Temporal focusing with spatially modulated excitation

Eirini Papagiakoumou,Dan Oron,Valentina Emiliani,Vincent De Sars

doi:10.1364/oe.17.005391

Abstract

Temporal focusing of ultrashort pulses has been shown to enable wide-field depth-resolved two-photon fluorescence microscopy. In this process, an entire plane in the sample is selectively excited by introduction of geometrical dispersion to an ultrashort pulse. Many applications, such as multiphoton lithography, uncaging or region-of-interest imaging, require, however, illumination patterns which significantly differ from homogeneous excitation of an entire plane in the sample. Here we consider the effects of such spatial modulation of a temporally focused excitation pattern on both the generated excitation pattern and on its axial confinement. The transition in the axial response between line illumination and wide-field illumination is characterized both theoretically and experimentally. For 2D patterning, we show that in the case of amplitude-only modulation the axial response is generally similar to that of wide-field illumination, while for phase-and-amplitude modulation the axial response slightly deteriorates when the phase variation is rapid, a regime which is shown to be relevant to excitation by beams shaped using spatial light modulators. Finally, general guidelines for the use of spatially modulated temporally focused excitation are presented.

Highlights

Generation of complex three-dimensional optical excitation patterns is a requirement in many practical applications, including optical imaging, photoactivation and lithography
We have considered the effects of 1D and 2D spatial modulation of temporally focused excitation patterns on the excitation pattern itself and on the axial resolution
The 1D analysis sets a limit on the amount of scanning necessary in order to fully cover a 2D area while maintaining the depth resolution of point scanning

Summary

Introduction

Generation of complex three-dimensional optical excitation patterns is a requirement in many practical applications, including optical imaging, photoactivation and lithography. For a 1D illuminated line (perpendicular to the grating lines), essentially combining temporal focusing with spatial focusing along one spatial dimension, depth resolution is equivalent to that of point scanning [13, 14], recovering the z−2 decay for a two-photon absorption process These two cases have a closed-form analytic solution. The excitation pulse spectrum is discretized, and the propagation from the grating to the image plane is solved separately for each spectral component using the angular spectrum of waves approach [16], taking into account the finite aperture of the objective lens This yields a four-dimensional distribution of complex field amplitudes Aimage(x, y, z, ω). When considering the sectioning capabilities of temporal focusing, we compare below to two cases: a standard point scanning multiphoton microscope, and a line-scanning two-photon microscope [18]

Temporal focusing with 1D patterned excitation

Temporal focusing with 2D patterned excitation

Conclusion