Improved depth resolution in video-rate line-scanning multiphoton microscopy using temporal focusing.

By introducing spatiotemporal pulse shaping techniques to multiphoton microscopy it is possible to obtain video-rate images with depth resolution similar to point-by-point scanning multiphoton microscopy while mechanically scanning in only one dimension. This is achieved by temporal focusing of the illumination pulse: The pulsed excitation field is compressed as it propagates through the sample, reaching its shortest duration (and highest peak intensity) at the focal plane before stretching again beyond it. This method is applied to produce, in a simple and scalable setup, video-rate two-photon excitation fluorescence images of Drosophila egg chambers with nearly 100,000 effective pixels and 1.5 microm depth resolution.

[1]  M D Duncan,et al.  Scanning coherent anti-Stokes Raman microscope. , 1982, Optics letters.

[2]  Oscar E. Martínez,et al.  3000 times grating compressor with positive group velocity dispersion: Application to fiber compensation in 1.3-1.6 µm region , 1987 .

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

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

[5]  Michal Linial,et al.  Gigantic optical non‐linearities from nanoparticle‐enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems , 1996 .

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

[7]  Yaron Silberberg,et al.  Nonlinear scanning laser microscopy by third harmonic generation , 1997 .

[8]  Buist,et al.  Real time two‐photon absorption microscopy using multi point excitation , 1998 .

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

[10]  Wilson,et al.  3D microscopy of transparent objects using third‐harmonic generation , 1998, Journal of microscopy.

[11]  W. R. Wiley,et al.  Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering , 1999 .

[12]  T Nielsen,et al.  High efficiency beam splitter for multifocal multiphoton microscopy , 2001, Journal of microscopy.

[13]  E. Neher,et al.  Highly nonlinear photodamage in two-photon fluorescence microscopy. , 2001, Biophysical journal.

[14]  Y. Silberberg,et al.  Third-harmonic generation with cylindrical Gaussian beams , 2004 .

[15]  Y. Silberberg,et al.  Scanningless depth-resolved microscopy. , 2005, Optics express.