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Real-time image acquisition
One of the problems commonly encountered in traditional single-beam multiphoton microscopy
is scanning speed. Increasing speed requires increasing laser power on the specimen. However,
the laser power deliverable to a biological specimen is limited by photodamage and photobleaching1, 2.
Thus, an effective approach to overcome these limitations is the use of parallel excitation
and acquisition. Our novel multiphoton system employs 64 beams: compared to a conventional
multiphoton microscope, our multispot approach allows either to acquire images much faster,
or to greatly improve the signal-to-noise ratio
Principle of operation
The Ti:Sa laser beam is split into 64 beamlets by a diffractive optics elements (DOE).
Every beamlet is focused by the microscope objective, resulting in a 8 × 8 matrix of excitation
focal spots. A pair of galvanometric mirrors provides scanning of the specimen in the x-y plane,
whereas a piezoelectric transducer (PZT) coupled to the objective allows scanning in the z direction.
A dichroic mirror (DM) redirects the emitted fluorescence light to the detection system.
Fluorescence light is detected by 64 photomultiplier tubes (PMT). Onboard electronics perform signal
amplification, analog/digital conversion and store operations
Simple, efficient and uniform
The core of our patent-pending multi-spot system is a single optical element (DOE),
that splits the source laser beam into 64 beamlets3. Due to its inherent simplicity, the system
is very simple to align, stable, and does not require painstaking adjustments by the user. Moreover,
the high DOE splitting efficiency allows to illuminate the sample with a high laser power for every
focal spot. Laser power is evenly distributed within the spots, thus providing uniform illumination
of the entire scanned area
Scanning depth
Our system employs a multianode photomultiplier array in a descanning mode to detect
fluorescence light. In contrast with CCD-based systems in a non-descanning mode, our solution allows
deeper sectioning, thanks to a lower cross-talk between image pixels and a better collection of scattered
fluorescent light4
Multiple choices
Users can set multiple parameters to best suit their needs. To gain penetration depth,
with 8x8 DOE the laser power can be increased to more than 12 mW for every spot5, whereas it is also
possible to use a 4x4 DOE and the laser power can be increased to more 45 mW each beam. The excitation
wavelength can be chosen depending on the dyes used. Scanning speed can be adjusted to more than 57 frames
per second at high resolution (512x512 pixels image)
Everything inside a box
Our multiphoton microscope is a completely one-box turn-system: the laser source,
detector, acquisition electronics and software are included, thus providing the user with everything
he/she needs to perform his/her experiments. The system is completely software-controlled, and does
not require further adjustments
References
1 König et al. Cellular response to near-infrared femtosecond laser pulses
in two-photon microscopes. Opt. Lett. 22:135-136 (1997)
2 Patterson and Piston. Photobleaching in Two-Photon Excitation Microscopy.
Biophys. J. 78:2159-2162 (2000)
3 Sacconi et al., Multiphoton multifocal microscopy exploiting a diffractive
optical element, Opt. Lett. 28:1918-1920 (2003)
4 Kim et al., Multifocal multiphoton microscopy based on multianode photomultiplier tubes, Opt. Exp. 15:11658-11678 (2007)
5 The power deliverable to the specimen depends on objective transmission and the wavelength dependent output
power of laser.
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