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>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Jim, you've just referred to backscatter confocal imaging as 
>something akin to darkfield confocal, and someone else earlier today 
>compared it to reflection interference microscopy. Which mode of 
>microscopy is backscatter imaging more like then? Is possible to 
>interpret the contrast in a backscatter image? I've tried 
>backscatter imaging a few times with adherent fibroblasts, and the 
>images do show interesting features, but how am I to tell what 
>causes one region of the image to look bright and another to look 
>dark? It's a very complicated light pattern often.
>
>John Oreopoulos

All good questions John.

To start with, the bad news: the BSL (actually it really is reflected 
light!) from the interface of the coverslip to the medium gives a 
HUGE signal. So huge that it actually sets up interference patterns 
with the incoming light. So I don't try to do BSL close to this 
interface.

And you DO have to use the Anti-flex pol techniques to eliminate a 
background signal from light reflected back at all the out-of-focus 
optical surfaces in the system. (This light is always there, but is 
usually removed by the barrier filter on the basis of its wavelength.)

I use the term darkfield because it fits: the undiffracted ray 
doesn't enter the detector, only scattered light does. More to the 
point it produces an image that is almost identical to widefield 
darkfield.

The thing that is different is that, as lasers are far more coherent 
than WF light sources, you get a lot of interference effects in 
confocal BSL than in widefield darkfield when imaging features that 
are large and smooth with respect to the wavelength of light.

So it  works best with objects that are small and at least a few 
microns away from the nearest glass-medium interface.

The contrast is simple: Brightness depends on particle size (until 
the particle is big enough to be "resolved") and varies with the 
square of the difference of its RI from that of the medium. It is 
slightly annoying that features that have a lower RI look just like 
those having a higher RI but if what you want is "structure," the BSL 
image can't be beat. And its free. No additional light need strike 
the specimen.

Cheers,

Jim P.

>On 6-Nov-07, at 5:16 PM, James Pawley wrote:
>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Hi all,
>>
>>Yes, scanning the specimen does make transmission confocal almost 
>>possible: as long as the refractive index of the specimen is 
>>entirely constant. (i.e., the specimen is dead and stained)
>>focal plane and the coverslip on the "in" direction than they are 
>>on the "out" direction, then even if you initially  line up the 
>>illumination optics to coincide with the detection path, you will 
>>find that this overlap will change as the specimen is moved past 
>>the spot.
>>
>>Consequently, the amount of signal actually passing through the 
>>detection pinhole depends not only on how much absorbing material 
>>is located at the focus of the laser but also (negatively) on the 
>>degree of misalignment. i.e. the output signal is no longer 
>>dependent on events occurring at the focal spot and therefore the 
>>resulting data no longer can be thought of as coming from an 
>>optical section.
>>
>>There have been suggestions that this "dynamic misalignment" 
>>problem could be avoided if the detector were not a fixed pinhole 
>>but a mini-image recording device (say a 5x5 CCD). As this spot was 
>>displaced from the axis by the inhomogeneities of the specimen, it 
>>would be tracked, the centroid of the spot located and its 
>>intensity measured. Sort of a movable pinhole to track the 
>>displaced spot to keep the whole system "confocal".
>>
>>This would be neat (In fact is was one proposed uses for the 
>>"EM-CCiode" detector proposed by me and others) but it would be 
>>really complicated to sort out the data. Probably for this reason, 
>>it has not been done.
>>
>>The other reason is that it really isn't clear what useful 
>>information would be obtained from the transmitted light signal 
>>that could not be obtained far more easily by detecting 
>>Backscattered light. Using any laser line, BSL will give you a 
>>lovely, fully-confocal  darkfield image that shows all 
>>inhomogeneities as white.
>>
>>For more  information, you might look at the chapter on 
>>Transmission Confocal by Carol Cogswell in the Second edition (but 
>>not the Third) of the Handbook.
>>
>>Cheers,
>>
>>Jim P.
>>
>>--
>>               **********************************************
>>Prof. James B. Pawley,               		            Ph.  608-263-3147
>>Room 223, Zoology Research Building, FAX  608-265-5315
>>1117 Johnson Ave., Madison, WI, 53706 [log in to unmask]
>>3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada
>>Info: http://www.3dcourse.ubc.ca/	     Applications due by March 15, 2008
>>	       "If it ain't diffraction, it must be statistics." Anon.


-- 
               ****************************************
Prof. James B. Pawley,               		   Ph.  608-263-3147 
Room 223, Zoology Research Building,                         FAX  608-262-9083
250 N. Mills St., Madison, WI, 53706  [log in to unmask]
"A scientist is not one who can answer questions but one who can
question answers."  Theodore Schick Jr., Skeptical Enquirer, 21-2:39