Optical microscopes and white light / laser light

[Steve Drake]

I have a few questions regarding an optical microscope and their white light sources...

So white light generally first hits a diffuser, some kind of ground glass lens. What is the purpose of this?

Then the light goes through a field diaphragm, which we can open and close. I have heard that closing of the field diaphragm can slightly increase the spatial coherence of the light, why is this?

Lastly, why is white light used and not laser light? Wouldn't the almost perfect spatial and temporal coherence of laser light mean we get much better images? With that said, how does white light form an image as good as it does being so spatially and temporally incoherent?

Sorry for the barrage of questions, just been trying to wrap my head around this stuff.

Thanks

 

[Dr. Courtney]

The diffuser provides uniform illumination.

Laser light is monochromatic. White light will reveal all the colors of the object under the microscope.

Some specialized imaging applications do use laser light.

But white light is better when the detector is the human eye.

 

[Andy Resnick]

I have a few questions regarding an optical microscope and their white light sources...
So white light generally first hits a diffuser, some kind of ground glass lens. What is the purpose of this?

The diffuser plate is there in case the user doesn't know how to establish Kohler illumination. I have all my students read and walk through the process as written out here:

http://www.well.ox.ac.uk/_asset/file/zeiss-guide-to-microscopy-from-the-very-begiining.pdf

We remove the diffusers, as they waste light.

Then the light goes through a field diaphragm, which we can open and close. I have heard that closing of the field diaphragm can slightly increase the spatial coherence of the light, why is this?

There's some confusion here: the field diaphragm controls the field of view that is illuminated by the source. The aperture diaphragm controls the numerical aperture of the condenser illumination. Stopping down the aperture diaphragm will increase the contrast and depth of field and decrease the resolution. To be sure, slightly stopping down both diaphragms will slightly improve the optical performance (less glare, for example) because you aren't pushing the limits of the design.

Lastly, why is white light used and not laser light? Wouldn't the almost perfect spatial and temporal coherence of laser light mean we get much better images? With that said, how does white light form an image as good as it does being so spatially and temporally incoherent?

Widefield imaging works best with incoherent light. Kohler illumination is perfectly incoherent, phase contrast and DIC imaging start with incoherent light and increase the coherence in a controlled way to achieve optical processing. Using 'raw' laser light for wide-field imaging results in terrible images- there is an intolerable amount of speckle due to the low spatial coherence of the beam. Using a spatial filter will increase the spatial coherence and give better images than the raw light, but that's a pointless exercise. Again, if you are not doing widefield imaging (confocal, near field, etc.) then laser light is better as it can be better focused and coincidentally can be used with fluorescence techniques.

 

[Steve Drake]

Hi Courtney, Andy, thanks for your replies. Some questions,

Widefield imaging

I am not 100% familiar with this term, is optical microscopy widefield imaging?

Using 'raw' laser light for wide-field imaging results in terrible images- there is an intolerable amount of speckle due to the low spatial coherence of the beam.

By laser light I meant very high temporally and spatially coherence lasers.

there is an intolerable amount of speckle due to the low spatial coherence of the beam

I thought speckle only comes from a coherent source interacting with a random array of scatterers, can you clarify what you mean please?

Essentially what I am trying to understand is if you replaced a traditional white light source on an optical microscope with a laser, and sent it through a collimator, what kind of image would you obtain vs white light?

Thanks

 

[Andy Resnick]

I am not 100% familiar with this term, is optical microscopy widefield imaging?

'Widefield' means that the field of view is larger than a point, it's used to distinguish 'normal' microscopy from say, scanning methods.

By laser light I meant very high temporally and spatially coherence lasers.

A note of caution- the 'raw' laser beam does indeed have high temporal coherence, but typically only moderate spatial coherence- it's set by the size of the exit window. To increase the spatial coherence of the laser light, you spatially filter the beam- essentially creating a new source that is much smaller in extent, by using a pinhole aperture.

I thought speckle only comes from a coherent source interacting with a random array of scatterers, can you clarify what you mean please?

That's close enough- the point is that the size of the speckle is related to the coherence area. Clearly, magnifying the object will also magnify the speckle.

Essentially what I am trying to understand is if you replaced a traditional white light source on an optical microscope with a laser, and sent it through a collimator, what kind of image would you obtain vs white light?

Again, it depends of the details of the sources and how they are aligned within the optics. Goodman's "fourier optics" book has a fantastic image comparing the two approaches, but I couldn't find it on the interwebnet. Here's an alternative:

http://www.nature.com/nphoton/journal/v6/n6/images/nphoton.2012.90-f3.jpg