http://confocal-microscopy-list.275.s1.nabble.com/Measuring-PSFs-using-water-dipping-objective-tp7578494p7578504.html
objective. Is there a way of correcting the PSF for use with a dipping
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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: +86 10-6276 7155
> 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
>>
>