High speed phase distortion measurement and compensation for focusing in space and time

ABSTRACT Random scattering and aberrations severely limit the imaging depth in optical microscopy. We introduce a rapid, parallel wavefront compensation technique that ef ficiently compensates even highly complex phase distortions. Using coherence gated backscattered light as a feedback signal, we focus light deep inside highly scattering brain tissue. We demonstrate that the same wavefront optimization technique can also be used to compensate spectral phase distortions in ultrashort laser pulses using nonlinear iterative feedback. We can restore transform limited pulse durations at any selected target location and compensate for dispersi on that has occurred in the optical train and within the sample. Keywords: Fluorescence microscopy, adaptive optics, imag ing through turbid media, ultrafast optics 1. INTRODUCTION Optical microscopy has become an indispensable tool in biol ogical sciences and medical a pplications since it can probe in vivo biological structures with molecular sensitivity [1]. However, with increasing imaging depth aberrations and random scattering deteriorate image quality, signal strength and signal to noise ratio (SNR) [2,3]. Thereby even nonlinear techniques like Two-photon microscopy, known for its superior penetration, are limited to less than 1mm depth in most biological tissues. But aberrations and scattering are deterministic processes that can be measured and compensated with fine enough wavefront control. Thereby high quality imaging can be restored deep inside highly scattering media, which has important implications to research fields that involve tissue imaging. Here we introduce a parallel wavefront optimization technique that allows fast compensation of complex aberrations. The concept is related to coherent optical adaptive technique, which was developed in the Hughes Laboratories to compensate atmospheric turbulence [4]. The basic idea can be explained as follows: two beams, one of them being phase modulated at frequency Z , are brought to interference at the sample locat ion. The signal from the target, which can be a strong scatterer or a fluorophore, is then also modulated at frequency Z , but will be likely out of phase with the input modulation signal. This phase difference is used as a feedback signal to maximize the interference at the target location. Multiple optical modes can be optimized in parallel using an array of phase modulators, each oscillating at a unique frequency. The technique has been successfu lly applied to Two-Photon imaging, restoring diffr action limited imaging at depths of up to 800 P m in biological tissue with exceptionally high SNR and dramatic gains in signal intensity [5]. However, it would be desirable to measure wavefront corrections at low light levels and without exciting fluorescence. Thereby phototoxic effects and photo-bleaching of fluorophores are minimized and more fluorescence is available for the actual imaging. To measure wavefront corrections without relying on fluorescence feedback signals, we combine optical coherence microscopy with the aforementioned parallel wavefront optimization technique [6]. We use the amplitude of low coherence backscattered light as the feedback signal for the wavefront optimization. Coherence gating and a confocal pinhole are used to confine the feedback signal in 3D. As a result, we can rapidly focus light through fixed brain tissue of up to 500 micron thickness. In analogy to spatial phase aberrations that prevent the form ation of a diffraction limited spot, spectral phase distortions can widen an ultrashort laser pulse (temporal width 10fs or le ss) beyond its transform limite d value in time. Ultrashort laser pulses can already experience significant dispersion when solely traveling through optical elements like lenses, making the pulse much wider when it arrives at the target location. Restoring transform limited pulse widths, however, is crucial in applications of ultrashort laser pulses like nonlinear microscopy, photochemistry and laser processing.