40x vs. 60x
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Hello all,
I have checked the intensity loss by imaging a fluorescent plastic block with both LUMPlanFI/IR 40x/.8 W and UplanApo/IR 60x/1.2 W through Z axis.The drop of 60x is the way faster that 40x.As I know with the higher NA I should have better light collection.
My question is:
Could it be because of the fact of higher NA higher sensitivity to the mismatch and higher SA?
or these two lenses are not comparable as 40x is a dipping lens?
Any information is highly appreciated.Thanks
Sarah
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Search the CONFOCAL archive at
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Brightness = 10 to the 4th x (Numerical
Aperture squared/Magnification squared)
So far three lens:
63x/1.4NA = 9.65
100x/1.4NA = 3.8
40x/1.3 NA = 18.9 thus 4x brighter than 100x and
twice that of 60x.
From Inoue and Spring, Video Microscopy, The Fundamentals,
2nd Edition page 137, 138
Mike Ignatius
Molecular Probes/Invitrogen
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sarah Kefayati Sent: Friday, May 09, 2008 2:55 PM To: [hidden email] Subject: 40x vs. 60x Hello all,
I have checked the intensity loss by imaging a fluorescent plastic block
with both LUMPlanFI/IR 40x/.8 W and UplanApo/IR 60x/1.2 W through Z axis.The
drop of 60x is the way faster that 40x.As I know with the higher NA I should
have better light collection.
My question is:
Could it be because of the fact of higher NA higher sensitivity to the
mismatch and higher SA?
or these two lenses are not comparable as 40x is a dipping
lens?
Any information is highly appreciated.Thanks
Sarah
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In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
= Sarah, your 40x/0.8 and 60x/1.2 are likely to be about the same brightness under ideal conditions (see Mike's post). On the other hand, the 40x/0.8 dipping lens is likely to be more resistant against spherical aberrations as you image deeper into the sample (it is designed to image through a couple of millimeters of water anyway, so a few microns more or less won't make much difference), while the 60x/1.2 will be much more sensitive to spherical aberration (and loss of luminosity), because it is corrected to get the optimal image near the coverslip surface. If you are imaging deeper than that, you may improve your results by adjusting the correction collar that is probably present on that type of lens, but if you are imaging a thick slice, then you can't avoid SA at all positions, unless perhaps you get a spherical aberration correction device from 3I (http://www.intelligent-imaging.com/spherical/main.php). I have no special interest, and don't know how well it works, but the article referenced (and linked to) in that page may be worth reading. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On May 9, 2008, at 2:55 PM, Sarah Kefayati wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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In reply to this post by Sarah Kefayati
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 >Brightness = 10 to the 4th x (Numerical >Aperture squared/Magnification squared) > >So far three lens: > >63x/1.4NA = 9.65 >100x/1.4NA = 3.8 >40x/1.3 NA = 18.9 thus 4x brighter than 100x and twice that of 60x. > >From Inoue and Spring, Video Microscopy, The Fundamentals, 2nd >Edition page 137, 138 > >Mike Ignatius > >Molecular Probes/Invitrogen Hi all, I think that Mike quotes Inoue and Spring correctly. (My copies of this book are at the UBC Course.) However, some sources quote a the fourth power NA dependence and this is really only warranted if the image of the illumination source that should be present in the BFP of the objective is of sufficient size to fill the BFP. This is not always true. In addition, it really only refers to the "apparent brightness to the eye", rather than some overarching "measure of optical goodness." Whatever you choose, I sometimes feel that these equations are a bit misleading as they seem to have led some to conclude that lower mag must be better for fluorescence. Period.. At a fixed NA, the diameter of the BFP is inversely proportional to the objective magnification. Once you know the size and shape of the image of the source formed by the illumination system in the BFP, you can figure out what fraction of it will pass through the BFP of each lens. (On an inverted scope with a dry lens, you can see the relation between the source image and the lens NA by holding a piece of lens tissue a few cm above the lens. If the illumination is Kohler-aligned, you you should see an image of the arc. The dark circular border represents the rated NA of the objective.). More than 85% of any light passing the BFP will pass the lens and illuminate the object. However, in the same way that the area of the BFP (and hence the amount of light passing through it) is inversely proportional to the square of the magnification, then the area of the specimen that is illuminated is also inversely proportional to the square of the magnification. So assuming that the same image of the arc is present in the BFP of both 40x and 100x lenses, the photons striking each square micrometer of the specimen will be the same. Assuming a uniformly-fluorescent specimen, the amount of signal that makes it back to a given pixel of the CCD camera, will be inversely proportional to the objective magnification. So superficially, the 40x will be about 6x brighter than the 100x. However, when we are recording a digital image, this is not the end of the story. Nyquist sampling theory says that we want the dimensions of each pixel of the CCD (referred to the specimen plane) to be about 2.5x smaller than the resolution limit of the optical system. This means that, for a given NA (and wavelength), there exists a "best" total magnification from the specimen to the plane of the CCD, and if your 40x lens doesn't provide enough then you should increase the magnification of the camera-coupling lens until Nyquist is happy. Alternatively, if your CCD pixels are two small, you can "bin" them 2x or 3x to make effective pixels that are 2 or 3 times larger in linear pixel dimensions. In any case, as long as the CCD pixels are the same size, when referred to the specimen, the NA is the same, and the image of the arc fills the BFP of the lower mag objective fairly uniformly, then signal recorded in one pixel of the CCD will be the same. (i.e., the objective mag is irrelevant because the total mag is fixed by Nyquist.) So why would you choose one over another? The 4Ox will look brighter "by eye" although not necessarily 38x brighter or even 6x brighter, because the image of the arc in the BFP usually not the optimal size. The 40x will also have a larger field of view making it easier to find the "good" part of the specimen. On the other hand, the image will be smaller and harder to "see" and you will bleach a larger area of the specimen while you are scanning around. Finally, if you fail to use the field diaphragm to limit the illumination to include only that part that is imaged by the CCD, you will have a lot more light reflecting around off optical surfaces, contributing to background haze and reducing image contrast. In addition, as it is much more difficult to correct all of the many aberrations over the larger field of view of the 40x, it will probably only have "diffraction-limited" performance over the central area. By the same token, there may be fewer optical compromises in the 100x objective, although the 2.5x shorter focal length may impose limits on the working distance. Many people will base their choice on whichever lens comes closest to Nyuquist sampling with their favorite CCD. (Cameras based on the popular SONY chips have 6.7 micrometer pixels, while those using the E2V 512x512 EM-CCD chip, preferred by many for disk scanning and TIRF, has 15 micrometer pixels.) All of this says nothing about spherical aberration that occurs when one focuses a dry or an oil lens into fluorescent plastic etc. Cheers, Jim Pawley ********************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 [hidden email] 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications still being accepted for Waitlist "If it ain't diffraction, it must be statistics." Anon. >From: Confocal Microscopy List >[mailto:[hidden email]] On Behalf Of Sarah Kefayati >Sent: Friday, May 09, 2008 2:55 PM >To: [hidden email] >Subject: 40x vs. 60x > >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >Hello all, > >I have checked the intensity loss by imaging a fluorescent plastic >block with both LUMPlanFI/IR 40x/.8 W and UplanApo/IR 60x/1.2 W >through Z axis.The drop of 60x is the way faster that 40x.As I know >with the higher NA I should have better light collection. > >My question is: > >Could it be because of the fact of higher NA higher sensitivity to >the mismatch and higher SA? >or these two lenses are not comparable as 40x is a dipping lens? > >Any information is highly appreciated.Thanks > >Sarah -- -- |
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