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.

Figure 1

a Experimental scheme of fluorescence microscopy by single cycle ultrasound pulse guided DOPC. b Experiment setup. λ/2, half wave plate; PBS, polarizing beam splitter; BB, beam block; BS, non-polarizing beam splitter; BE, beam expander; M, mirror; BP, band-pass filter; LP, long-pass filter; L1, f = 35 mm lens; L2, f = 50 mm lens; D, fluorescence detector; Stage, 3-axis motorized translation stage. The pixel size of both the SLM and the CMOS camera is 8 microns. The distance from the sound focus to the SLM is 305 mm.

Figure 2

a Measured transverse PSF through 2 mm thick tissue phantoms (μ_s=6.42/mm, g=0.9306). b Measured axial PSF. c and d are the corresponding images resampled with bicubic interpolation. e–g Gaussian fitting of the measured PSF.

Figure 3

a Direct optical imaging of the fluorescence hole array without tissue phantoms. b Direct optical imaging of the fluorescence hole array surrounded by 2 mm thick tissue phantoms (μ_s=6.42/mm, g=0.9306). c Image acquired with ultrasound pulse guided DOPC through tissue phantoms. The laser power on the sample was 25 mW during sound modulation and 10 mW during fluorescence excitation. d Bicubic interpolation of c. e 2D convolution of a with Fig. 2 c. The tissue phantoms were aligned parallel to the yz plane in Fig. 1 b.