Fluorescence imaging has revolutionized biomedical research over the past three decades. Its high molecular specificity and unrivaled single molecule level sensitivity have enabled breakthroughs in a variety of research fields. For in vivo applications, its major limitation is the superficial imaging depth as random scattering in biological tissues causes exponential attenuation of the ballistic component of a light wave. Here we present fluorescence imaging beyond the ballistic regime by combining single cycle pulsed ultrasound modulation and digital optical phase conjugation. We demonstrate a near isotropic 3D localized sound-light interaction zone. With the exceptionally high optical gain provided by the digital optical phase conjugation system, we can deliver sufficient optical power to a focus inside highly scattering media for not only fluorescence imaging but also a variety of linear and nonlinear spectroscopy measurements. This technology paves the way for many important applications in both fundamental biology research and clinical studies.

In this work, we report a novel high capacity (number of degrees of freedom) open loop adaptive optics method, termed digital optical phase conjugation (DOPC), which provides a robust optoelectronic optical phase conjugation (OPC) solution. We showed that our prototype can phase conjugate light fields with ~3.9 x 10−3 degree accuracy over a range of ~3 degrees and can phase conjugate an input field through a relatively thick turbid medium (μsl ~13). Furthermore, we employed this system to show that the reversing of random scattering in turbid media by phase conjugation is surprisingly robust and accommodating of phase errors. An OPC wavefront with significant spatial phase errors (error uniformly distributed from – π/2 to π/2) can nevertheless allow OPC reconstruction through a scattering medium with ~40% of the efficiency achieved with phase error free OPC.

Biological tissues are rarely transparent, presenting major challenges for deep tissue optical microscopy. The achievable imaging depth is fundamentally limited by wavefront distortions caused by aberration and random scattering. Here, we report an iterative wavefront compensation technique that takes advantage of the nonlinearity of multiphoton signals to determine and compensate for these distortions and to focus light inside deep tissues. Different from conventional adaptive optics methods, this technique can rapidly measure highly complicated wavefront distortions encountered in deep tissue imaging and provide compensations for not only aberration but random scattering. The technique is tested with a variety of highly heterogeneous biological samples including mouse brain tissue, skull, and lymph nodes. We show that high quality three-dimensional imaging can be realized at depths beyond the reach of conventional multiphoton microscopy and adaptive optics methods, albeit over restricted distances for a given correction. Moreover, the required laser excitation power can be greatly reduced in deep tissues, deviating from the power requirement of ballistic light excitation and thus significantly reducing photo damage to the biological tissue.immunology | neuron imaging | nonlinear imaging | lymphocyte imaging | nonlinear iterative feedback

In vivo imaging at high spatiotemporal resolution holds the key to the fundamental understanding of complex biological systems. Integrating an optical phase-locked ultrasound lens into a conventional two-photon fluorescence microscope, we achieved microsecond scale axial scanning, which enabled high-speed volumetric imaging. We applied this system to multicolor volumetric imaging of fast processes, including calcium dynamics in the cerebral cortex of behaving mice, and transient morphology changes and trafficking of immune cells.