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X Must-Have dark field vs phase contrast

dark field vs phase contrast

What Advantages and Disadvantages about dark field vs phase contrast?

What Advantages and Disadvantages about dark field vs phase contrast?

No one system is perfect, and dark field microscopy may or may not appeal to you depending on your needs.

Some advantages of using a dark field microscope are:

Extremely simple to use
Inexpensive to set up (instructions on how to make your own dark field microscope are below)
Very effective in showing the details of live and unstained samples
Some of the disadvantages are:

Limited colors (certain colors will appear, but they’re less accurate and most images will be just black and white)
Images can be difficult to interpret to those unfamiliar with dark field microscopy
Although surface details can be very apparent, the internal details of a specimen often don’t stand out as much with a dark field setup.

Below are contrasting examples of dark field (left) versus bright field (right) illumination of lens tissue paper. Note how they both create a different style of image.

Dark field illumination Bright field illumination

Admit it, by now you’re curious to check out your own dark field! You can create one with minimal time and effort. Just read on…

dark field vs phase contrast

How to made the dark field vs phase contrast?

How to made the dark field vs phase contrast ?

It is very easy to make dark field vs phase contrast yourself. What you have to do is place an opaque round stop in the condenser. An easy way is to cut a piece of black paper and put it on a filter in your filterholder. You can put the stop on a piece of clear acetate sheet. You can even try to draw the stop on it with black paint. The most important thing is to have it big enough to stop all light going directly into the objective. Only the light that is reflected by the objects in the sample reaches the objective then. Stronger objectives are more difficult because their NA is often too high. The NA of your condenser should always be higher then the NA of the objective. If patch-stops of 8, 10, 12 and 15mm are made you can’t go wrong really. For objectives of around x10 the middle sizes prove best.If you like to make the patchstop as precise as possible: The best way is to set up as normal (brightfield), remove the eyepiece and close/open the substage iris until it is *just* visible. Then, either bending your neck over double, or carefully removing the condenser, measure the diameter of the iris diaphragm as it is now set. A pair of calipers is useful here. This diameter is that for the patch stop. Very often, to be on the safe side it is best to add about 10% to this figure, this avoids leakage, especially if you have no means of centering the stop in the filter holder. If you have a phase contrast condenser, the largest phase contrast annuli often make excellent patch stops for darkfield!The real connoisseurs must have recognized the skills of Klaus Kemp in the arranged (cleaned) diatom slide photographed by Mike Samworth.

dark field vs phase contrast

What is dark field vs phase contrast?

Brightfield microscopy uses light from the lamp source under the microscope stage to illuminate the specimen. This light is gathered in the condenser, then shaped into a cone where the apex is focused on the plane of the specimen. In order to view a specimen under a brightfield microscope, the light rays that pass through it must be changed enough in order to interfere with each other (or contrast) and therefore, build an image. At times, a specimen will have a refractive index very similar to the surrounding medium between the microscope stage and the objective lens. When this happens, the image can not be seen. In order to visualize these biological materials well, they must have a contrast caused by the proper refractive indices, or be artificially stained. Since staining can kill specimens, there are times when darkfield microscopy is used instead.

In darkfield microscopy the condenser is designed to form a hollow cone of light , as apposed to brightfield microscopy that illuminates the sample with a full cone of light. In darkfield microscopy, the objective lens sits in the dark hollow of this cone and light travels around the objective lens, but does not enter the cone shaped area. The entire field of view appears dark when there is no sample on the microscope stage. However, when a sample is placed on the stage it appears bright against a dark background. It is similar to back-lighting an object that may be the same color as the background it sits against – in order to make it stand out.

dark field vs phase contrast

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