Re: Measuring PSFs using water dipping objective

Posted by Peng Xi-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Measuring-PSFs-using-water-dipping-objective-tp7578494p7578501.html

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Hi Stephan,
     To measure the PSF you should learn from Stefan --- Stefan Hell
who invented STED nanoscopy. They measure PSF on a daily basis, as
they need to overlap confocal PSF with a doughnut shape PSF in X, Y,
and Z direction. Now that I have also stepped in this field, I can
tell you how to do it.
     Basically, the confocal PSF can be measured with  fluorescent
bead. Attach the bead on the surface of coverglass with poly-L-Lysin.
Use Mowoil as an embedding medium. Add antifade reagent such as DABCO,
because during PSF measurement you often stick to a very small region,
therefore the sample undergoes large photobleaching. Also, if you want
PSF more close to real situation, you may try smaller beads, as in
fluorescent mode your measured PSF is a convolution of real PSF with a
nanopartile with a certain size. You have to deduct the particle size
from it
(Diameter of real PSF)^2=(Diameter of measured PSF)^2 -(Diameter of bead)^2.
     If you are able to remove your bandpass filter (I assume you are
using fluorescent confocal) before the detector pinhole, you can also
try to see whether you are able to use gold nanoparticle reflection to
get PSF. Then you don't have photobleaching, and as long as your gold
nanoparticle is smaller than your theoretical resolution, you don't
need to count the size of nanopartile, because the reflection is only
on the tip of the bead, not the waist. :)
     A good reference for such sample preparation:
http://www.springerlink.com/content/t8ntp60137050851/#section=614830&page=1

     Good luck!

Sincerely,
Peng Xi
Ph. D.    Associate Professor
Dept. of Biomedical Engineering, College of Engineering
Peking University, Beijing, China
Tel: [phone]
Email: [hidden email]
http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-20-13-14100
On Thu, Jun 14, 2012 at 3:28 AM, Zac Arrac Atelaz <[hidden email]> wrote:

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>
> I can think of two possible issues afecting the result, very thick layer of agar being cracked by the objective, very high power output from the laser setting free the particles, if you keep the power low and change the wavelength you can avoid that one. I hope this can help you. Regards Gabriel OH
>  > Date: Wed, 13 Jun 2012 20:30:29 +0200
>> From: [hidden email]
>> Subject: Re: Measuring PSFs using water dipping objective
>> To: [hidden email]
>>
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>> Hi Stephan,
>>
>> > I am looking for a good protocol to measure PSFs on a confocal microscope
>> > using water dipping objectives (objectives used for physiology, i.e. no
>> > cover glass!).
>> > I tried using fluorescent beads embedded in 5 % low melting point agarose,
>> > but it appears that the beads are moving, probably due to slight swelling of
>> > the agarose which is immersed in the water.
>>
>> I did something like that recently, using 80 nm diameter nanogold
>> particles embedded in low-melt agarose and imaged in reflected light
>> mode on a Zeiss LSM 780 NLO with dip-in lenses. The advantage over using
>> a fluorescent bead is that the reflected signal is bright and of
>> narrowly defined spectral range (we wanted to define chromatic
>> aberration of our objectives, not get an experimental PSF for
>> deconvolution). Particle movement didn't seem to be too much of an
>> issue, perhaps because I could scan fast thanks to the high signal. Or
>> possibly, the agarose mixture was a bit different to yours. Or, it might
>> not have mattered for our question and I have forgotten. If I scrape my
>> neurons a bit harder I recall that the near-IR laser made the particles
>> wobble a bit, maybe because of local heating. Let me know if you want
>> details of the protocol.
>>
>> Michael
>