Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
There's the theory and there's practice.
For flat cells (e.g. fibroblasts) adhered to a glass substrate in cell
culture media our experience has been higher resolution and more light with
a higher NA oil objective than with a lower NA water objective.
At 09:33 AM 09/07/06 -0400, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>I don't have a copy of the book yet either (soon) but am curious how
>others adjust the collar on a water lens. I have found it difficult
>at best. I have been unable to convince myself that the water is
>better than the oil within 10um of hte coverslip, but I am willing to
>be convinced (especially for the money we paid for that lens). Is
>the procedure is the same for all brands of lenses? Dave
>
>Dr. David Knecht
>Department of Molecular and Cell Biology
>U-3125
>91 N. Eagleville Rd.
>University of Connecticut
>Storrs, CT 06269
>860-486-2200
>860-486-4331 (fax)
>
>
>On Sep 6, 2006, at 3:41 PM, Reece, Jeff (NIH/NIEHS) [E] wrote:
>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Yes, of course the reflection off the interface is a useful
>>marker. The
>>marker can be seen even better by just switching to a different
>>emission
>>filter that lets in more excitation light, so why is it necessary
>>to see
>>it in a channel that is supposed to be designed for fluorescence?
>>
>>I'm one of these folk that likes to test theory when it is
>>important to
>>know, and there's a big apparatus with lasers and blinking lights
>>across
>>the hall. And I actually know how to adjust the dang collar. So what
>>exactly does "essentially touching the glass" mean? <500nm? Sorry,
>>haven't gone out to buy the book yet. :)
>>
>>Cheers,
>>JEff
>>
>>
>>>-----Original Message-----
>>>From: James Pawley [mailto:[log in to unmask]]
>>>Sent: Wednesday, September 06, 2006 2:01 PM
>>>To: [log in to unmask]
>>>Subject: Re: High NA objectives for confocal microscopy
>>>
>>>---------------------- Information from the mail header
>>>-----------------------
>>>Sender: Confocal Microscopy List
>>><[log in to unmask]>
>>>Poster: James Pawley <[log in to unmask]>
>>>Subject: Re: High NA objectives for confocal microscopy
>>>--------------------------------------------------------------
>>>-----------------
>>>
>>>Search the CONFOCAL archive at
>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>>>Search the CONFOCAL archive at
>>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>
>>>>To reduce the reflections due to the laser light on the
>>>coverglass using the
>>>>Olympus FV300 we typically would put an ND filter in place
>>>to reduce the
>>>>light to between 5 and 20% of the incident power. Then we
>>>would use the AOTF
>>>>control to choose our power. Typically for live cell work we
>>>use 0.1% of the
>>>>488 nm line of a 40 mW Ar laser. So attenuating to 20% gives
>>>us 5x more
>>>>sensitivity with the AOTF now controlling the power from 0%
>>>to 20% rather
>>>>than 100%. It turns out AOTFs are good attenuators but not
>>>great blockers of
>>>>laser light so this will block reflections from both the 488
>>>nm and the 514
>>>>nm lines (as well as weaker lines). I'm not sure other
>>>confocal microscopes
>>>>still have ND filters in the light path but this is certainly a
>>>>cheap solution.
>>>>
>>>>Claire Brown
>>>
>>>It might be worth pointing out that, in the event that you don't need
>>>to collect a fluorescent signal in its presence. (i.e., you are
>>>interested in structures farther into the specimen), the reflection
>>>from the coverslip interface can be a useful marker for the location
>>>of the this interface.
>>>
>>>And in relation to the subject of the original post, using an NA 1.4
>>>objective will make it worse, at least it will as long as the laser
>>>light fills the full input pupil (practically, that the laser beam
>>>diameter is larger than that of the "glass" as the back of the
>>>objective). This is because the fraction of light reflected by the
>>>glass/medium interface depends not only on the RI's of these two
>>>layers but also on the angle. As the angle of incidence increases
>>>beyond that corresponding to NA 1.2, the fraction reflected increases
>>>markedly, reaching 100% at NA larger than about 1.33.
>>>
>>>To emphasize what Guy and others have said: unless the structure of
>>>interest is essentially touching the glass, there is NO advantage to
>>>using "the larger NA". a) because it really isn't larger when you
>>>account for all the light lost to reflection at the interface and b)
>>>because SA decreases the resolution so much that the peak brightness
>>>of a point object is lower.
>>>
>>>Some folk persist in thinking that this isn't so, perhaps because a)
>>>large objects (i.e, those not near enough to the resolution limit to
>>>be blurred by SA) will appear a little brighter, b) they have not
>>>taken the time to adjust the collar on their water objective for the
>>>coverslip thickness (not so important for an oil lens as the oil and
>>>the coverslip have about the same RI.) and consequently, the image
>>>from the water lens is aberrated and hence dim or 3) they have read
>>>somewhere that the brightness in widefield fluorescence varies with
>>>the fourth power of the NA. The idea is that both the illumination
>>>and the collection of light vary with the square of the NA. This is
>>>true for the illumination as long as the image of the arc in the BFP
>>>actually fills the pupil, that the "high-NA light" is not lost by
>>>reflection at the coveslip interface and you don't use a very small
>>>field diaphragm aperture. It is not true for the collection if the
>>>larger NA produces SA.
>>>
>>>If you are working with living cells, get the water lens and learn
>>>how to adjust it.
>>>
>>>This topic is so important that it rates an entire chapter in the new
>>>handbook as well as being discussed in many other chapters. There is
>>>also a long discussion of the construction and features of notch
>>>filters, particularly whose fabricated using the new "hard" coatings.
>>>
>>>Cheers,
>>>
>>>Jim P.
>>>--
>>> ****************************************
>>>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
>>>"He who can get you to believe absurdities, can get you to
>>>commit atrocities."
>>> Voltaire.
____________________________________________________________________________
Michael Cammer Analytical Imaging Facility Albert Einstein Coll. of Med.
URL: http://www.aecom.yu.edu/aif/
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