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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.
>>