Re: TIRF depth calibration

Posted by Mark Cannell on
URL: http://confocal-microscopy-list.275.s1.nabble.com/TIRF-depth-calibration-tp5769934p5771787.html

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Hi All,

FCS might be a good method to solve what may be a very difficult  
measurement. I'm not sure how methods that actually alter the local  
refractive index' (e.g. beads that are of significant to size to the  
TIRF field or inclined coverslips) can give the 'correct' answer  
though. Furthermore, measuring the distance is of little value unless  
the optical properties of  your testing sample are exactly the same as  
the experimental. For example, measuring TIRF depth in water does not  
give the TIRF depth inside attached cells (because their refractive  
indexes are different). Since the TIRF field is determined by  
Maxwell's equations, could one not just calculate the TIRF field since  
you can can measure the incident angle of the input light and it's  
divergence quite accurately and *may* know the refractive indices in  
the system?

My 2c

Cheers Mark


On 25/11/2010, at 5:20 AM, John Oreopoulos wrote:

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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:
>
>> *****
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>>
>> 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
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>>>
>>> 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   [phone]
>>>> Fax     [phone]
>>>> www.till-photonics.com
>>>