Olympus BH2 Series Podręcznik - Strona 6

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Figure 9 – Place green/yellow filter into filter receptacle

Congratulations, you have now properly setup your

BH-2 microscope for phase contrast viewing.

Using Phase Contrast

The hard part is now done. From here on out, to use

phase contrast, just be sure to always match the

number on the selector dial on the front of the phase

contrast condenser with the magnification of the

selected phase contrast objective. That is basically all

there is to it. Don't forget to return the selector dial on

the phase contrast condenser to the "0" setting

whenever you wish to switch back to regular brightfield

viewing.

Periodically Verify Centering of the Phase Annuli

It is good practice to re-check the centering of the

phase annuli before starting a phase contrast

observation session. With practice, you will come to

see that once the phase annuli have been centered,

they rarely need to be re-adjusted.

About the Phase Contrast Filter

Note that it is necessary to use the green/yellow filter in

the illumination system, in order to provide light of the

proper wavelengths with which the phase contrast

objectives were designed to operate. This is critical if

you wish to obtain optimal phase contrast results. This

filter is particularly important when you wish to

perform photomicrography, since that's when image

quality is most critical. The inevitable green/yellow cast

that the filter imparts may be removed from the images

by converting them to grayscale.

For routine viewing, however, you may find that you

prefer to omit the green filter from the illumination

system, knowing that the phase contrast effects will be

less than ideal, but the resulting color rendition will be

much closer to normal.

Phase Contrast on the Olympus BH-2 Microscopes

Phase Contrast Artifacts

Certain visual artifacts will inevitably appear when

viewing specimens under phase contrast, which can

sometimes make it difficult to accurately interpret the

resulting images. The bad news is you really cannot get

rid of these artifacts. You will find that certain

specimens are ideally suited for phase contrast,

whereas others are better suited to regular brightfield

viewing, because of these artifacts.

Halos: The most obvious phase contrast artifacts are the

halos which you will see surrounding your specimens.

These halos are caused by some of the diffracted light

from the specimen passing through the phase ring in

the objective. Ideally, only the background illumination

should pass through the phase ring, and only diffracted

light should pass through the areas inside and outside

the phase ring. In positive phase contrast imaging (i.e.,

when using PL or PLL phase contrast objectives), this

effect causes larger objects have a brighter edge,

whereas in negative phase contrast (i.e., when using

NM or NH phase contrast objectives), this effect causes

them to have a darker edge.

Shade-Off Effect: Another phase contrast artifact is

known as the Shade-Off Effect.

homogeneous parts of the image show up at the same

brightness level as the background (i.e., the same as the

surrounding medium). This occurs because although

the light experiences a phase shift as it passes through

these regions of the specimen, only minimal diffraction

occurs and the angle it scatters is therefore limited,

causing it to pass through the phase ring and therefore

not experience the desired interference.

Contrast Inversion: A third phase contrast artifact is

known as Contrast Inversion. In the case of positive

phase contrast, objects with a high index of refraction

situated next to objects with a low index of refraction

will appear brighter than the background, instead of

darker. This happens because in these cases the phase

shift is not the usual quarter wave that should occur,

and instead of the expected destructive interference

occurring, constructive interference occurs instead. The

opposite of this is true for negative phase contrast.

How To Contact the Author

Please feel free to direct any questions or comments

regarding this document (or BH-2 microscopes in

general) to the author as listed on the cover page of this

document.

Revision 1

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