Re: microbead calibration to set psf?

Posted by James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/microbead-calibration-to-set-psf-tp2560403p2564213.html

Hi all,

I think that Eric is right. Most specimens do have suitable small features inside of them. Indeed, standardless, bootstrap (blind) deconvolution depends on this in a way fact (although it can also extract PSF info from the images of larger features as long as they have sharp edges).

I would just like to add that 3D confocal images made by collecting the light that is backscattered by small refractive index anomalies are a particularly good source of point-like features.

If you have never collected a BSL image, all that is necessary is to use some sort of beam splitter that will pass at least half of the light at laser wavelength and remove the barrier filter. It is true that this may reveal a signal largely consisting of light reflected from the various glass-water and glass air interfaces in your optical system but the confocal pinhole will eliminate most of these if they are not located near image planes. The most annoying of these reflections will be that from the glass-water interface on which the cell may be resting. So focus a few microns into the cell.

There you will find a bewildering array of small scattering features, the images of which should be suitable for determining the excitation PSF. And all for free. No extra exposures needed, as it is quite possible to collect the BSL and fluorescent signals simultaneously and in a non-interfering manner

Such as optimized collection system is diagrammed in Figure 2.7 of the Handbook.

Cheers,

Jim Pawley

              **********************************************
Prof. James B. Pawley,                                          Ph.  608-263-3147 
Room 223, Zoology Research Building,                                  FAX  [phone]
1117 Johnson Ave., Madison, WI, 53706                                [hidden email]

3D Microscopy of Living Cells Course, June 13-25, 2009, UBC, Vancouver Canada

Info: http://www.3dcourse.ubc.ca

Applications still being accepted as space is available.

"If it ain't diffraction, it must be statistics." Anon.

Sorry for the self promo but on this subject maybe some of you could be interested in the following appproach?

It is indeed difficult to get proper beads but also to get them into place if one considers that, for deep imaging, the specimen is also part of the optical system!
Cheers
Eric

 

1: Biophys J. 2003 Dec;85(6):3991-4001.

Image-adaptive deconvolution for three-dimensional deep biological imaging.
de Monvel JB, Scarfone E, Le Calvez S, Ulfendahl M.
Center for Hearing and Communication Research, Karolinska Institutet, Stockholm,
Sweden. [hidden email]
Deconvolution algorithms are widely used in conventional fluorescence microscopy,
but they remain difficult to apply to deep imaging systems such as confocal and
two-photon microscopy, due to the practical difficulty of measuring the system's
point spread function (PSF), especially in biological experiments. Since a
separate PSF measurement performed under the design optical conditions of the
microscope cannot reproduce the true experimental conditions prevailing in situ,
the most natural approach to solve the problem is to extract the PSF from the
images themselves. We investigate here the approach of cropping an approximate
PSF directly from the images, by exploiting the presence of small structures
within the samples under study. This approach turns out to be practical in many
cases, allowing significantly better restorations than with a design PSF obtained
by imaging fluorescent beads in gel. We demonstrate the advantages of this
approach with a number of deconvolution experiments performed both on
artificially blurred and noisy test images, and on real confocal images taken
within an in vitro preparation of the mouse hearing organ.



Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience

Karolinska Institutet

Postal Address:
CFH, M1:02
Karolinska Hospital,

SE-171 76 Stockholm, Sweden

Work: [phone],
Cell: [phone]
Fax: [phone]

email: [hidden email]
http://www.ki.se/cfh/


----- Original Message -----
From: Guy Cox <[hidden email]>
Date: Tuesday, March 31, 2009 4:08 pm
Subject: Re: microbead calibration to set psf?
To: [hidden email]

> Europium chelates are phosphorescent rather than luminescent -
> that is to say, they have a very long - millisecond - fluorescence
> lifetime. That should still work in widefield but would be
> hopeless for confocal. Pretty much unfadeable, which is a plus.
>
> Tetraspeck bead are for calibrating colocalization rather than PSF
> - having multiple fluorochromes on one bead must reduce the
> intensity at any one wavelength. As I said before, I've used bead
> from Bangs Labs and I've had good images from beads down to 60nm.
> (Though at that scale it requires care and patience).
>
>
> Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox CRC Press / Taylor & Francis
> HYPERLINK
> "http://www.guycox.com/optical.htm"http://www.guycox.com/optical.htm______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
> ______________________________________________
> Phone [phone] Fax [phone]
> Mobile [phone]
> ______________________________________________
> HYPERLINK "http://www.guycox.net/"http://www.guycox.net
>
>
>
> _____
>
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of David Knecht
> Sent: Tuesday, 31 March 2009 11:57 PM
> To: [hidden email]
> Subject: Re: microbead calibration to set psf?
>
>
> I am looking for some very bright 100nm beads. I tried to look
> for the beads you mention and it looks like Duke Scientific no
> longer exists and is now part of hte Thermo conglomerate. The
> closest thing I found to what you describe is the Europium chelate
> 100nm beads. Is that what you were referrring to? THey provide
> very little data on fluorescence properties and they are unusual
> in apparently being broad excitation spectrum but there is no
> emission spectrum in their literature I can find. Since they
> fluoresce maximally at 613nm it would seem they would be a poor
> fit for most FITC, TRITC, CY3 or CY5 type filter sets. Can you
> clarify your experience with them? Dave
>
> On Mar 31, 2009, at 3:36 AM, Mariette P.C. Kemner - van de Corput
> wrote:
>
> I have good experience with fluorescent beads from Duke
> scientific. The
> 100nm beads are very bright and are perfect for determining the
> PSF. I use
> the red, green and blue 100nm beads to determine the PSF in three
> channelsand to deconvolved my 3 colour FISH images.
> I have trouble finding the 100nm and 200nm TetraSpeck beads on my
> slidebeacuse they are rather weak in intensity. So that's why I
> switched to the
> single coloured 100nm beads from DS. The 500nm TetraSpeck beads
> are very
> good and I use them to determine the chromatic shift in 4
> channels. Once
> made, bead slides can be used for a long period of time.
>
> Mariette
>
> ____________________________________________
>
> Dr. M.P.C. Kemner-van de Corput,
> ____________________________________________
>
> MGC - Dept. of Cell Biology & Genetics
> Erasmus Medical Center
> Dr. Molewaterplein 50, 3015 GE Rotterdam
> POB 2040, 3000 CA Rotterdam, The Netherlands
>
> Office: H-Ee751; tel: [phone]
> Lab: H-Ee710; tel lab: +31 10 704.3315
> tel secr: +31 10 704.3169
> ____________________________________________
>
> HYPERLINK
> "http://www2.eur.nl/fgg/ch1/cellbiology/"http://www2.eur.nl/fgg/ch1/cellbiology/http://www.thesis.kemner.biz/
> ____________________________________________
>
> Op Di, 31 maart, 2009 1:59 am, schreef Matiar Jafari:
>
>
> hey again,
>
>
>
> I'm trying to find out how and what the best way to setup my own psf
>
>
> is in AutoQuant. I know i need microbeads and im wondering what brand
>
>
> and what product i need and then how i need to shoot it and load it.

>
>
> Also what diameter does it need to be. A cat# would be great too also
>
>
> i don't know if it matters on what im shooting but just to put it out
>
>
> there im shooting gfp slices. Sorry if this is vague. Any help would
>
>
> be greatly appreciated. Thanks in advance
>
>
>
> Thank You
>
>
> --
>
>
> Matiar Jafari
>
>
>
>
> Dr. David Knecht
> Department of Molecular and Cell Biology
> Co-head Flow Cytometry and Confocal Microscopy Facility
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)
>
>
>
>
> No virus found in this incoming message.
> Checked by AVG.
> Version: 7.5.557 / Virus Database: 270.11.33/2031 - Release Date:
> 30/03/2009 5:56 PM
>
>
>
> No virus found in this outgoing message.
> Checked by AVG.
> Version: 7.5.557 / Virus Database: 270.11.33/2031 - Release Date:
> 30/03/2009 5:56 PM
>
>

--