Light microscopy is one of, if not the most enabling tools in the field of biomedical research. Depending on your application, different modes of illumination are desirable for specimen viewing. This video will demonstrate two optical modes of transmitted light illumination, phase contrast, and DIC or differential Interference contrast microscopy.
Hello, I'm Victoria San Frolic from the core Optical Imaging facility at the University of Texas Health Science Center in San Antonio. Today I will show you how to properly view specimens using phase contrast and differential interference contrast microscopy. So let's get started.
Standard Brightfield microscopy is not adequate for viewing transparent and colorless specimens. Phase contrast microscopy is often used to produce contrast for transparent non light absorbing biological specimens. The technique was discovered by Zurich in 1942 who received the Nobel Prize for his achievement.
The phase contrast microscope is a bright field light microscope with the addition of special phase contrast objectives, which contain a phase plate or ring, and a condenser annulus instead of a diaphragm. The annulus is usually located in the condenser turret and the size of the annulus needs to be selected for different objectives. After setting up Kohler illumination, choose the proper phase objective and then rotate the appropriate phase annulus into position in the condenser.
Once the objective phase ring and condenser annulus have been properly aligned to be concentric and overlapping, we can view the specimen and properly adjust the focus. So let's demonstrate what phase contrast looks like through the microscope. Here's a look at a rat liver slice.
Under phase contrast. This method enhances the appearance of biological material by changing the contrast from different shades of gray to shades from black to white. You will notice however that specimens are surrounded by a halo, which obscures fine structure.
This halo is an artifact of illumination by the phase annulus. Now you'll notice when we switch to brightfield, this specimen is very difficult to view. Switching back to phase contrast, we can clearly see the structure and shape of the cells in the rat liver Tissue Since its introduction in the late 1960s.
Differential interference contrast or DIC microscopy has been popular in biomedical research because it produces high resolution images of fine structures by enhancing the contrasted interfaces, the image produced is of a very thin optical section. In fact, the ability of DIC to produce optical sections has made it a useful mode of transmitted light illumination to a company confocal microscopy. The DIC microscope is a bright field light microscope with the addition of the following elements, a polarizer between the light source and the condenser.
A DIC beam splitting prism in the condenser tart, A DIC beam combining prism just in back of the objective and an analyzer in the infinity space before the two blends. In order to obtain the best performance from DIC optics, it is important to first align the light microscope for Kohler illumination and then place the DIC elements in the optical path. Light from the source passes through a polarizer and is then split by the first prism.
These paired beams travel close to one another. If the pair both passed through the same material in the specimen, then as they're recombined by the second prism, they do not interfere with each other and therefore appear gray. However, at an edge of a structure, one beam of the pear passes through different material from its partner and is altered.
When these beams are recombined by the second prism, they will interfere either constructively producing a bright spot or destructively producing a dark spot. Here's a look at diatoms under DIC contrast. You will notice that fine detail of the diatom structure is readily visible.
However, if the DIC optics are removed, these details disappear. The contrast across the specimen is bright on one side and darker on the other. This gives the impression of topography, but in fact, this is an illusion.
The direction of the shadow cast effect can be reversed by turning the polarizer through a crossover point to the opposite Contrast. We've just reviewed the optical modes of phase contrast and DIC. Just to remind you, phase contrast elimination enhances the contrasted specimens from blacks to whites and is useful for viewing specimens that are both colorless and transparent.
DIC also generates contrast into specimen. It does so by creating a high resolution image of a thin optical section. So that's it.
Thanks for watching and good luck with your microscopy.
