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

Ian brings up a good point--there is a "richness of variables" beyond
temporal pulsewidth which can impact the 2-photon efficiency of a given
experimental system. Both chirp and pulse shape come into play to
different degrees depending on the non-linear absorbing moeties
involved. See below:

Buist, A.H., Muller, M., Ghauharali, R.I., Brankenhoff, G.J. Squier,
J.A. Bardeen, C.J., Yakovlev, V.V., and Wilson, K.R. “Probing
microscopic chemical environments with high-intensity chirped pulses.”
/Opt. Lett./ *24*, 4, 244-246. 1999.

Bardeen, C.J., Yakovlev, V.V., Squier,J.A. and Wilson, K.R. “Quantum
Control of Population Transfer in Green Fluorescent Protein by Using
Chirped Femtosecond Pulses.” J. Am. Chem Soc. 120, 13023. 2001.

Bardeen, C.J., Yakovlev, V.V., Squier, J.A., Wilson, K.R., Carpenter,
S.D., and Weber, P.M. “Effect of pulse shape on the efficiency of
multiphoton processes: Implications for biological microscopy.” /J.
Biomed. Opt./ *4*, 3, 362-367. 1999.

Brixner, T., Damrauer, N.H., Niklaus, P. and Gerber, G. “Photoselective
adaptive femtosecond quantum control in the liquid phase.” /Nature/
*414*, 57-60.

Lozovoy, V.V., Pastirk, I., Walowicz, K., and Dantus, M. “Multiphoton
intrapulse interference. II. Control of two- and three-photon laser
induced fluorescence with shaped pulses.” /J. Chem. Phys/. *118*, 7,
3187-3196.

Best,
Karl

Ian Read wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Arthur,
>
>The idea of pulse stretching is an interesting one, and the experimental
>implications are different depending on your coupling strategy.  As a
>general rule, fiber coupling using femtosecond pulses is too complicated to
>be practical.  That's not to say that it can't be done, it's just more
>difficult than it's worth.  In this special case, the concept of 'pre-
>chirping' is not straight forward because the peak-power levels are so high
>that non-linear effects begin to show up that aren't easily compensated
>for.  One can avoid these effects by keeping the average power low but then
>you run into trouble with coupling efficiency into the fiber, and getting
>enough energy to your sample (which is why you wanted to fiber couple to
>begin with)  Another disadvantage of fiber coupling is that re-optimizing
>the 'pre-chirp' fiber apparatus is usually necessary following a wavelength
>change, making tuning the system a chore.
>
>Direct coupled systems, as you point out, will also exhibit some pulse-
>stretching but not nearly as much.  Exactly how much stretching depends
>upon how much, and what type of material is used in the various optical
>components making up the beam path.  If these parameters are known, then
>one can very accurately calculate the resultant pulse width.  Trouble is
>that microscope objective designs are proprietary and this information is
>not readily available.  However, some investigators have measured the pulse
>width through different microscopes so we can guess at typical values.
>>From the data presented here on the listserver from Amplitude systems:
>
>Input pulse: 100 fs
>Output pulse: 200 fs
>
>Input pulse: 200 fs
>Output pulse: 220 fs
>
>The calculated dispersion assuming 800 nm is 5000 fs^2.  A number typical
>of most microscope set-ups.  This number changes with wavelength - as the
>wavelength gets shorter, the dispersion goes up.  (If interested, I can
>provide you with an excel spreadsheet to compute these for yourself)  The
>real question is how does this effect the 2-photon absorption efficiency?
>One can also calculate this difference in terms of fluorescence generated
>for a given pulse width.  For the first case, the resulting pulse is 200 fs
>leading to a fluorescence intensity of 75 arb. units.  For the second,
>where the resulting pulse is 220 fs the intensity is 60 arb. units.
>(Calculations outlined by Peter T.C. So, in Methods of Cellular Imaging, A.
>Periasamy, p. 147) So it is not correct to assume that 'less pulse-
>strecthing means more signal'  There are more factors at play that
>determine the optimum parameters.  In fact, even with highly dispersive
>microscope systems, pulse-stretching can be compensated for by only small
>increases in the average power.
>
>Feel free to contact me with any questions:
>Ian Read
>Spectra-Physics
>650-966-5346
>
>

--
Karl Garsha
Light Microscopy Specialist
Imaging Technology Group
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
405 North Mathews Avenue
Urbana, IL 61801
Office: B650J
Phone: 217.244.6292
Fax: 217.244.6219
Mobile: 217.390.1874
www.itg.uiuc.edu