Microscope: Aperture, Light Source Impact on Image Contrast/Depth/Light

[superwolf]

How will changing the aperture of the condenser of a microscope and the power of the light source change the image when it comes to contrast, depth of field and amount of light that reaches the camera?

 

[Monique]

I think this tutorial can be very helpful to you: http://www.olympusmicro.com/primer/anatomy/condensers.html

 

[superwolf]

Nice.

Allowing less light to go through the aperture of the condenser, we get more contrast. Price: smaller area in focus and lower depth of field. Open aperture: Better DOF. Bigger area in focus. Lower contrast. Can compensate with more intensity from the light source if the image becomes too dark.

Correct?

 

[Andy Resnick]

Not exactly. First off, let's state that the microscope is aligned either for critical or Kohler illumination conditions. For Kohler illumination, the source is imaged at the back pupil plane of the condenser and the front focal plane of the condenser is coincident with the sample plane (Critical illumination is essentially equivalent, but the source is imaged onto the sample plane).

Then, the numerical aperture of the condenser, which is adjusted by the aperture diaphragm (located at the back pupil plane), sets the contrast of the image (under conditions where the NA of the objective is much larger than the NA of the condenser). Low condenser NA corresponds to higher low-frequency contrast but lower resolving power. The illuminated area is set by the fireld diaphragm,which is located at a plane conjugate to the sample plane: the field diaphragm is imaged onto the sample plane.

Under critical/Kohler conditions, Lamp brightness doesn't affect image quality (within reason- the color balance can also be affected), and surprisingly, condenser aberrations do not affect image quality either.

The DOF is set by the objective lens, not the condenser. Likewise, field flatness is set by the objective lens.

Are you having trouble with a particular application?

 

[superwolf]

Thanks!

Are you having trouble with a particular application?

No, I'm just trying to get things correct in my light microscope lab report.

 

[superwolf]

Are these statements correct?

1. If the resolution of a light microscope is too low, one can decrease the wavelength of the lightsource, or increase the NA.

2. Phase contrast mode is related to bright field in that the background is bright and features causing light to scatter give raise to image contrast, but in addition one makes use of the phenomenon of phase shifting to give additional contrast.

3. Bright field mode gives better resolution than phase contrast mode.

4. Living cells and other aquatic materials are almost transparent when illuminated because the refractive index is close to that of the surroundings of the specimen.