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Greetings Jason,
    There are a number of variables which play into the excitation
efficiency and detection efficiency on a multiphoton imaging system.
The average power required to achieve an acceptable signal:noise ratio
will depend on these factors.  One issue has to do with the multiphoton
absorption cross-section of the fluorophore being used.  Conventional
dyes used in fluorescence and confocal microscopy will not necessarily
excite with an equivalent efficiency under multiphoton illumination.
The future will doubtless bring greater availability of MP friendly
probes (quantum dot conjugates are one example) with large 2 photon
absorption cross sections.  Unfortunately at this time, many dyes with
large nonlinear coefficients are only soluble in organic solvents and
are not appropriate for use with aqueous environments.  Thankfully this
isn't always the case.  The difference in 2-photon absorption efficiency
is like night and day in some comparisons, though.  The concentration of
the fluor has a big impact too.  Low expression of GFP can be quite a
headache in the context of multiphoton imaging.
    Another factor which will impact the performance of the system is
the detection scheme.  Is it a direct detection rig, or are descanned
detectors being used?  Spectral detection vs. filter based?  Direct
detection with high quality emission filters will yield the best signal
for most applications.
    The pulse-width at the sample will impact on 2-photon absorption
efficiency at a given average power.  Pre-compensation for pulse width
broadening is frequently used to ensure the shortest pulses possible at
the sample plane.  The phase distribution of the laser pulse as well as
the frequency distribution or "chirp" of the pulse can have a large
impact. The chirp is affected by pulse pre-compensation schemes,
instrumentation for manipulating phase distributions is not common on
conventional imaging systems.  Instruments for measuring these
parameters at the sample plane are just now being put to use on
"real-life" imaging systems.  In some cases, multiphoton excitation may
be less selective than continuous wave illumination and high background
fluorescence from various moieties in the sample can contribute to image
degradation.  This depends in part on the frequency bandwidth of the
excitation pulses at the sample.  It is important also that the laser is
tuned to the appropriate wavelength for the fluorophore being used, and
it can be difficult to know what wavelength that is.  The idea that it
is twice the continuous wave excitation value is not necessarily the case.
    The amount of laser power going into the scan head is pretty much
meaningless unless you are positive everything is aligned optimally.
Misalignment will result in very little excitation at the sample despite
large amounts of power going into the scanning system. Different
objectives have different transmission curves in the near IR. Different
beam expanders will have a large impact on average power at the
objective.  It is important to have some idea of the actual power at the
objective focus.  Also, the numerical aperture of the lens being used,
the zoom level, and the dwell time of the laser have an impact on the
amount of laser energy to which the sample is being exposed in a given
amount of time.
    So what is the practical laser power to be used for imaging?  It
depends on many factors, more factors than in a continuous wave
situation.  Three milliwatts (or lower) is a nice number for a well
characterized system, with optimized detection and bright optics, and a
well behaved sample using a good probe.  Under 10 milliwatts is
relatively non-invasive where long term imaging of living samples is
concerned, depending on the zoom level and the scan speed and the NA of
the objective.  Under 30 milliwatts is doable for short exposure time
lapse, low zoom, low NA or high-speed imaging or turbid, thick, or
scattering samples.  These power values assume pulsewidths around 150
fs.  Instantaneous optical breakdown and related complications start to
occur in many samples at much above 30 milliwatts, and the threshold for
obvious physical damage will depend on the sample and the parameters
outlined above.  In other words, if you can't get a decent image at low
power levels, it doesn't necessarily mean that your system isn't
working; not all situations are amenable to imaging at power levels
comparable to those used with continuous wave illumination at this point
in time.  It is good to have a nice standard sample that you know works
well in the event you have to demonstrate this to someone.  As far as
apparent technological shortcomings go, I tend to place blame on the
available dyes/fluorescent proteins.  I am optimistic that the
availability of more efficient non-linear probes will help expand the
window for non-invasive imaging in coming years.

Best Regards and Happy New Year,
Karl

Jason Goh wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear All,
>
>Hope you've all had a good holiday season.  I am currently working on a new type of two-photon microscope and am having troubles with signal-to-noise.  I was wondering if anyone has a handle on the practical laser powers necessary for two photon excitation (preferably at the sample but going into the microsope will do).  Thanks in advance for your help.
>
>Regards,
>Jason
>
>----------------------------------------------------------
>Dr Jason Goh
>Applied Optics Group
>School of Electrical & Electronic Eng.
>University of Nottingham
>TEL:  +44 (0) 115 951 5615
>FAX:  +44 (0) 115 951 5616
>
>
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--
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