Re: TIRF depth calibration

Posted by Graham Wright on
URL: http://confocal-microscopy-list.275.s1.nabble.com/TIRF-depth-calibration-tp5769934p5775891.html

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Thanks to everyone for the input and apologies for missing the earlier
discussion in my search (wrong search terms).

So, for the people that have tried any of the varied methods, how different
are the empirical results to the calculated depth, based on the angle, given
by some of the commercial systems?

Thanks again,
Graham

---
Graham Wright, PhD

Microscopy Unit Manager
Institute of Medical Biology
8A Biomedical Grove, #06-06 Immunos, Singapore 138648

W:  http://www.imb.a-star.edu.sg/imu/

On 25 November 2010 18:04, Lauran Oomen <[hidden email]> wrote:

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>
> Another publication related to this topic is this one:
>
> http://spiedl.aip.org/getabs/servlet/GetabsServlet?prog=normal&id=JBOPFO000011000001014006000001&idtype=cvips&gifs=yes&ref=no
> Mattheyses, A.L. and Axelrod, D.
> Direct measurement of the evanescent field profile produced by
> objective-based total internal reflection fluorescence
> J. Biomed. Opt., Vol. 11, 014006 (2006)
>
> Did not try it (yet), however.
>
> Best wishes
>
> Lauran Oomen
> --------------------------------------------------------------
> Lauran Oomen
> Manager Digital Microscopy Facility (C.2.023)
> NKI-AVL
> Plesmanlaan 121
> PO Box 90203
> 1006 BE Amsterdam
> The Netherlands
>
> phone +31 205126080
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of John Oreopoulos
> Sent: woensdag 24 november 2010 17:21
> To: [hidden email]
> Subject: Re: TIRF depth calibration
>
> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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>
> And for the more adventurous types out there, you can measure your
> penetration depth using fluorescence correlation spectroscopy (FCS) if you
> have the right hardware:
>
> Harlepp, S., et al., Subnanometric measurements of evanescent wave
> penetration depth using total internal reflection microscopy combined with
> fluorescent correlation spectroscopy. Applied Physics Letters, 2004. 85(17):
> p. 3917-3919.
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> 9078 Leslie Street, Unit 11
> Richmond Hill
> Ontario, Canada
>
>
> On 2010-11-24, at 10:37 AM, Christophe Leterrier wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > There is also a simple method in Fiolka et al. Miocroscoc. res Tech 2007
> :
> > http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf
> >
> > <http://www.neuro.nano-optics.ethz.ch/publications/fiolka.pdf>involving
> an
> > inclined coverslip with attached fluorescent beads. I didn't try it
> myself
> > so I can't tell if it works, but seems simpler than AFM or microtubules
> > method.
> >
> > Christophe
> >
> > On Wed, Nov 24, 2010 at 16:32, John Oreopoulos
> > <[hidden email]>wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Sebastian, I remember reading a publication from about ten years ago
> that
> >> talked about mounting a pencil in a piezoelectric micro-manipulator and
> >> sticking a fluorescent bead on the end of the pencil tip. This was very
> >> similar to the AFM method. Is it the same thing?
> >>
> >> Graham, there is a fairly detailed discussion on this topic from a few
> >> years ago on the archive that talks about a few other ways to measure
> the
> >> actual TIRF penetration depth (as opposed to calculating it based on an
> >> assumed refractive index and crudely measured incident angle):
> >>
> >>
> >>
> http://lists.umn.edu/cgi-bin/wa?A3=ind0702&L=CONFOCALMICROSCOPY&E=quoted-printable&P=827770&B=--Apple-Mail-181-588547976&T=text%2Fhtml;%20charset=ISO-8859-1
> >>
> >> I've tried the AFM method as well - it works, but my main complaint with
> >> this and some of the other protocols is that they require complicated
> and
> >> expensive equipment, and can be difficult to get right. The method
> involving
> >> fluorescent microtubles by Jorg Enderlein's group is a fairly new one
> that
> >> is elegantly simple, but again requires you to have access to some
> fairly
> >> special reagents that might not be found in every lab. A few months ago
> I
> >> came across yet another older method that had evaded my previous
> searches on
> >> the topic. This one is similar to the others that involve imaging
> >> fluorescent microbeads, but I like this because all it requires is a
> >> microscope with a motorized drive on the z-axis:
> >>
> >> Steyer, J.A. and W. Almers, Tracking single secretory granules in live
> >> chromaffin cells by evanescent-field fluorescence microscopy.
> Biophysical
> >> Journal, 1999. 76(4): p. 2262-2271.
> >>
> >> John Oreopoulos
> >> Research Assistant
> >> Spectral Applied Research
> >> 9078 Leslie Street, Unit 11
> >> Richmond Hill
> >> Ontario, Canada
> >>
> >>
> >> On 2010-11-24, at 6:54 AM, Sebastian Rhode wrote:
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> *****
> >>>
> >>> Hi Graham,
> >>>
> >>> one method which definitly works (I tried it out by myself) is the use
> of
> >> an
> >>> combined TIRF-AFM setup. You just have to couple fluorescent beads to
> the
> >>> tip of your AFM an record pictures while approaching/or moving away the
> >>> coverslip surface. Unfortunately an AFM is really expensive.
> >>>
> >>> So I found some other methods, which might work as well --> see
> >>> TIRF_Introduction.pdf, which I send to you directly (LIST server does
> not
> >>> accepted this pdf-file).
> >>>
> >>> One methodes uses an objective piezo-drive and a pencil and the second
> >> one
> >>> stained beads or a stained solution with intransparent beads.
> >>>
> >>> I case of questions, feel free to contact me directly.
> >>>
> >>> Cheers,
> >>> Sebastian
> >>>
> >>>
> >>> Dr. Sebastian Rhode
> >>> Project Manager
> >>> Research & Development
> >>>
> >>> TILL Photonics GmbH
> >>> Lochhamer Schlag 21
> >>> D- 82166 Gräfelfing, Germany
> >>> Phone   +49 (0)89 895 662-120
> >>> Fax     +49 (0)89 895 662-101
> >>> www.till-photonics.com
> >>
>