microbead calibration to set psf?
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Matiar Jafari |
microbead calibration to set psf?
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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 |
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Matiar Jafari |
Re: microbead calibration to set psf?
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Thank you Tom for your response.
Well im shooting dendritic spines in CA1 and i dont know if the size of the bead matters. Thank You --- Matiar Jafari On Mon, Mar 30, 2009 at 5:09 PM, Donnelly, Tom <[hidden email]> wrote: > If you want to roll your own, use tetraspec beads 100nm and a 1000:1 > lower concentration of 200nm beads. Dry them onto a coverslip and mount. > > Molecular probes, aka Invitrogen, has a prepared bead slide that works > as well. > > Why don't you ask Autoquant? They should be the experts in collecting > psf's with their software. > > Tom > > Tom Donnelly > Applied Precision, Inc. > 1040 12th Ave. N.W. Issaquah, WA 98027-8929 > (425)829-9414 (425)557-1055 fax > [hidden email] > http://www.api.com/lifescience/DeltaVision.html > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Matiar Jafari > Sent: Monday, March 30, 2009 4:59 PM > To: [hidden email] > Subject: microbead calibration to set psf? > > 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 > > > This email message, together with any attachments, is for the sole use of the > intended recipient(s) and is the confidential information of Applied Precision > Inc. If you are not the intended recipient, your review, use, disclosure, > copying or dissemination of this email message or its attachments, or the > information contained therein, is strictly prohibited. If you are not the > intended recipient or if you think this email was sent to you in error, please > notify the sender by reply email and delete this message and its attachments, > as well as all copies, from your system. > > > -- Matiar Jafari |
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M. van de corput |
Re: microbead calibration to set psf?
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In reply to this post by Matiar Jafari
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 channels and to deconvolved my 3 colour FISH images. I have trouble finding the 100nm and 200nm TetraSpeck beads on my slide beacuse 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: +31 10 704.3949 Lab: H-Ee710; tel lab: +31 10 704.3315 tel secr: +31 10 704.3169 ____________________________________________ 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 > |
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In reply to this post by Matiar Jafari
Autoquant doesn't usually need an experimental psf, though maybe it can use one. To get a reasonably true psf (that is, free from the influence of the bead) the bead needs to be well below the resolution limit. I'd suggest 100nm if you are using immersion objectives. Some deconvolution software can use larger beads and mathematically extract the psf from the resulting image. Since I've always done 'blind' deconvolution with Autoquant I don't know if it can do that. Check your manual. It it can it's best to go that way - you'll get better signal/noise.
Bang's Labs have just about every form of bead. Pick one that is close to the excitation you'll be using. To prepare a slide dilute the suspension with distilled water and let a drop dry on a #1.5 coverslip. Mount it (bead side down ...) with your favourite mountant. 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 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Matiar Jafari Sent: Tuesday, 31 March 2009 9:59 AM To: [hidden email] Subject: microbead calibration to set psf? 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 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 |
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Knecht, David |
Re: microbead calibration to set psf?
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In reply to this post by M. van de corput
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:
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) |
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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 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? On Mar 31, 2009, at 3:36 AM, Mariette P.C. Kemner - van de Corput
wrote:
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. No virus found in this outgoing message. |
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Eric Scarfone |
Re: microbead calibration to set psf?
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Sorry for the self promo but on this subject maybe some of you could be interested in the following appproach?
1: Biophys J. 2003 Dec;85(6):3991-4001. > 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 > 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 +61 2 9351 3176 Fax +61 2 9351 7682 > Mobile 0413 281 861 > ______________________________________________ > 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? > > > 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 > 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 > > Office: H-Ee751; tel: +31 10 704.3949 > 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 > > > > > 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. > Version: 7.5.557 / Virus Database: 270.11.33/2031 - Release Date: > 30/03/2009 5:56 PM > > |
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Knecht, David |
Re: microbead calibration to set psf?
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In reply to this post by Guy Cox
Bangs doesn't seem to make alot of colors or sizes of PSF sized beads. They list 60nm and then the next smallest is 190nm. Is that too large for PSF applications? Have you used the Suncoast Yellow, which is the only thing in a yellow/red window they have? Dave
On Mar 31, 2009, at 10:11 AM, Guy Cox wrote:
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) |
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In reply to this post by Knecht, David
David,
Thermo still lists the beads:
Just checked it, is that what you need?
Zoltan
On Tue, Mar 31, 2009 at 2:56 PM, David Knecht <[hidden email]> wrote:
-- Zoltan Cseresnyes Facility manager, Imaging Suite |
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Jean-Pierre CLAMME-2 |
Re: microbead calibration to set psf?
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In reply to this post by Matiar Jafari
Hi, I just used PS-Speck Beads from Molecular Probes and they worked just fine for me. They are brighter than the Tetra Speck but single color. JP Confocal Microscopy List <[hidden email]> wrote on 03/30/2009 04:59:05 PM: > 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 |
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Glen MacDonald-2 |
Re: microbead calibration to set psf?
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In reply to this post by Guy Cox
Hi Dave,
An easy way to find the focal plane of the smaller beads is to mix in a few beads of a larger size. Then you can easily find the surface of the slide or the coverlslip on which you applied the psf beads. Regards, Glen On Mar 31, 2009, at 7:11 AM, Guy Cox wrote: > 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 > 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 +61 2 9351 3176 Fax +61 2 9351 7682 > Mobile 0413 281 861 > ______________________________________________ > 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 >> channels >> and to deconvolved my 3 colour FISH images. >> I have trouble finding the 100nm and 200nm TetraSpeck beads on my >> slide >> beacuse 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: +31 10 704.3949 >> Lab: H-Ee710; tel lab: +31 10 704.3315 >> tel secr: +31 10 704.3169 >> ____________________________________________ >> >> 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 Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] ****************************************************************************** The box said "Requires WindowsXP or better", so I bought a Macintosh. ****************************************************************************** |
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James Pawley |
Re: microbead calibration to set psf?
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In reply to this post by Eric Scarfone
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, Room 223, Zoology Research Building, 1117 Johnson Ave., Madison, WI, 53706 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! 1: Biophys J. 2003 Dec;85(6):3991-4001. Image-adaptive deconvolution for three-dimensional deep biological imaging. Karolinska Institutet SE-171 76 Stockholm, Sweden > -- |
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In reply to this post by Knecht, David
Dave,
The problem is that a
bottle lasts a VERY long time! My 60nm beads certainly came from Bangs -
my 100nm ones were given to me by Carol Cogswell many years ago. I've not
tried anything in the longer wavelength range since I've not had any reason
to. I think Bangs have been taken over so their range may have been
reduced since I last dealt with them. 190nm is certainly a bit large for a
1.4NA lens.
Guy
Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht Sent: Wednesday, 1 April 2009 12:31 AM To: [hidden email] Subject: Re: microbead calibration to set psf? On Mar 31, 2009, at 10:11 AM, Guy Cox wrote:
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. No virus found in this outgoing message. |
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Dear All, I am looking for a supplier of Ultra Clean Cover glass (#1.5 and/or
#2) (Ultra Clean stands for chemically clean, i.e. treated with a piranha solution
(Sulfuric acid + hydrogen peroxide), etc. Second, would you be willing to share your experience on the high
efficiency transfection of HELA cells (>40%) at low confluency (30-40%),
which HELA reagent would reveal the lowest cytotoxicity. Thank you, Vitaly NCI-Frederick, 301-846-6575 |
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Florian Eich |
Re: Ultra clean cover glass and HELA transfections
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Hello Vitaly,
during my work in the lab I found Metafectene Pro a very good transfectant for HELA cells, regardless of the confluency. But it mainly depended also on very clean plasmid-preps which sometimes was more difficult than the transfection. Also worth a try if you are unsatisfied with "normal" transfection would be to do Elektroporation with Amaxa, this also worked nicely for my experiments, and since you have the cells in suspension when you do the transfection you can select the number you seed and then get the desired confluency. To try Amaxa, just contact them. In Germany they sometimes supply you with a machine to test their system. No commercial interest for both companies mentioned. Good Luck Florian |
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