Testing two Olympus "relief phase contrast" objectives on an Olympus inverted microscope
Comparison with Hoffman modulation contrast (and DIC)

By Daniel Nardin, France

In 2009, I was intrigued by Olympus objectives marked 'RP' on eBay and I wanted to know more about them.
They correspond to a technique called "relief phase contrast" whose description I found on an advertising brochure (ref. 1). Intrigued, I acquired them and I tested this technique before comparing it to others found useful under the same circumstances. I used an inverted microscope with plastic culture dishes which do not work with interference contrast.

I) Material

I am equipped with Olympus microscopes and for a long time, my main model is  the BHS. With the help of internet auction sites, I have added accessories and also bought other second-hand microscopes.

I bought in particular in the United States a few years ago, an Olympus CK2 inverted microscope. It is 110V but I added a 110-220V transformer for use in France!

 CK2 Olympus

I used it first with objectives A4Pl, A10PL with the phase rings supplied and a LWD20PL (this last one has a collar for regulation of thickness of object cover 0 - 2mm).


As remarked at the start of this article, last year I
bought two Olympus objectives:

10x achro RP NA 0,25 and
40x LWDCDPlanFRP NA 0,55

Note that the 40x is designed for a thickness of object cover of 1mm!
While the 10X is for a classic 0,17.

objectif RPC olympus

I made rings for phase corresponding to those in the original condenser of the CK2 but this one had an insufficient opening for the 40X.


Now at the same time, I bought second hand two partial Hoffman Modulation Contrast sets for the Nikon Diaphot. So I have a whole set of lenses and two Nikon condensers of NA 0,5.

One was associated with the objectives for contrast of Hoffman:
Plan 10x 0,25
LWD20x 0,40
LWD40x 0,50

It is now my usual set on this microscope.
     

objectifs HMC

 

The other condenser with four openings in turret, serves for do-it-yourself work and in particular for this relief contrast. I made phase rings with cardboard. 

condenseurs HMC diaphot adaptés CK2

A brochure associated with the Olympus CK microscope (ref 1) gave me a model to make the phase rings adapted to two objectives for this "Relief Phase Contrast ( RPC)".

principe RPC Olympus

It is necessary to note that the brochure dedicated to the more recent microscopes CKX (ref 2) describes a different technique "Relief Contrast" which seems very close to the contrast of Hoffman ( HMC).
 relief contrast 

Did the patents of this technique become exploitable by Olympus? I have no details of the story, but my tests by setting up the two techniques cover two generations of equipment.


2) Results of the tests

To study the techniques images were taken with a digital Nikon camera that show.

the classical oral epithelium  cells.

Pictures are all resampled for 500 pixels length.


1st series (scale 0,5 = 2 photosites for 1 pixel in the image):
Phase Contrast                                                                                             "Relief phase contrast" Olympus
10x








40x

comparaison phase et relief phase contrast

2nd series (this time the full field with the projection NFK2,5x eyepiece which was used, is kept):

Phase "Relief phase contrast" Olympus
10x phase 10x 10x relief phase
40x phase 40x 40x relief phase
At 40x, I also compared the images with Hoffman modulation and differential interference contrast, but I did not retain the same field after change of microscope.
40x HMC 40x 40xCI
Hoffman modulation contrast Differential interference contrast

2nd test subject: only using the 40x objectives, were observations of a drop of liquid floating of natural yogurt to see bacteria responsible for the processing of the milk: long lactobacteria  and chains of streptococci, among globules of fat
Phase Contrast Relief phase contrast
phase relief phase
40xHMC 40xCI
Hoffman modulation contrast Differential interference contrast

Now, three examples on the same line for easier comparison:
40x Phase 40x relief phase contrast 40x Hoffman
Phase Contrast Relief phase contrast Hoffman modulation contrast
Lastly, a comparison with the bacteria of the yogurt, this time, to the scale 1/2 
phase
Relief phase
HMC
Phase Contrast Relief phase contrast Hoffman modulation contrast

3) Discussion
These techniques are consumers of light. A good part of the cone of lighting is blocked but there is a phase plate or a modulator in the objective. 

The images in RPC have an overall similar look to phase contrast, but the halo is present only with the latter one. It gives a light impression of relief but which has nothing to do with that seen with DIC. This technique RPC seems to me to bring little benefit. It was nevertheless proposed recently (2007) by a working doctor in Romania Joerg Piper (ref. 3).

It seems that Zeiss (ref. 4) also used a similar idea for a while (Variable relief contrast = Varel) before developing in 2003 an interference contrast method suitable for plastic dishes i.e. PlasDIC. (Ref. 5)

Differential interference contrast ( DIC) is much preferred every time we can use it.

The Hoffman contrast works with the plastic material dishes incompatible with DIC. It is the current technique with my Olympus microscope, named "relief contrast" by Olympus.

The techniques based on an oblique illumination are however interesting because they have only a low cost and can be implemented by amateurs.

The PlasDIC would certainly be interesting to try but it is reserved for Zeiss equipment. I wonder however if it would not be possible to realize it with a prism IC for reflected light? Perhaps it is a new aspect to explore by do-it-yourself.

Daniel Nardin.  Please feel free to e-mail me  if you have any questions or comments.

 


Bibliography and Internet references :

(1) Olympus relief phase contrast Brochure CK :  http://www.iolympus.cz/mikroskopy/prospekty/Relief%20phase%20contrast.pdf

(2) Olympus relief contrast Brochure CKX:  http://www.iolympus.cz/mikroskopy/prospekty/CKX2.pdf

(3) Relief phase contrast  http://www.relief-phase-contrast.com/index.html
Piper  J. : Relief-Phasenkontrast - eine universell einsetzbare Modifikation des Phasenkontrasts mit verbesserter Bildqualitšt, Mikrokosmos 96 / 4, 223-229, 2007

(4) Zeiss Varel  
http://www.zeiss.de/C1256F8500454979/0/877D55B12C3A9095C1256F8E003506DE/$file/varel.pdf

http://www.smt.zeiss.com/C12567BE0045ACF1/Contents-Frame/B14DDA7D7E2CA1D9C1256FB6002A5EA1

(5) Zeiss PlasDIC  http://www.zeiss.de/C12567BE00472A5C/EmbedTitelIntern/Article-PlasDic_e/$File/GIT_Arcticle.pdf


 

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