Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi all,

I agree with Mario that saturation of the fluorescent dye can lead to a
significantly broadened bleached area. Being involved in FRAP, we have
recently been studying this effect in detail for fluorescein (and have
submitted a paper on our findings). Not only did we see a deformation
(broadening) of the central maximum (Airy disk), also the higher order
diffraction maxima gave a substantial contribution to the bleaching profile.

For example, and as an extreme case, when bleaching a spot with a 10 mW
stationary laser beam for (only!) 1 ms, up to the third diffraction ring
became clearly visible in the bleaching pattern with ~20% bleaching
efficiency compared to the central maximum. As a result, the radius of the
bleached spot was 4 times (!) larger than expected theoretically when only
taking the Airy disk and first order bleaching kinetics into account.
Furthermore, apart from the illumination power, the bleaching profile
appeared also to be strongly dependent on the bleaching time (or scanning
speed for a CSLM).

So broadening of the photobleached area is very well possible and it seems
advisable not to use saturating illumination intensities when doing
photobleaching experiments.

Best regards,

Kevin

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List
> [mailto:[log in to unmask]]Namens Mario M. Moronne
> Verzonden: donderdag 20 november 2003 23:23
> Aan: [log in to unmask]
> Onderwerp: Re: Laser spot size
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi All,
>
> Tiny point (small pun). The "parking" of the beam is also dependent
> on the lack of any motion of the film, not just the mirrors.
>
> I probably just missed it, but I don't remember seeing complete
> experimental details of how the bleaching was done. For example,
>
> 1. What was the residence time of the parked beam? This is important
> in terms of production and diffusion of dye reactive radicals, and
> possibly compounded by heating effects. Here is a case of convolving
> not only the optics but the propagation of chemically reactive species
>
> 2. The fluorophore film was deposited on a coverslip of what
> thickness? and imaged in what medium, with what RI? If the coverslip
> was not the correct thickness, then the exact focal plane could very
> well have been slightly out of position. To test for the correct
> focus, it would seem to make sense to perform a Z series bleaching
> experiment, bleaching at different points using different Z-positions
> to find the correct focus. However, this must be done accounting for
> the effects of power saturation as described below.
>
> 3. I believe someone mentioned the issue of linearity of bleaching
> with power delivered to the spot. This is certainly important as
> linearity becomes meaningless once the power level has reached the
> point where fluorophore excitation is saturated. Depending on the
> fluorophore extinction coefficient, full saturation can be reached
> with as little as 100-1000 uW with a 1.4 NA lens. Thus, the power at
> the peak of illumination might be well above saturation while the
> power at half max might also still be close to fully saturated and
> bleach nearly as effectively as at the peak. This could lead to a
> bleached area that is much broader than the theoretical spot size of
> the objective and have a profile quite different than what one might
> expect from an Airy disk.
>
> I think this is an interesting problem, but it would seem to me given
> the caveats previously discussed that bleaching a spot on a film may
> not be the best way to evaluate an objective's focal spot size.
>
> Regards,
> Mario
>
>
>
> >Search the CONFOCAL archive at
> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> >If your closed loop servo is oscillating it needs service...
> >
> >Mark Cannell
> >
> >Beat Ludin wrote:
> >
> >>Search the CONFOCAL archive at
> >>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>
> >>At 08:44 20.11.2003, you wrote:
> >>
> >>>Could there be any scattering of the beam while it is parked?
> >>
> >>
> >>I have a similar question: how stable is the position of the beam when
> >>its
> >>parked. I'm totally naive when it comes to galvo mirror control (i.e.
> >>I've
> >>never taken a closer look at how exactly it works) but I would
> expect the
> >>positioning to be a closed-loop system involving some sort of feedback
> >>mechanism. So systems like that can never stand completely still , i.e.
> >>they always oscillate around the set point. Could the amplitude of this
> >>oscillation be big enough to increase the apparent diameter of
> a "parked"
> >>beam? I really have no idea, but I guess there are people here who can
> >>answer this. Also, if there's some kind of electrical interference that
> >>might also introduce an oscillation. Just some additional hypotheses to
> >>explain the large apparent beam diameter...
> >>
> >>Beat
>
>
> --
> _________________________________________________________
> Mario M. Moronne, Ph.D.
> NanoMed Technologies LLC
> President and CTO
> ph (510) 528-2400
> FAX (510) 528-8076
> 1561 Posen Ave
> Berkeley, CA
> 94706
>
> [log in to unmask]
> [log in to unmask]
>