In 1896, a British microscopist, Julius H. Rheinberg demonstrated a form of optical staining to the Royal Microscopical Society and the Quekett Club.

This technique, called “Rheinberg illumination”, is a variation of darkfield illumination using colored glass or gelatin filters to provide colors and contrast to both the specimen and the background.

It offset the need for chemical staining which does not always provide the desired contrast, necessitates the killing of the specimen and often interferes with the live conditions and vital processes of the micro-organisms.

 

How does it work?

As said before, the Rheinberg illumination technique is a kind of darkfield illumination called optical staining. The substage condenser is arranged so that the light path from the lamp source will pass through the object at oblique angles. An objective’s numerical aperture is reduced with a light stop, iris or funnel stop. Consequently, the only light which can enter the objective is light refracted, reflected or diffracted by the object when the oblique rays strike the specimen. So, it appears bright on an black background increasing the visibility of  thin or small details.

If we put colored filters (Rheinberg filters) in the condenser filter holder, we shall get a colored background corresponding to the color of the filter inserted.

 

Mikropolychromar description

In the 1930s, the German optical company “Carl Zeiss Jena” developed such an optical staining apparatus called the “Mikropolychromar”. The optical staining it achieved presented an advantage over ordinary darkfield illumination in that specimens are not only differentiated from the background by their relative brightness but with contrasting colours.

By means of this optical contrast staining device, the finest details and structures could be revealed with a remarkable distinctness. In fact, the “Mikropolychromar” combined the advantages of brightfield and darkfield with striking colour contrast.

In the diagram below, we can see the path of the rays through the “Mikropolychromar”.

 

Section through the Mikropolychromar

Path of the light rays

Controls and light path rays :

• 1.         Aperture diaphragm for the marginal rays (12) with the nickel plated knob (1)
• 2          Aperture diaphragm for the axial rays (11)
• 2 a       Aperture diaphragm for the axial rays (11)
• 3.         Auxiliary diaphragm for adjusting the intensity of the axial rays (11)
• 4.         Colourless heat absorbing protective glass
• 5.         Frosted glass and UV protective shield
• 6.         Interchangeable colour glass rings which colour marginal rays (12)
• 7.         Interchangeable colour glass rings which colour axial rays (11)
• 8.         Milled head by means of which the apparatus can be swung out sideways:

That means with a slightly eccentric position it effects a transition to a semi-bright, semi-dark field illumination (Close to oblique illumination.)

• 9.         Rotation of the whole apparatus around optical axis
• 10.       Aplanatic condenser N.A 1.4 into which the Mikropolychromar is screwed
• 11.       Path of the axial rays
• 12.       Path of the marginal rays

 

  Mikropolychromar (1) fitted with its illuminating lamp (2). (Carl Zeiss Jena document )  :

 

 

 Mikropolychromar in its 1935/36 case (Document © Alain BERJONVAL)

 

Rheinberg rings, filters and light stops

Mikropolychromar apparatus

 

Annular Rheinberg filter rings.

(Document © Alain BERJONVAL)

 

How to use it?

 

Mikropolychromar fitted on a 1933 Zeiss F or G stand. Three levers controlled the three diaphragms.

(Document © Alain BERJONVAL)

 

The apparatus is introduced to and screwed onto the aplanatic N.A 1.4 condenser, then observations can be started immediately once the illumination has been adjusted. Manipulation is very simple, there are no difficulties or tricky adjustments. Different kind of illumination effects can be achieved in the shortest possible time with few operations.

Due to ease of illumination, I fitted a “Mikropolychromar” on a Leitz Ortholux I with this self-made Leitz dovetail apparatus:

 

(By courtesy of Mr. Roger Bluteau)

 

 

On Ortholux I

 

Left. Fitted beneath Ortholux I mechanical stage.

Right. Mikropolychromar fitted on condenser holder

   

A few trials

 

 

Conclusion

The Zeiss Mikropolychromar was developed during the 1930s, from an illumination technique demonstrated by Julius H. Rheinberg in 1896.

It combined in one apparatus all illuminating techniques known, (bright, darkfield illumination, and even oblique illumination), with optical staining.

It allowed the scientific user to deal with colourless and poor contrast specimens.

It anticipated the new illumination concepts such as phase contrast (Phaco), demonstrated by Zernike in the 1930s. (The first phase-contrast microscope prototype was made by Carl Zeiss Jena in 1936) and interference microscopy with a very simple technique based on Rheinberg illumination. Maybe, only the Leitz “Heine” condenser, can offer comparable versatility.

The purpose of this article is also to demonstrate the ability to use old illumination techniques like Rheinberg illumination or oblique illumination as a cheaper alternative to phase contrast and interference microscopy.

 

 

Bibliography

Carl Zeiss Jena (Zeiss documents)

Optical Microscopy by Michael W. Davidson & Mortimer Abramowitz

Leitz dovetail apparatus by : Mr Roger BLUTEAU

Photos and documents  :© A. BERJONVAL