Multiphoton excitation microscopy is becoming increasingly important as it offers several advantages over confocal laser (single photon excitation) scanning microscopy (CLSM) such as use of lesser damaging NIR wavelengths, reduced scattering, deeper imaging, no need for pinhole, higher light collection efficiency and excitation of UV dyes at NIR wavelengths; in addition, excitation and photobleaching are limited to the focal region.

Multiphoton microscopy shares one disadvantage with CLSM though- low image rate. Images must be reconstructed pixel by pixel, and with an image rate of 1-2/s, the technique is not suitable for investigating fast biological processes.

To speed up image acquisition, various techniques such as line scans, polygon mirrors, resonant scanners and microlens arrays have been used. The problems with the microlense array design are: low light throughput, non-uniform intensity foci and lens aberrations. This focal overlap is of particular concern in popular Nipkow disk scanning confocal microscopy whose sectioning ability is additionally compromised by cross-talk between detection pinholes.

In our approach a special beamsplitter, based on flat optical elements, divides an incoming laser beam into N beamlets that are coupled to the microscope and scanned simultaneously in the object plane, speeding up image acquisition by a factor of N; depending on the laser power, N can be as high as 256.

Several important features of the beamsplitter are:

higher (>75%) light throughput,

uniform intensity of beamlets,

intrinsic time-multiplexing due to slightly different optical paths,

absence of optical aberrations due to flat optical elements

convenient control on the polarization, degree of parallelization (number) of beamlets, inter-foci distance and field of view.

Selected References

Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system
G.J. Brakenhoff, J. Squier, T. Norris, A.C. Bliton, M.H. Wade, B. Athey                Journal of Microscopy 1996, 181 (Pt 3):253-9.

Real time two-photon absorption microscopy using multipoint excitation
A.H. Buist, M. Muller, J. Squier, G.J. Brakenhoff                                              Journal of Microscopy 1998

High-speed, two-photon scanning microscope
Ki Hean Kim, Christof Buehler, Peter T.C. So                                                  Applied Optics 1999

Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons
G.Y. Fan, H. Fujisaki, A. Miyawaki, R.-K. Tsay, Roger Y. Tsien, Mark H. Ellisman Biophysical Journal 1999

Multifocal Multiphoton Microscopy                                                               J. Bewersdorf, R. Pick, S. W. Hell                                                            Optics Letters 1998, 23, 655-57

High efficiency beamsplitter for multifocal multiphoton microscopy,                    T. Nielsen, M. Fricke, D. Hellweg, P. Andresen                                                J. Microscopy 2001, 201, 368-376.

Space-multiplexed multifocal nonlinear microscopy
S.W. Hell, V. Andresen                                                                              Journal of Microscopy 2001