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>The difference between 3, 4 or 5 orders of 
>magnitude is  simply the pixel size one chooses 
>for calculation.  1024 x 1024 gives 1,048,576 
>(actually that's 6 orders of magnitude), 
>512x512 gives a factor of 262,144, and 256 x 256 
>gives 65,536.    I'm not trying to labour a 
>trivial point, I'm making the important one that 
>you should NOT use more pixels than you need to 
>capture your information.  One of my more 
>spectacular 3D images was of an extremely 
>beam-sensitive specimen taken at 256x256x97. 
>Of course if you want to enter one of Mike 
>Davidson's competitions you'll need a high pixel 
>count (and a stable sample) but for cutting edge 
>science the moral is just that - cut.
>
>                                                           Guy


Perhaps I should explain my arithmetic.

The Airy Disk covers an a larger area of the 
specimen than that which is later represented by 
one, Nyquist-sampled pixel.

I estimate that Nyquist-sampled pixel is about 
10x smaller than the area covered by "those 
pixels in the Airy disk that have 'significant 
counts'".

The argument is as follows:

  If we accept the distance between the centre of 
the disk and the first dark ring as a reasonable 
guess of the "resolution," this distance should 
be represented by at least 2 pixels, and the 
distance across the central maximum (which is 
doing most of the damage) will be 4 pixels. If 
the pixel boundaries were set up symmetrically 
around the centre, one might  make a rough 
estimate of the disk as 4x4 pixels , however, 
there would be very few counts in the 4 pixels on 
the corners, so maybe 12 pixels total.

On the other hand, the distance from one 
dark-ring to the dark-ring on the other side 
should really be closer to 4.5 of or 5 pixels or 
a "gross first estimate of  area" of 20 or 25 
pixels. Against this we must realize that, in 
general, the disk won't be centered and, if even 
the centre pixel produces a recorded signal of 
say 20 counts, you don't have to get far from the 
centre for counts (and therefore damage to the 
specimen which is what we are talking about) to 
become fairly negligible.

So I am happy to guesstimate that the 
Nyquist-sampled PDF has about 10 pixels "with 
significant counts" hence 10 pixels in which 
"significant damage" will occur.

Every assumption could be disputed, but the "true 
number (if there is one) is not 1 and is not 100.

This leads to 1000 x1000/10 = 100,000.

Of course, if we actually have a real AIry disk, 
(Usually we don't. The peak intensity is seldom 
~30x higher than that of the first bright ring.) 
almost 20% of the power is still in the outer 
rings, and above arithmetic ignores the effect of 
this relatively low intensity light/damage. Real 
"Airy disk" spots focused inside real specimens 
probably have considerably more of the total 
light in the area of these "outer rings".

Regards,

Jim Pawley

>Guy Cox, Honorary Associate Professor
>School of Medical Sciences
>
>Australian Centre for Microscopy and Microanalysis,
>Madsen, F09, University of Sydney, NSW 2006
>
>-----Original Message-----
>From: Confocal Microscopy List 
>[mailto:[log in to unmask]] On 
>Behalf Of James Pawley
>Sent: Saturday, 28 September 2013 12:09 AM
>To: [log in to unmask]
>Subject: Re: Photobleaching of confocal microscopy
>
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>>Dear Listers,
>>
>>I have a basic question about the relationship between photobleaching,
>>pixel dwell time and intensity of excitation beam in confocal microscopy.
>>
>>Review paper "Optical Sectioning microscopy" in Nature Methods 2005
>>said about bleaching of confocal microscopy.
>  >
>>"The probability that a molecule bleaches depends on its exposure to
>>the excitation light. This is the product of the irradiance a molecule
>>receives and the time it receives it.
>>Thus, a molecule that receives irradiance I1
>>   of duration t1 is as likely to bleach as one that receives twice the
>>irradiance (2I1) for half the time (t 1/2)."
>>
>>Although this is well accepted among confocal microscopy user, I am
>>wondering if there are any data or paper to support this notion.
>>I also would like to know how exactly same the photobleaching of these
>>two cases are.
>>
>>If someone knows any information, we appreciate comments.
>>
>>Best Regards,
>>Shigeo Watanabe, HPK
>
>Dear Shigeo,
>
>Dave piston did a lot of work on bleaching rate 
>for 1- and 2-photon excitation about a decade 
>ago, using dye dissolved in oil and squished 
>into a very thin layer by prolonged pressure. He 
>found that above a certain power density in the 
>spot, the bleach rate was greater than what one 
>would assume from a linear extrapolation. Of 
>course, such experiments are complicated by the 
>fact that the intensity varied markedly across 
>the focused spot (so maybe it was being "super 
>bleached" in the centre of the spot but that 
>this was masked by emissions from the outer 
>parts of the spot where bleaching was only 
>linear) , but because his dye was uniform and 
>thin, at least he could avoid the complication 
>that dye might diffuse in or out of the exposure 
>area or that, although the dye might bleach in 
>the focus plane, signal might still be detected 
>from so-far unbleached dye located above or 
>below this plane where the intensity was lower 
>(because the spot was not yet fully focused and 
>hence larger in diameter and so less intense).
>
>On the other hand, bleaching behavior in oil is 
>likely to be different than that in water, 
>especially if the water contains dissolved 
>oxygen. (Bleaching is seldom a matter of photon 
>absorbed -> molecule damaged, it is more 
>commonly, photon absorbed, excited molecule 
>interacts with oxygen to produce reactive oxygen 
>species which may diffuse some distance before 
>interacting to produce some permanent chemical 
>change. One should note that this chemical 
>change will probably only occasionally involve 
>the original dye molecule.)
>
>Guy speaks of the peak intensity as being 3-4 
>orders of magnitude greater in confocal than in 
>wide field. I usually guess 5 orders (assuming 
>something close to Nyquist sampling, you are 
>illuminating about 10 pixels out of a raster 
>that I assume to be 1,000x1,000). The early 
>editions of The Handbook had chapters by the 
>Watt Webb group that showed that with most dyes, 
>you reached very close to saturation at about 
>1mW of laser power in a high-NA spot.
>
>In other words, I disagree with the "short 
>version of bleaching" in the Nature article that 
>you quote. It may be OK when the laser power in 
>the spot of around one µW, but not at one mW or 
>when one is working at low NA and therefore the 
>spot is bigger and so less intense.
>
>There are several chapters (particularly those 
>by Karsten Koenig, Alberto Diaspro and Jurek
>Dobrucki) devoted to bleaching in the third edition of the Handbook.
>
>Regards,
>
>Jim Pawley
>--
>James and Christine Pawley, 5446 Burley Place 
>(PO Box 2348), Sechelt, BC, Canada, V0N3A0, 
>Phone 604-885-0840, email <[log in to unmask]> 
>NEW! NEW! AND DIFFERENT Cell (when I remember to 
>turn it on!) 1-604-989-6146


-- 
James and Christine Pawley, 5446 Burley Place (PO 
Box 2348), Sechelt, BC, Canada, V0N3A0,
Phone 604-885-0840, email <[log in to unmask]>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146