|From Brightfield to
(Part 2) .....The Many Facets Of brightfield
A personal Odyssey
By Paul James
Brightfield'seffectiveness at revealing structural form in any semi-transparent specimen has almost as much to do with the relationship between mountant and specimen as with its dependency on diffraction etc.. Of course regardless of this and other things we are obliged to scrutinise the specimen beneath a coverslip because of conventional optic design parameters regarding objective performance. No matter for beneath the coverslip the rules no longer apply and therefore we can mount the specimen in any medium we choose to enhance refractivity differentials, or of course we can opt for 'dry' mounting. The sum total of variations of mounting mediums, each of differing refractivity for a given specimen, changes the overall contrast observed at the interface between the mountant and the specimen. Such properties can be used to advantage especially with specimens of the more transparent type like diatoms etc.. Of course the business of image contrast enhancement is not new. It can for instance be realised in obscure fields of research such as imaged with Schlieren photography, where very subtle changes in the refractive index of air/liquids brought about by pressure disturbances or heat, can be grossly exaggerated.
Clearly then the brightfield image is far more complex than it at first appears. It is basically a compromised fabrication from the effects of the properties of light and the interference of the specimen, with of course a little help from the substage condenser iris diaphragm. Unfortunately, in percentage terms, the imaged proportion of light transmitted to the eye is very small, but the remainder, the excess, impairs the discriminatory powers of vision. Yet this wholly unwanted remainder, the glare, cannot be easily removed. It is basically this latent thorn in brightfield microscopy which brought about modifications of lighting techniques and so brings me to my next point.
For me the most interesting aspect of the raw brightfield image is that it is actually the sum total of a multi layered combination of lighting effects which are now well known. Thus we can isolate from the brightfield image Oblique, Darkfield, and COL by the use of specific stops. If for instance a x20 objective is being illuminated with a cone of light from the condenser whose na is well in excess of the objective's, then clearly the excess would generate the darkfield effect but for the fact that the central lower na cone images by transmission simultaneously and therefore totally floods the Darkfield component. If on the other hand the central zone of the ascending light from the source is blocked by a centralised circular stop so allowing only the peripheral cone to enter the objective, then a noticeable improvement in contrast and detail of the specimen ensues which is a basic form of COL, but the darkfield component still exists in this scenario but again is flooded out in the COL imagery. Obviously to raise Darkfield both the COL and low na BF cones of the illuminant have to be blocked by the so called DF stop. Each of these 3 illumination techniques require only a little further tweaking to elicit maximum effect.
The asymmetry of the Oblique lighting technique puts it into a category of its own, since the others make use of symmetrically disposed stops. It is probably the oldest Brightfield lighting modification, possibly evolving from the earliest days of compound microscopy, and can be induced crudely, yet somewhat effectively, by blocking a little of the source below the condenser with a finger tip, which is probably how it came to be discovered. Oblique can be very effective, though judging the best position and shape of stop is a matter of trial and error and knowing where the condenser's anterior focal plane is for very best results. Despite its relative crude generation, the 'science' of oblique's unique image forming process isn't at all easy tounderstand. To my knowledge, the last specifically designed optic to raise Oblique illumination was manufactured in Hungary in the 1950's and appeared to be a standard substage condenser, but with inbuilt stops and semi silvered filters etc.. Despite its effectiveness throughout the entire amplification range of the microscope, it wasn't manufactured for very long, and by all accounts has become a rarity.
All these basic but most useful lighting techniques are brought about by selectively blocking off zones or sections of the source with or without the inclusion of colour or polar filters to improve matters. In the final analysis, the effectiveness of the chosen illuminant is dependent on both the thorough knowledge of technique and its matching the specimen. Practice makes perfect.
In conclusion the brightfield image is clearly a very complex one, yet can be 'tuned' so effectively by placing the simplest of stops to tease out the desired component. Brightfield's Phase Contrast and DIC are very effective too but are in one sense complex entities in themselves requiring separate optical parts or modifications that exaggerates low level phase shifts already present in cellular imagery. Whilst these can be very effective it has to be paid for. I will not expand on this here because they are not natural components of the raw brightfield image.
To be continued.......
|All comments welcome by the author Paul James
Microscopy UK Front Page
Published in the July 2009 edition of Micscape.
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