observed colour towards blue or yellow, depending on the sample structure, and on the orientation. This can make biological samples much clearer as different structures now can have different colours. These colours will change with orientation. Some samples work well with just the normal polarising microscope compensators.
For microscopes without a filter holder above the condenser polariser, some mylar film can simply be adhered to the bottom of the slide. With the gypsum compensator inserted, the stage is rotated until a suitably coloured image is obtained. The camera is then rotated to restore the desired image orientation on the screen. The camera white balance setting can also be used to further improve contrast, if needed.
Diatom - Nitzschia circumsuta BW-113, polarised illumination with dual compensators, 63x objective, Omax 1.3 MP camera
Sensitive Tint the Low Cost Way
Many microscopists do not have access to a polarising microscope but they do have some Polaroid filters. The polarising caps for the eyepiece and light source, mentioned above, are easy to add. Turn one filter until the background view is dark.
The nearest inexpensive match I have found to a full wave gypsum sensitive tint plate is 3 layers of the 6 micron mylar film, mentioned above. One layer of clear Scotch Packaging Tape, adhered to a slide and set at 45 degrees is almost as good. Other sources of plastic include packaging, report covers and oven bags. If filters are cut from the same sheet, set them all in the same orientation. This filter stack can be placed above the lower Polaroid and rotated until the background has a magenta or pink colour. Adding or removing mylar or plastic layers and rotating the slide will produce the right colour. Use the camera white-balance to help with contrast. Because traditional compensators are narrow and expensive they are normally only inserted in a 45 degree slot just above the objective.
Part of the thin plastic cover of a CD case can be used as a sensitive tint filter. The plastic is brittle and prone to cracking. Cutting almost horizontally with a fine saw places the least stress on the plastic, otherwise cracks may form. The plastic cover can be tested between crossed polars before cutting. Choose the area which gives a suitable range of colours as the cover is rotated. Most CD covers are not evenly birefringent, but areas can be found which are. Fit above the lower polariser and rotate to suit.
For whole-slide macro photography I took many photos for students using a circular polariser below the sample. The intention was to produce a coloured general view of the slide. The camera lens side of the polariser faced the sample slide. It provided a polariser and a 1/4 wave retarding plate in one component. A plain polariser (analyser) was sometimes used above the slide. A circular polariser could be used instead, with the camera lens side facing the slide. The 1/2 wave length total retardation produces a darker background and stronger sample colours. There is sometimes less apparent rotational variation of colours as two orders of the same colour may be seen.
White industrial hairnet, Kyowa stereo microscope, two circular polarisers, Omax 1.3 MP camera
The picture field width is 4 mm. The hairnet is advertised as "Non-Woven, 100 % Spun-bonded Polypropylene". The fibres are about 13 microns (0.013 mm) in diameter. There is a pattern of small glue dots holding everything together. In transmitted, polarised, 1/2 wave retarded light, clear polypropylene fibres, with the same composition and diameter, all show the same yellow colours, according to my colour meter. Where they cross, the colours tend towards a brown-yellow with less red and a little blue present. There is little change in colours with orientation. The larger and clear glue spots are coloured various shades of blue and cyan in this lighting.
This setup can be duplicated with the polarising microscope by using a 1/4 wave compensator above the objective and a circular polariser (instead of the normal polariser), below the sample slide. A rare and expensive 1/2 wave compensator plate would also work. Sample birefringence will still be seen but it will be almost extinction free. Two orders of yellow are seen here which makes the sample appear to be insensitive to fibre orientation.
Dark-Field, Rheinberg and Circular-Oblique Illumination
I cut out some plastic disks, from a black plastic binder cover, using some hole punches. For the larger disks I cut around a coin or a washer of the right size. I used Blu-Tac to hold the coin steady while cutting with scissors. The disks range in size from 5 mm to 16 mm. I made some perspex filter holders, described below, to allow the disks to be inserted in the condenser slot above the polariser.
The diaphragm is used to control the illuminated annulus size. The annulus of light comes to a point focus at the sample. With the right sized disk a second annulus of light is formed just outside the objective lens field of view. Only scattered light from the sample enters the objective lens. The sample is usually bright, sometimes with diffraction colours, and the background is dark.
Some test dark-field stops, oblique stops and a 6 micron Mylar compensator
The larger stops suit the more powerful objectives, although they are quite critical to align for good dark-field work. A 10X to 20X objective is a good choice for initial work. The stops are placed on a tabular perspex filter holder and secured with a spot of Blu-Tac. The filter holder is inserted into the condenser slot of the microscope. The Blu-Tac allows the stop to be moved and centred with the aid of the Bertrand-lens. Removing an eyepiece is another option for aligning the stop. Careful condenser focusing and centering will be needed. If no dark-field illumination is seen, open the condenser diaphragm and raise or lower the condenser. Removing the eyepiece and looking down the tube will help with centering the disk-stop and choosing the right size. The bright illuminated spot should only just be obscured. Use a 10X objective for the centering adjustments.
A Leitz microscope dark field kit has stops 6, 11, 16 and 19 mm in diameter for the 4X, 10X, 20X and 40X objectives. A good fit to this data is y = 7*ln(x)+22 where x is the numerical aperture and y is the required stop-disk diameter. There is no need to be too exact and the data here is a guide only.
A modern Olympus microscope uses dark-field stops averaging 11, 17, 20, 23 and 25 mm in diameter for 4X, 10X, 20X 40X and 60X objectives. The corresponding fit to this data is y = 6.5*ln(x)+26 where x is the numerical aperture and y is the required stop-disk diameter.
My very old Olympus POS polarising microscope has disk-stops of about 13 mm for the 4X and the 10X objectives. A 20 mm diameter stop is suitable for a 40X objective, about 3 mm less than calculated for a modern Olympus condenser. Better results are obtained with the condenser set close to the sample slide.
An item of interest to me is a commercial dark-field condenser which has easy to use centering adjustments. Some use reflecting optics to produce a ring of light which converges on the sample. Others use a disk, as already described. A typical NA range from 0.7 to 0.9 suits higher powered objectives.
LED Dark-Field Illuminator
I have also been experimenting with a small LED ring-light mounted just under the stage aperture. The LED ring-light mounts on the fold-away condensing optics and therefore uses the condenser adjustments. The tiny LEDs are recycled from faulty light-bulbs. They are mounted between tiny rectangles of circuit board which are glued inside a short 24 mm ID aluminium ring. 8 LEDs are wired in pairs with 390 k𝛀 dropping resistors. The LEDs are controlled with a 4 channel rotary switch so all 16 combinations of 4 lights are possible. This means that not only dark-field illumination is possible but many combinations of oblique illumination are as well. A fixed aperture above the LEDs protects the microscope optics from too much stray light.
So far the conclusion is that good dark-field images can be obtained for survey work. These results are, however, no better than using a circular disk in the condenser. The range of oblique illumination is more interesting, for photographic work. The LED ring-light also makes a very good dark-field and oblique illuminator for a stereo microscope.
Using a blue disk and a yellow backing produces Rheinberg illumination. Scattered light becomes yellow and the unscattered background becomes blue. Other colours can be used. Use darker colours such as blue or red for the disk and yellow, green or clear for the backing, as these colours will appear as highlights. The photos below were taken on my Kyowa polarising microscope using a 10X objective and an Omax 1.3 MP camera. The image widths are approximately 1mm. The pine-needle slide is old and the Canada Balsam is starting to crystallise. The epidermis ruptured during microtoming and some cell contents smeared to the left. Some of these details were not obvious with normal lighting.
Pine Needle cross section - Rheinberg illumination using a blue disk and a yellow backing
Augite Andesite - Rheinberg illumination using a blue disk and a yellow backing
Old red/blue stereo glasses are a good source of filter material. Coloured Cellophane is another. Cellophane may need to be stacked to increase colour density. Marker pens can also be used. For the stop you can use the hole in a suitable washer as a guide. Alternatively, drill a hole in a suitable template. Personally, I often use a simple clear filter with a blue stop in the centre. The sample highlight colours are not altered.
Circular-oblique illumination is less critical. An annular ring of light illuminates the sample but offsetting the disk means that the image has shading and relief, like in a good portrait. One disk has a semicircular cutout at the edge to provide two levels of oblique lighting and some shading for contrast. The setup is not dissimilar to the lighting setups used for portraiture and movies. More than one light source (the ring and the cutout) and some delicate shading (slightly off centre) can produce a nice image.
With the condenser top-lens near to the slide the illumination is usually dark-field. With the condenser higher still the illumination becomes circular-oblique as some annular light now enters the objective. This lighting works best with the stronger objectives. An improvement in resolution is usually observed with this illumination. Some of the references at right describe both techniques. Microscopy-UK has many articles of interest.
Diatom - pores, dark-field illumination, 63x objective, Omax 1.3 MP camera
Diatom - pores, circular-oblique illumination, 63x objective, Omax 1.3 MP camera
One of the many methods for diatom preparation uses hydrogen peroxide, potassium dichromate, potassium permanganate, hydrochloric acid, sulfuric acid and nitric acid. In the late 1970s I replaced a similar process with my Plasma Asher for the inorganic analysis of blood samples. It was very effective for many toxicology samples and a number of laboratories around the world used similar schemes. This 2010 reference outlines the use of Plasma Ashing to directly process diatom samples on a microscope cover slip.
In 2009 we used Plasma Ashing to prepare a diatom sample directly on an electron microscope stub. The stub was set downstream from the RF plasma so no heating occurred. I have since modified the Plasma Asher so that the only reagent is oxygen generated from a small fuel cell. Air can also be used. The plasma slowly converts all organic material to carbon dioxide, without any heating. Both diatom references, below, used Plasma Ashing for sample preparation.
My Stereo Microscopes
I have two old stereo microscopes. One is a Kyowa stereo microscope with 10 and 30 times magnification. I added a bike LED front light shown at right for strong incident illumination. I also have a Cooke stereo microscope which is capable of both incident and transmitted light viewing. The magnifications are 15.6, 44 and 125 times. This microscope has been slightly modified for polarised light viewing. The latter setup was particularly useful for looking at the crystallinity of plastic films. Strong LED illumination was needed for this application.
Stereo Microscope Photography
The 1.3 MP Omax camera comes with an adapter, so it can fit an eyepiece tube of my Kyowa stereo microscope. In this setting the camera takes better photos than my 2 MP JEPCAM web camera. It can handle a wide range of light levels easily, as the hp35 calculator display photo below shows.
Hewlett Packard hp35 calculator display digit
This microscope is occasionally fitted with two circular polarisers set up so the total retardation is about 275 nm, or 137.5 nm per polariser. The photo below shows a mixture of cotton fibres, synthetic fibres and hair using this lighting.
Redwood hall dust sample, 4 mm field-width in polarised light with 275 nm retardation
Flat samples should be tilted upwards at the right by 7.5 degrees, if the camera replaces the left eyepiece. The sample will now be in focus from edge to edge. I have made a tilting sub-stage from an old computer access cover. I have also made a diffuse backlight from a small $2.75 LED key-ring light. This now runs off any USB port.
At 10X the horizontal field of view is 12 mm and at 30X it is, of course, 4 mm. For a good summary of stereo microscope photography Micro Tech Lab concisely covers the main issues.
Note that a normal compound microscope, using its lowest power objective, is a very nice incident-light microscope. All that is needed is to arrange some lighting. I use a single 11.8 volt recycled white LED mounted under the microscope arm so it illuminates the sample. In the photo below a bonding wire has partially failed creating a thermal intermittent. Occasionally one LED in a string of many will fail due to thermal stress and the forces applied when the fluorescent epoxy is added on top. The "hand made" construction of LEDs shows there is a long way to go in reliable design. Many small torches now have small "integrated" multiple LEDs with short bonding wires, so there is hope for better light bulbs. This view, using the 3.5x objective, is about 3mm wide.
White LED bonding wire fault
Digitech 5 MP USB Microscope
USB Microscope Upgrade
The focussing mechanism was a bit loose so I disassembled it and inserted shims to tighten it up. I placed the shims between the supporting screws and the body of the focussing mechanism. I extended the stand to a height of 280 mm using some 16 mm diameter polished aluminium tubing. This allowed the full camera focus range to be used. It is quite a good long working distance microscope. I added an old x-y stage to help with sample positioning. I drilled a new hole into the microscope base tapped at 40 TPI to take a 1/8 inch Whitworth bolt. I also drilled 1 mm diameter positioning holes.
For low magnifications, the addition of two $7 flexible LED lights has greatly improved the lighting. They have magnetic bases and a rectangular metal plate holds them in place. The built-in lighting produced reflections on some objects. I added 47 ohm resistors to the lighting circuit as the brightness was more than I needed. Plates made from some double sided circuit board were added below the battery holders. The 47 ohm resistors were each soldered into a notch, cut into one edge, to connect the copper faces together. This has improved the battery life from about 3 hours to more than 30 hours of continuous use.
ImageJ and Scale-Bars
This microscope is now a reasonably professional instrument. I can use ImageJ instead of the supplied software. I use the ImageJ scale bar tool along with two white marks added to the camera focusing wheel. The left hand mark is placed on the right edge of the focussing wheel when it is turned fully to the right. The right hand mark is placed on the left edge of the focussing wheel when it is turned fully to the left. The field width is 1600 Pixels. For various settings of the focus the field width in mm is:
The scale-bar shown below uses the highest magnification setting in transmitted light. The interval between bars is 0.01 mm.
Leitz Stage Micrometer
I have created a shortcut so I can just press "0" to start viewing.
The transparent shield on the nose of the microscope could be used to temporarily capture small insects for observation. It can also protect samples from the wind when doing field work. This only applies at the listed magnifications.
Pentax Macro Camera
My Pentax macro setup is shown at right. The bellows lens extension on the Pentax digital camera uses a reversed 28mm lens as the objective. Turning the lens around means the optical paths through the lens are similar to those in normal photography. The lens functions as the designer intended and image quality is high. Longer focal length lenses can be used for lower object magnifications. Normal microscope objectives can also be used. I use a special microscope objective which has a built-in diaphragm for depth of field control.
Illumination is provided by variety of low cost white LED light sources and diffusers. One LED power supply has been modified to run at 1.5 volts, using a switched mode oscillator. This is efficient and the light output only varies a little as the battery voltage falls. A second spot-light is a gooseneck LED lamp which plugs into a rechargeable USB lithium battery pack. I have two lights which can move with the camera while another pair of lights are stationary.
Canon Macro Cameras
I have macro adaptors for Canon A75, A590 and G5 cameras. These cameras fit onto a dedicated LED illuminator and closeup lens shown at right. This setup is small and is suitable for field use. The Plasma-Ashed lemon leaf and carrot leaf images were taken with a Canon A75 on this illuminator.
The G5 camera also mounts on my old Leica enlarger stand. I use a range of small LED lights and diffusers to provide illumination. In the photo a bubble level ensures all of the image is in focus. Alternatively placing a flat mirror on the sample table should result in a reflected and centered image of the camera lens. The camera is now parallel to the sample table, regardless of any other tilts.
Canon Powershot G5 macro camera
For photographing items, such as prints or books, I have improved the lighting which is now powered from a USB power supply. A heavy perspex window helps to flatten pages. To save the binding a book only needs to be partly opened, as shown below. A black card obscures any items above the window, except the camera lens. This eliminates reflections and improves contrast.
Two unusual Diatoms from New Zealand: Tabularia Variostriata a new species and Eunophora Berggrenii. Margaret A. Harper*, David G. Mann and John E. Patterson, Diatom Research (2009), Volume 24 (2), 291-306.
New diatom taxa from the world’s first Marine Bioblitz held in New Zealand: Skeletomastus a new genus, Skeletomastus coelatus nov. comb. and Pleurosigma inscriptura a new species. Margaret A. Harper*, John E. Patterson, John F. Harper, Acta Botanica Croatica 68 (2) 2009.
Oxygen Plasma Asher. J E Patterson. Analytical Chemistry, 51, 1978, 1087-1089
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Diatom - Pleurosigma angulatum, dark-field image, 40X objective, 2 MP JEPCAM
Diatom - Pleurosigma angulatum, oblique illumination, 63x objective, 2 MP JEPCAM
Leaf stem - Nerium Oleander, polarised illumination with dual compensators, 10X objective, Omax 1.3 MP camera
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Pentax macro camera
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Digitech 5 MP USB camera, Barrytown sand sample
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Cook, Troughton and Simms M6100 Greenough stereoscopic microscope
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