Focused fluorescence excitation with time-reversed ultrasonically encoded light and imaging in thick scattering media

Abstract

Scattering dominates light propagation in biological tissue, and therefore restricts both resolution and penetration depth in optical imaging within thick tissue. As photons travel into the diffusive regime—typically 1 mm beneath human skin, their trajectories transition from ballistic to diffusive due to increased number of scattering events, which makes it impossible to focus, much less track, photon paths. Consequently, imaging methods that rely on controlled light illumination are ineffective in deep tissue. This problem has recently been addressed by a novel method capable of dynamically focusing light in thick scattering media via time reversal of ultrasonically encoded (TRUE) diffused light. Here, using photorefractive materials as phase conjugate mirrors, we show a direct visualization and dynamic control of optical focusing with this light delivery method, and demonstrate its application for focused fluorescence excitation and imaging in thick turbid media. These abilities are increasingly critical to understanding the dynamic interactions of light with biological matter and processes at different system levels, as well as their applications for biomedical diagnosis and therapy.

Wavefront Shaping and Its Application to Enhance Photoacoustic Imaging

“…To overcome these limitations, researchers have developed virtual guide stars so that diffused photons emerging or propagating through the ROI can be specifically tagged or preferentially detected. An example is focused ultrasound modulation based on the acousto-optic effect [40,71], where the ultrasonically frequency-shifted photons are phase conjugated and make their way back to the ultrasonic focus-it is, thus, also the optical focus. Similar ultrasonically determined optical focusing can also be achieved if the ultrasonically frequency-shifted photons are extracted and its strength is used as the feedback signal for iterative optimization [27,82,83].…”

“…For that purpose, researchers have proposed injected or embedded probes, such as small optical sensors [66,67], fluorescence particles [68,69], and nonlinear beads [57], ultrasound mediation [40,58,61,70,71], as well as absorption perturbation [22,25,46,48,[72][73][74][75][76]. Among them, PA signal has been proved very attractive [22,25,28,47,48,53,55,73,74,76], as it is noninvasive, relatively deeply-penetrating, and it can pinpoint the focal spot accurately (the acoustic and optical foci overlap).…”

Frequency-swept time-reversed ultrasonically encoded optical focusing

“…3,4 In TRUE focusing, a focused ultrasonic (US) pulse, applied inside a turbid sample, frequency modulates (or encodes) light within the acoustic volume. A phase-conjugated version of the encoded wavefront is then generated, using either analog or digital phase conjugate mirrors (PCMs).…”

“…[3][4][5][6] Digital PCMs, consisting of a camera and a spatial light modulator (SLM), are attractive for higher energy focusing. [4][5][6] To record the encoded wavefront, a reference beam interferes with the encoded light on the camera, causing the intensity of the interferogram to beat at their difference frequency. 6 The encoded wavefront is extracted from the beat, and then its phase-conjugated version is reproduced using the SLM.…”

“…[4][5][6] However, one of the challenges of applying digital TRUE focusing for imaging was the long time (several seconds) taken to generate a single optical focus. 5,6 The low signal-to-noise ratio (SNR) of the encoded-light detection typically mandates that multiple frames of interferograms be recorded and averaged to obtain a single encoded wavefront.…”