Fluorescence imaging beyond the ballistic regime by ultrasound-pulse-guided digital phase conjugation

Abstract

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.

One-Wave Optical Phase Conjugation Mirror by Actively Coupling Arbitrary Light Fields into a Single-Mode Reflector

“…The PCM conjugates the phase on the mirror surface, and causes the reflected light to rewind through the path that the input light has travelled. This "time-reversal" property of PCMs has been tried in various optical applications such as aberration canceling [2,3], pulse compression [4,5], laser resonators [6,7], holography [8], and the suppression of multiple light scattering in biological tissues [9][10][11][12][13][14]. More direct demonstrations have also been performed using acoustic waves [15] and microwaves [16], where conventional electronics can directly measure and generate the phase profile in real time.…”

Effects of digital phase-conjugate light intensity on time-reversal imaging through animal tissue

“…Therefore, some technique to suppress scattering effects is necessary for practical applications of light in these fields. In recent years, a principle of scattering suppression using the time-reverse capability of phaseconjugate light has been proposed [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. Various attempts based on this principle have been undertaken, such as the time-reversed ultrasonically encoded (TRUE) method [1,[4][5][6][7][8].…”

Frequency-swept time-reversed ultrasonically encoded optical focusing

“…4-6 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.Here, we propose a method called frequency-swept TRUE focusing, which takes advantage of the multiple recorded frames used for encoded-light detection to accelerate TRUE focal scanning. By sweeping the frequency of both the ultrasound and the light at the same time, we achieve simultaneous recording of multiple wavefronts, corresponding to different positions along the acoustic axis, without sacrificing SNR and using the same number of camera frames.…”

“…4-6 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.…”

“…[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.…”