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I am getting more confused the more I think about this. If you use a
lower NA lens, you get a larger excitation spot in X,Y and Z, but
presumably a symmetric increase, and you have the same NA for emission
so you collect a lower resolution (larger) voxel worth of fluorescent
light at each point in the scan and need to open the pinhole to collect
it all. If you underfill a high NA lens, you create a lower NA
excitation spot, but you collect the fluorescence at full NA of the
objective. I am still unclear about the implications of this last
point but it seems important and I had never thought about it. Also,
presumably, these two methods of decreasing NA are going to give you
different results. Does the excitation spot profile increase equally
in X, Y and Z if you underfill the objective? I had the impression
that it increased in Z but not as much in X, Y. My mental analogy was
sending an infinitely thin laser beam straight through the objective
which would presumably emit the same thin beam. That would mean you
would lose axial resolution but not so much lateral. If the effect is
non-uniform, then the "airy disk" becomes asymmetric and now closing
the pinhole presumably has complex effects on X-Y vs Z resolution. Dave
On Wednesday, December 18, 2002, at 12:13 AM, Guy Cox wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Juat to ad a bit to Jim's comment, with which I agree entirely ..
>
> From the details of your post you are obviously using a Leica system
> and the way their software calculates '1 Airy' is a little eccentric.
> Only the value it sets with the beam expander set at 6 is the actual
> maximum-aperture Airy disk value - if you use a lower beam expansion
> it recalculates for the smaller illumination NA and therefore sets a
> larger '1 Airy' To paraphrase Star Trek "It's logic, but not as we
> know it".
>
> The beam expander value the software sets should be correct for the
> spefied lens in confocal mode, but if you are doing multiphoton work
> the Ti-S laser beam comes in at a larger diameter and you should use
> one settting less on the expander (3 if it sets 6, or 0 if it sets 3).
>
> Guy
>
>
>>> i was wondering if anyone could tell me the relationship the beam
>>> expander, and the pinhole have on z-section thickness. i understand
>>> turning the beam expander off gives a less confocal image, and using
>>> it
>>> at setting 3 or 6 decreases the z-section thickness, respectively.
>>> i'm
>>> trying to figure out what effect that has on setting the pinhole on
>>> one
>>> or two airy (or whatever airy). i also understand decreased airy
>>> yields decreased z.
>>>
>>> is there a mathematical equation to determine the z section based on
>>> mag, beam expander, and pinhole?
>>>
>>> thank you for your help in advance.
>>>
>>> gary
>>>
>>> open minds open worlds
>>>
>>> Gary Laevsky
>>> Department of Molecular and Cell Biology
>>> University of Connecticut
>>> U-125, 75 N. Eagleville Rd.
>>> Storrs, CT 06269
>>>
>>> v (860) 486 - 3449/1895
>>> f (860) 486 - 4331
>>
>> Hi Gary,
>>
>> Depending on the size of the BFP of the particular objective used,
>> not expanding the beam, may result in the BFP not being filled with
>> laser light. If this occurs, the objective operates as though it had
>> the NA that IS filled by the light bundle, and a lower NA produces a
>> larger spot in the x,y and z directions and also one that has (much)
>> less peak intensity.
>>
>> As the objective does operate at full NA on the light coming back
>> from the specimen, the "resolution" in this direction should be only
>> set by diffraction and your pinhole should be set accordingly
>> (probably to one Airy-unit).
>>
>> However, if you have underfilled the objective on purpose,
>> specifically to increase the optical section thickness (for instance,
>> when looking at a Ca-indicator dye in a living tissue-culture cell),
>> then you would do well to also increase your pinhole diameter. The
>> extra signal will reduce statistical noise, making your
>> Ca-concentration measurement more accurate. As the x-y spatial
>> resolution is probably limited more by diffusion of the Ca than by
>> the optics, its reduction will not be as important.
>>
>> There are lots of equations but not usually in the terms you suggest.
>> Just remember that x-y resolution is limited by NA, z-resolution by
>> (NA)*2 and the beam expander can reduce NA. (see discussion of how to
>> measure the NA that your optical system is actually working at, see
>> posting under: "Re: measuring laser power for all" in the archive
>>
>> 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]
>> "A scientist is not one who can answer questions but one who can
>> question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39
>
>
> Assoc. Prof. Guy Cox, ooOOOOOOoo
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>
Dr. David Knecht
Department of Molecular and Cell Biology
U-125
75 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)
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