Workshop on contrast enhancement

Saturday 9th April 2022

Quekett members can watch a video of this workshop.

Phil Greaves welcomed the participants and then gave a brief introduction to the various methods that have been used to enhance the contrast of images seen through a microscope without staining. He emphasised the importance of setting up the illumination correctly and went through the steps to achieve Köhler illumination. This is designed to produce bright and uniform illumination, which is especially important for taking photographs through a microscope

Phil Greaves’ introductionPhil Greaves’ introduction

You can see the slides from Phil’s introduction here:

Click the arrows to move through the slides. Click the symbol at bottom right for a larger version.

Stephen Parker gave us a detailed introduction to dark-ground illumination, which can be achieved with simple stops or with specialised dry and immersion condensers.

Stephen ParkerStephen Parker

You can see the slides from Stephen’s presentation here:

Click the arrows to move through the slides. Click the symbol at bottom right for a larger version.

Carel Sartory gave a presentation on Rheinberg illumination, which developed from dark-ground illumination. The dark central stop can be replaced by a coloured filter, to show a specimen against a coloured background. The stop can be surrounded by a contrasting colour filter, which adds colour to the specimen. As with dark-ground, the size of the stop needs to be an appropriate size for each objective. Carel has recently found that the Rheinberg discs do not have to be close to the iris diaphragm in the substage condenser; they also seem to work when placed on the light output on the base of the microscope.

Carel showed his images and specimens live on various cameras, but he has kindly collected several of them into this PowerPoint:

Click the arrows to move through the slides. Click the symbol at bottom right for a larger version.

Quekett members can watch a video of the 2021 Christmas Lecture “Julius Rheinberg – His contribution to microscopy” by Carel.

There is an article on the website by Carel on Making Rheinberg illumination discs.

Chris Thomas gave us a thorough introduction to phase contrast, which normally requires a special condenser and special objectives. The condenser contains annuli that only allow light to pass through a bright ring, one annulus for each objective magnification. The objectives contain a dark ring, and the annuli have to be aligned with the rings using a phase telescope or a Bertrand lens that provide a good view of the rear focal plan in the objective. Phase contrast can be positive (subject darker than the background) or negative (subject lighter than the background).

Chris ThomasChris Thomas

You can see the slides from Chris’s presentation here:

Click the arrows to move through the slides. Click the symbol at bottom right for a larger version.

Chris also showed a video of pond life using dark-ground, annular lighting and negative (anoptral) phase contrast:

It is possible to obtain phase contrast without buying special objectives and condensers, and Graham Matthews has written an article on DIY Phase Contrast.

Phil Greaves gave a talk on differential interference contrast (DIC), which can produce spectacular images but is very expensive. It requires a special condenser containing Wollaston prisms, and second Wollaston prisms above the objectives.

Phil GreavesPhil Greaves

You can see the slides from Phil’s presentation here:

Click the arrows to move through the slides. Click the symbol at bottom right for a larger version.

There were also some gossip exhibits, but not enough time for members to see them all.

Alan Wood brought the phase condenser and 4 phase objectives from his Olympus BHT microscope and mounted them on the Club’s Olympus BHC microscope so that he could demonstrate negative and positive phase contrast.

Alan Wood’s exhibitAlan Wood’s exhibit

Differences in refractive index in the specimen cause phase shifts in the light, and phase contrast makes these visible by changing them to differences in intensity.

Olympus made 4 different types of phase contrast objectives for the BH-2 and other microscopes of that generation; PLL (positive low low), PL (positive low), NM (negative medium) and NH (negative high). Positive phase contrast makes the subject darker than the background, and the phase of diffracted light is retarded by 90°. Negative phase contrast makes the subject lighter than the background, and the phase of undiffracted surround light is retarded by 90°

Olympus phase objectivesOlympus 10× PLL, PL, NM and NH phase contrast objectives

Olympus phase ringsPhase rings in Olympus 10× objectives

PLL and NH phase contrastNegative high (NH) and Positive low low (PLL) phase contrast

Alan also showed two ways to obtain dark-ground illumination for stereomicroscopes and macro photography that do not use optics.

One method uses an inverted LED ring-light with the aperture filled with black card. The other method uses an LED stage plate with a disk of black card obscuring most of it, leaving just a bright ring.

In both cases, the slide or specimen is placed on a piece of black foam board (with a central hole to let light through) resting on an inverted pudding basin from which the base has been cut out.

Construction of dark-ground illuminatorDark-ground illuminator, showing (left) black disc, (centre) inverted basin with base removed, and (right) the complete illuminator

If the lens of your stereomicroscope does not have sufficient working distance to accommodate a pudding basin, adding a 1.5× supplementary objective may allow it to fit.

Olympus SZ4045 stereomicroscope with dark-ground illuminationDark-ground illuminator using an LED stage plate

Zoea larva of porcelain crabZoea larva of porcelain crab (Porcellana platycheles), slide by N.B.S.

Dark-ground mosquito larvaLarva of a mosquito (Culex pipiens), slide by T. Gerrard & Co.

Dandelion seedSeed of dandelion (Taraxacum officinale)

Report and photographs by Alan Wood

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