Real time two‐photon absorption microscopy using multi point excitation

In this communication we present the development of a real time two‐photon absorption microscope, based on parallel excitation with many foci. This pattern of foci is created by a two‐dimensional microlens array. The fluorescence is detected by direct, non descanned detection on a CCD camera. Due to the parallel nature of both excitation and detection it is possible to speed up image acquisition significantly. This makes the instrument especially suitable for studying living specimens and/or real time processes. The optical design of the instrument is discussed and an imaging example is given. We specifically address the relation between the axial sectioning capability and the distance between the illumination foci at the sample.

[1]  P. So,et al.  Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes. , 1997, Optics letters.

[2]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[3]  Hans J. Tiziani,et al.  Chromatic confocal microscopy with microlenses , 1996 .

[4]  Joseph R. Lakowicz,et al.  Multiphoton excitation of the DNA stains DAPI and Hoechst , 1996 .

[5]  S. Hell,et al.  Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index , 1993 .

[6]  B. Athey,et al.  Real‐time two‐photon confocal microscopy using a femtosecond, amplified Ti:sapphire system , 1996, Journal of microscopy.

[7]  O Nakamura A two-photon scanning fluorescence microscope with deep UV excitation and near UV detection , 1995 .

[8]  N. Nanninga,et al.  Three‐dimensional imaging in fluorescence by confocal scanning microscopy , 1989, Journal of microscopy.

[9]  S. Hell,et al.  Multifocal multiphoton microscopy. , 1998, Optics letters.

[10]  Clemens Storz,et al.  NONLINEAR ABSORPTION EXTENDS CONFOCAL FLUORESCENCE MICROSCOPY INTO THE ULTRA-VIOLET REGIME AND CONFINES THE ILLUMINATION VOLUME , 1994 .

[11]  W. Webb,et al.  Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity. , 1994, Applied optics.

[12]  Jean-Claude Diels,et al.  Ultrashort Laser Pulse Phenomena , 1996 .

[13]  Vladislav V. Yakovlev,et al.  Ultrafast lasers and biological applications: from two-photon to molecular relaxation imaging , 1998, Photonics West.

[14]  B. Dong,et al.  Beam shaping in the fractional Fourier transform domain , 1998 .

[15]  J N Mait,et al.  Kinoform-based Nipkow disk for a confocal microscope. , 1995, Applied optics.

[16]  D. Kleinfeld,et al.  Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy , 1994, Journal of Neuroscience Methods.