Re: Objective for confocal

Posted by vb-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Objective-for-confocal-tp590639p590651.html

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For what conditions the high NA oil immersion lenses (w/o the correction collar) are optimized, room temperature or Live Cell Imaging (at 37 deg)? I recently compared NA 1.45 versus NA 1.49 (with the correction collar), and haven't noticed any difference on fixed samples of 125-150 nm fluorescent virus particles (at RT).

 

vitaly

nci-frederick

 

  

----- Original Message -----

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To: [hidden email]

Sent: Friday, October 26, 2007 8:07 PM

Subject: Re: Objective for confocal

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I quite agree with what Julio said.  There is one more

point, though.  The NA 1.46 lens is presumably a 

TIRF lens, and so may be handy if one later wants to

add TIRF capabilities.  As such, it will probably also

have a correction collar, and if this is used correctly it

will significantly improve performance.   At high NA,

minor differences in coverslip thickness and room

temperature will noticeably impact on resolution

unless they are corrected for.

 

                                                          Guy

 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez
Sent: Saturday, 27 October 2007 9:10 AM
To: [hidden email]
Subject: Re: Objective for confocal

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello Ann, 

there may be different opinions on this, but here is my take:

With a confocal microscope, you can add magnification by zooming in and matching the image size (pixel dimensions) for optimal resolution. This means you can get 100 x magnification with a 40x at zoom 2.5, or a 63x at zoom 1.6.

What the 100x/1.46 gives you in theory is greater resolution (20-30% more), since resolution increases with the square of the numerical aperture. In practice, you may not always achieve this extra resolution, unless you have quite bright samples that are relatively thin and properly mounted, in order to minimize spherical aberrations. 

You also need to consider the type of objective and the application. PlanApos are corrected for more colors, and therefore are best for precise colocalization of up to four dyes. Fluars or similar objectives are corrected for fewer colors, but tend to be brighter than PlanApos, because they are simpler lenses with less glass... these are often better for live-cell imaging, or for samples with fewer colors...   for a more precise description, you may want to check the tutorial on lenses and chromatic aberrations here:

http://micro.magnet.fsu.edu/primer/java/aberrations/chromatic/index.html

We typically use objectives in the 40x-60x range for most of our confocal applications (in addition to a 10x or 20x for low-mag work, and possibly a long-working distance 40x for thick/unusual samples). We rarely use a 100x, except maybe to look at yeast cells, or some very fine cellular detail.  A 100x/1.46 lens will often be less bright than a 40x/1.3, which means that your gain in resolution may be offset by increased bleaching. In addition, a 40x is more versatile since you can image a wider field of view at zoom 1, and then get a close-up at zoom 2-3 if you need more mag, without having to switch lenses.

A 100x/1.46 lens, on the other hand, would be quite nice on a widefield microscope... also, this type of lens with high NA is what you need for applications such as TIRF (total internal Reflection microscopy)...

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

Tel: Office: 206-667-1215/ Lab: 206-667-4205

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On Oct 26, 2007, at 1:09 PM, Yang, Ann-Fook wrote:

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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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