Re: Objective for confocal

Posted by Nowell, Cameron on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Objective-for-confocal-tp590639p590641.html

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Hi List,
        This is always a fun topic:)

I should quantify the test we carried out here.


The tests were carried out on the following lenses on an Olympus FV1000 single photon, non-spectral confocal
- 63x NA 1.42 UPlanApo UIS2
- 100x NA 1.4 UPlanApo UIS2


The reason for the test were that we had a lab pushing to get a 100x objective because of the increased magnification. The tests were carried out to demonstrate that the 100x lens would be more detrimental to their samples (ie bleaching) and would not provide any greater resolution.

The tests were carried out on drosophila embryos that were mounted in glycerol

The end result was that laser power had to be boosted, leading to faster bleaching, to get an equivalent image with the 100x when compared to the 60x. This is all based on our confocal and result may be different for other machines and lens combinations.



Cheers


Cam





-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stanislav Vitha
Sent: Tuesday, 30 October 2007 2:26 AM
To: [hidden email]
Subject: Re: Objective for confocal

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Cameron,
I will be inetrested in the test report on the two Olympus lenses. Were your tests done in the wide-field or confocal mode?
Our Olympus confocal had a 60x/1.42 PLAPON objective that turned out to be substantially worse in resolution than we expected from theoretical calculations. After testing several more of the same type we switched to the 100x/1.4 Universal Plan Apo (Thanks to our local Olympus office I was able to pick the best one among several of those objectives). It showed much better resolution and less chromatic aberration across the visible spectrum. I can e-mail test results to those who are interested (both mirror slide and sub-resolution beads were used).

Regarding the signal intensity - I thought that in the point-scanning confocal mode, there should not be a big difference in excitation intensity and signal detected between different magnification objectives of the same NA - the size of the illuminated spot is determined by NA, as well as the amount of fluorescence signal collected. Since all the collected fluorescence signal is focused to a single spot in the pinhole plane, there again should not be much difference between, say, 60x and 100x objective, providing the pinhole size is set to the same Airy size
and all other things are the same. Am I missing something fundamental?  
 
Stan

Dr. Stanislav Vitha      [hidden email]
Microscopy and Imaging Center
Texas A&M University
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On Sat, 27 Oct 2007 11:08:29 +1000, Nowell, Cameron <[hidden email]> wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hi Ann,
>           To follow up from Julio's comments. The higher NA will give
you improved resolution. A 1.42 NA object theoreticatly can resolve down to 210nm at 488nm exitation. That holds true for what ever the magnification the objective has.
>
>We have compared a 63x 1.42 NA to a 100x 1.42 NA objective. Both were
Olympus UIS2 PlanApo objectives. Both could resolve the same structures.
But the big difference was the amount of light that was needed to capture an image with the 100x objective. On average 7x more light input was required to get image with the 100x lens that was equivalent to the 63x lens.
>
>So my advice woudl be to get a 63x 1.42 NA objective, or better yet a
>40x
1.4NA if you can find one (they are being made by Nikon i think but not sure about others).
>
>Of course this is only important if you want to b e able to resolve
>very
small structures. If you do not want to be resolving down to 210nm then your 40x oil and 63x water imersion objective will be just fine.
>
>I can send you (and anyone else on the list) a copy of the reoprt with
the comparison of the 63x vs the 100x lens if you want.
>
>
>Cheers
>
>Cam
>
>
>Cameron Nowell B.Sc (Hons)
>Microscopy Imaging and Research Core Facility Peter MacCallum Cancer
Centre

>7 St Andrews Place
>East Melbourne, Victoria 3002
>
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>Mobile: [phone]
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>________________________________
>
>From: Confocal Microscopy List on behalf of Yang, Ann-Fook
>Sent: Sat 27/10/2007 6:09 AM
>To: [hidden email]
>Subject: Objective for confocal
>
>
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hi All,
>
>I am new in confocal microscopy.
>We have a 40x/1.3 oil and a 63X/1.2 water objectives. Is there any

advantage to add a 100X/1.46 Oil objective?

>
>Ann Fook Yang,
>Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada
>EM Unit/ Unite EM Edifice K.W. Neatby Building, 960 Carling Av /960
>Boulevard Carling, Ottawa,Ontario K1A 0C6
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>[hidden email]
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