by Dr. Robert Berdan
July 4, 2017
Above is a basic student microscope which costs between $100-$300 used. This is a good starter microscope but it only provides basic white illumination. If you own such a microscope you can enhance its ability by adding Polarizing filters, Rheinberg filters and creating Darkfield and Oblique illumination for very low cost.
When you buy your first microscope and begin looking at live cells by scraping the inside of your cheek (see bottom pictures), or view pond organisms the first thing you notice is that they hard to see let alone photograph. The problem is contrast - most of the small animals are "bags of water" and appear translucent. To overcome this you can fix the cells and stain them, but looking at dead cells is never as satisfying as viewing live ones. There are other methods to enhance contrast optically that are low in cost which I describe below.
Submandibular glands, tissue section stained with Eosin and Heamtoxylin. These type of microscope slides can be purchased and are great for teaching Histology, but preparing such slides requires a microtome, and staining expertise. Most student microscopes will work well with prepared slides. Bright field illumination 200X.
Haematopinus suis (female) 40X Rheinberg illumination using central blue filter and outer red filter.
To observe live cells or arthropods there are various methods in microscopy to increase contrast and make them easier to see. Some of these methods like Phase contrast, and Differential interference contrast are expensive and only some research quality microscopes have them. In this article I will describe Rheinberg lighting, Darkfield and Oblique lighting which are easy and simple to implement. If you take photographs it also helps to work with an image editing program like Adobe Photoshop to clean up the photos, remove debris, adjust white balance, stack the images, merge serveral images and enhance contrast. I shoot RAW files in my camera in order to have maximum flexibility in processing the images. The main challenges in getting sharp pictures with your microscope are eliminating camera vibration and using a fast shutter speed to stop the rapid movement of some organisms.
Pediculus humanus captis male (Head louse) photographed with Rheinberg lighting, 40X
Naididae - a freshwater oligochaete with 2 anterior eyes (Nais Pseudobtusa ?) viewed with Rheinberg lighting 40X
Rheinberg filters are named after Julius Rheinberg who proposed his Multiple Colour Illumination Method to increase contrast of unstained specimens in microscopy in 1896 at a Quekett Microscopical meeting. A set of Rheinberg filters can be purchased on E-bay starting around $25 and you can easily make them your self with coloured acetate or a colour laser printer.
Above on the left are three ordinary colored filters 32 mm diameter, and to the right are a set of Rheinberg filters which I purchased from E-Bay for about $40 US. The central disc colour if black creates a black background (Darkfield) and while the outer colour bends as it passes through parts of the specimen to light up some features. You can make any combination of colours. I find that using complimentary colours e.g. blue-yellow, red-cyan, green and magenta work best, but its fun to experiment. The filters provided by E-bay are about 1\8 inch thick Plexiglas and if you put them on top of the light source I recommend you place them on top of a clear glass or UV filter to absorb the heat. The best place to put them is directly under the condenser, but not all condensers have a filter holder. In general I find they work well only with low power microscope objectives 1.3X, 2.5X, 4X and 10X. Some condensers work better then others I have a special condenser for my low power objectives (Olympus). You can make these filters yourself and I recommend Mike Shaw's Book "How to Make Multi-coloured Filters: Rheinberg, Polarizing, Darkfield and Oblique" for those that would like to try it - you can purchase his book from Amazon.ca. Mike describes how to use a compass cutter, but one can also just use a pair of sharp scissors or sharp knife. Coloured acetate sheets can be purchased from office supply stores, Amazon and many other places.
Rheinberg filters work by creating a narrow outer beam of light (red) and a central stop of colour. The central stop results in the background colour and the outer ring of colour is refracted (bent) by the specimen. Diagram courtesy of Berklee education.
Above is a Nauplius larvae for a Copepod (Cyclops) using Rheinberg lighting - central green disc and yellow outer disc. I used a 10X microscope objective. Total magnification 100X
Above is the same Nauplius larvae photographed with Phase contrast (Positive Low) with a 20X objective. total magnification 200X.
Above diagram showing how to insert Polarizing filters in your microscope - courtesy of Zeiss.
Above is a Nauplius larvae photographed between Polarizing filters. The larvae has a single red eye and in the dark produces a red light beam (OK I am kidding I added the light beam with Photoshop :-) ). The muscles are birefringent so they glow between polarizers and the red eye is clearly visible. 150X.
Above shows a ribbon worm (Nemertean) photographed with different Rheinberg filters which are shown in the lower left corner of each frame. 4X objective.
Wool fibers photographed with 10X Objective using a Yellow and Blue Rheinberg filter
Bottle brush pores (Fern pollen) 100X using a Rheinberg Filter with central blue and outer red colour.
Pediculus humanus captis male (Hair louse) photographed with Rheinberg lighting, 40X stitch of 3 images.
Pine Wood section with Rheinberg filter, red outer and blue inner. 40X
Diatoms from prepared slide 200X Rheinberg lighting - blue central filter with Orange outer filter
Diatoms with Rheinberg Illumination - black central disk and outer blue disc
Rotifer with Darkfield illumination (clear disk with back inner circle). A coin placed on a clear or blue filter works to create Darkfield though you will need to experiment with coins of different diameter with different objectives.
To create oblique illumination in your microscope is easy. You can simply place a black or opaque object to cover about half the light source or if your condenser has a filter tray, swing it so it blocks some of the light. Vary the position and you will see the subjects take on a 3D appearance. You can combine this with the Rheinberg filters to create some interesting effects. I sometimes use a glass slide with black electrician tape. Other microscopists experiment with different shapes of opaque substances to create the oblique lighting - see some of my web links below for examples.
Rotifer showing a 3D relief, below the Rotifer is a strand of Spirogyra algae. I used a Rheinberg filter with light blue central stop and pushed my filter tray part way to cover the light and create oblique illumination. Oblique illumination can resemble Differential Interference Contrast (DIC) microscopy which costs thousands of dollars to add to a microscope. Still - I like DIC better and hope to get DIC for my microscope in the future.
Voticella growing in a patch on algae, photographed with Negative Phase contrast microscopy - 200X
Hoffman Modulation - a form of oblique illumination with polarized light. I have one objective 40X that I can produce this simulated 3D effect but again it's not a substitute for DIC microscopy. The images are also in appear in grey scale with little or no colour.
One of my cheek cells at 400 X Hoffman Modulation Contrast. To get cheek cells use the edge of a microscope slide and scrape it inside your mouth then spread out the cells on another slide and add a coverslip. In bright light illumination they will be hard to see - try oblique illumination. Cheeck cells are easy to see with phase contrast as shown below. There are two types of phase contrast: Postive which makes refractile objects dark and Negative Phase contrast which makes refractile objects bright- see below.
My cheek cells viewed in Negative Phase contrast 200X. In Positive Phase contrast the nucleui and mitochondria appear dark - see below. Phase contrast is an expensive microscope add on.
Cheek cells with Positive Phase contrast microscopy approx 175X.
All my photographs are taken with a Nikon D800, 500, Canon 80D or 5D Mark II using free software called Digicam control to capture the images to my laptop. In the future I will be experimenting with Polarized light combined with Rheinberg illumination, fluorescence and other methods to enhance the birefringence of specimens like Diatoms and Rotifers. There is an incredible variety of microorganisms that live in ponds, streams, lakes and rivers around my home so I have an abundant source of specimens to view. RB
References and Additional Links.
Julius Rheinberg (1896) Original Note on Coloured Illumination "Rheinberg filters" - PDF
E-bay where you can purchase Plexiglas Rheinberg filters
Rheinberg filters by Mike Shaw - also see his book on how to make them yourself
How to make Rheinberg filters by Mike Shaw
Easy to make contrast filters for the microscope by Wim van Egmond
Making Rheinberg Illumination discs - shows how to determine the central disk diameter
Rheinberg Illumination by Chuck Huck
Oblique Illumination by Dave Walker
Paedia Optic Multifilter - simulates Rheinberg and other lighting types digitally - neat but expensive
Rheinberg Illumination by Molecular Expressions Microscopy
Rheinberg Illumination using fiber optic lights
How to make microscope filters - www.microbehunter.com
What is Rheinberg illumination? -microbehunter.com
Oblique Illumination in microscopy - Nikon
Oblique Illumination in microscopy - Olympus
Microbehunter Magazine Issue 48 PDF - digital condensor simulates Rheinberg Illumination
Rheinberg Illumination - Berklee
Slide Presentation of different microscopy methods to create contrast by Weismann - PDF
Hoffman Modulation Contrast at Olympus Microscopes
Hoffman Modulation Contrast at Wikipedia
Phase Contrast Microscopy at Wikipedia - compares cheek cells in brightfield and phase contrast
Differential Interference Microscopy - Wikipedia
Dr. Michael Shribak - developing microscopy methods to boost birefrigence - see his Rotifer photographs
Polarized light imaging of birefringence and diattenuation at high resolution and high sensitivity - for Geeks only
Also see my other articles on Microscopy:
The Art & Science of Photomicrography with Polarized Light
Microscopic Life in Ponds and Rainwater
Photographing Through a Microscope Photomicrography - Inner Space
Robert Berdan is a professional nature photographer living in Calgary, AB specializing in nature, wildlife and science photography. Robert offers photo guiding and private instruction in all aspects of nature photography and Adobe Photoshop training.
Email at: firstname.lastname@example.org
Web site: www.canadiannaturephotographer.com
Phone: MST 9am -7 pm (403) 247-2457.
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