The Two Biggest Problems With Biological Tissue Examination Techniques Using Phase Contrast Microscopy
Histology is the study of cells and tissues with the aid of microscopic devices. While there are various biological tissue examination techniques using phase contrast microscopy utilized today, all of them are victims to the two biggest problems of interpreting phase contrast images: halos and shade-off.
Halos and Shade-Off – Image Distortion Produced from Phase Contrast Microscopy Halos and shade-off are natural occurrences produced by phase contrast but they are nevertheless commonly referred to as image distortions. These two form patterns that do not directly match thickness value and refractive index between the biological tissue and the rest of the sample.
SHADE-OFF – Another type of image distortion in phase contrast microscopy, shade-off is commonly seen when specimens are large and possess extended phases. Shade-off also appears more frequently than halos in images of flattened tissue culture cells. Shade-off occurs when the intensity of an image is negatively affected by phase advancement or retardation, overlap size of the condenser annulus, size and absorption of the phase plate. Also, there are times when images simply end up with intensities that aren’t in parallel with the optical path difference of the sample.
Shade-off is also sometimes known as zone-of-action since shade-off occurs when zones in the center with the same thickness values produce a different light diffraction with those produced by the zones found at the sample’s boundaries and edges.
HALOS – In phase contrast microscopy, circular phase-retarding occurs in your microscope’s objective phase plate. Besides producing surround waves, it also inevitably transmits a tiny degree of diffracted light from the biological tissue sample. That ring of diffracted light is what people refer to as halo. This problem is made even worse because the one of the surround waves projected by your microscope’s phase subcondenser is smaller than the actual size of your microscope’s plate ring. Because of this, the halos can sometimes make it more difficult for the user to view the specimen.
There are certain types of specimens in which phase contrast halos become more apparent. When samples are large and possess low spatial frequency like cells and its nuclei, halos become more prominent. Light energy redistribution is also important. Sometimes, light can distributed in such a way that most of it serves a destructive purpose rather than a constructive purpose; in other words, light energy distribution can make halos more trouble than they usually are. When the images have large halos with high contrast, they can create confusion and cause the user to misidentify and misinterpret the images.
Halos are not however entirely evil. There are times when users in fact welcome their presence such as in the case when microorganisms like bacteria are being observed. Bacteria are small and practically invisible to the naked eye and even to ordinary microscopes. Phase contrast microscopy however uses the varying thickness of the specimen to create contrasts and distinguish the microorganism from its surroundings. Halos are also helpful in negative phase contrast microscopy: with it, scientists make use of dark halos to see low frequency images more clearly.
There are various ways to reduce the problems caused by halos in phase contrast microscopy. Adjusting light diffraction and phase shift may reduce the size of the halo surrounding your image. Another and simpler way of reducing halo woes is by changing the refractive index of the area surrounding the specimen by using agents like serum albumin, mannitol, or even glycerol. Depending on the sample you’re observing, the use of such agents could generate a reverse contrast image, illuminating dark colored specimen without greatly changing background intensity.
One more way of reducing the halo effect in an image is by using custom-designed phase objectives that use smaller rings. These are called apodized phase contrast objectives and when in use, they can make images of your biological tissues appear with enhanced clarity and great detail.
Use of amplitude filters can also reduce halo size and intensity. When they are attached adjacent to your microscope’s phase film, light transmission is modified to ensure that little or no halos are produced
Halos and shade-off will always ultimately depend on the optical and geometrical properties of your microscope’s phase plate and of course the characteristics of your biological tissue specimen. Halo and shade-off are also greatly affected by the objective magnification you’re using; generally speaking, it’s safer to use lower magnification when observing biological tissue specimens with phase contrast microscopy.
Now that you know the two biggest problems with biological tissue examination techniques using phase contrast microscopy and how to solve them, you’ll be able to make more accurate observations and hypotheses critical to your study.
