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Yes, nm.
Julio, you are right about lateral resolution. My reasoning is that
although the optical performance does not depend on the structure, 150 nm
is supposed to be good enough to sample structures of 500 nm. I would like
to avoid too much oversampling.
I don´t know how Biorad calculated that 2.6 zoom is the maximum advisable
for this objective (it is a shame, but I never worried before about
sampling rate, resolution, etc)
Many thanks for your help.
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello All,
>
> To avoid more confusion, you both meant nanometers, not micrometers, when
> talking about xy-, z-resolution and z-stepsize, correct?
> E.g., "optimal" xy resolution is 200 nm, not microns, with pixel size at
> 80
> nm with Nyquist, 160 nm z-stepsize, etc.
>
> Zoltan
>
>
> On 2/9/08, Julio Vazquez <[log in to unmask]> wrote:
>>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal = Hi Pedro,
>>
>>
>> I am not sure how you or Biorad are calculating the optimal pixel sizes,
>> or what the specific constraints for the Biorad 1024 are. For a
>> 60x/1.4NA
>> objective, the theoretical resolution in x/y at 580 nm (if you are using
>> a
>> red mito tracker) is around 200 microns (I am using the formula d = 0.5
>> x
>> lambda/ NA). Therefore, your optimal pixel size, if you use a Nyquist
>> factor
>> of 2.5, should be around 80 microns. Obviously, you may get slightly
>> different values depending on how your choice of wavelength.
>>
>>
>> Regarding the sampling along the z axis, it is not so much the
>> dimensions
>> of the structures (mitochondria) that matter, but the optical properties
>> of
>> your scope. The axial resolution is about half the x/y resolution, and
>> therefore 160 micron spacing between sections would be about optimal.
>> If
>> you are trying to look at very fine structures, oversampling a little
>> (both
>> in x, y, and z) may not be a bad idea, as long as your sample can take
>> it
>> and doesn't bleach...
>>
>>
>> I don't think it as an issue that your x/y pixel dimensions are
>> different
>> from your z section spacing. The PSF itself is not spherically
>> symmetrical
>> (but elongated along z) and your sampling along x/y and z should be set
>> accordingly. Secondly, when you load your images into whatever program
>> you
>> use for doing the 3-D measurements, the image should be calibrated to
>> reflect the actual x,y and z dimensions. As long as you used Nyquist
>> sampling, and you have good signal-to-noise, you should be able to get
>> accurate measurements.
>>
>>
>> For deconvolution, we've had good results with both measured PSF and
>> calculated PSFs; blind should work fine too, but I haven't tried. If you
>> have some reference beads, you could image them under conditions similar
>> to
>> those used to image your sample, deconvolve them with the same method,
>> and
>> do some measurements. That should give you an idea of how well your
>> deconvolution alrgorithm is performing. If it doesn't look good, you can
>> always re-deconvolve your data with a different method.
>>
>>
>>
>>
>>
>>
>> Julio.
>>
>>
>>
>> --
>> Julio Vazquez, PhD
>> Director of Scientific Imaging
>> Fred Hutchinson Cancer Research Center
>> 1100 Fairview Ave. N., mailstop DE-512
>> Seattle, WA 98109-1024
>>
>>
>> Tel: Office: 206-667-1215/ Lab: 206-667-4205
>> FAX: 206-667-6845
>>
>>
>> [log in to unmask]
>> http://www.fhcrc.org/science/shared_resources/imaging/
>>
>>
>>
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>>
>>
>>
>>
>>
>> On Feb 9, 2008, at 9:30 AM, Pedro J Camello wrote:
>>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>
>> I´m trying to image a 3D map of mitotracker-stained cells. I´m using a
>> Biorad 1024, 60x, NA 1.4. My first attempts generated acceptable images
>> with iris 1.6 -2 (close to the theoretical optimal iris indicated by
>> Biorad with this objective).
>>
>>
>> My 2 questions are:
>>
>>
>> 1) Using zoom 2.6 (the theoretical optimum zoom for this objective) I
>> get
>> pixel size 166 µm. Since published images with my cell type (pancreatic
>> acini) indicate that most of the mitochondria are around 500 nm diameter
>> (similar to the measured resolution of mi micro with iris 2) my intend
>> is
>> to sample at 150 nm to meet Nyquist. However, this creates a non-cubic
>> voxel (166x166x150). Will be this a problem to make posterior 3D
>> visualization and volume measurements?
>>
>>
>> 2) If I need deconvolution to posterior processing and measurements, is
>> it
>> essential PSF measurements or will blinded methods be enough?
>>
>>
>> Thanks in advance
>>
>>
>> --
>> Dr Pedro J Camello
>> Dpt Physiology
>> Faculty of Veterinary Sciences
>> University of Extremadura
>> 10071 Caceres
>> Spain
>> Ph: 927257100 Extension 1321/1290
>> Fax:927257110
>>
>>
>>
>
>
> --
> --
> Zoltan Cseresnyes
> Facility manager, Imaging Suite
> Dept. of Zoology University of Cambridge
> Downing Street, Cambridge
> CB2 3EJ UK
>
> Tel.: (++44) (0)1223 769282
> Fax.: (++44) (0)1223 336676
>
--
Dr Pedro J Camello
Dpt Physiology
Faculty of Veterinary Sciences
University of Extremadura
10071 Caceres
Spain
Ph: 927257100 Extension 1321
Fax:927257110
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