Super‐Low‐Dose Functional and Molecular Photoacoustic Microscopy

Abstract Photoacoustic microscopy can image many biological molecules and nano‐agents in vivo via low‐scattering ultrasonic sensing. Insufficient sensitivity is a long‐standing obstacle for imaging low‐absorbing chromophores with less photobleaching or toxicity, reduced perturbation to delicate organs, and more choices of low‐power lasers. Here, the photoacoustic probe design is collaboratively optimized and a spectral‐spatial filter is implemented. A multi‐spectral super‐low‐dose photoacoustic microscopy (SLD‐PAM) is presented that improves the sensitivity by ≈33 times. SLD‐PAM can visualize microvessels and quantify oxygen saturation in vivo with ≈1% of the maximum permissible exposure, dramatically reducing potential phototoxicity or perturbation to normal tissue function, especially in imaging of delicate tissues, such as the eye and the brain. Capitalizing on the high sensitivity, direct imaging of deoxyhemoglobin concentration is achieved without spectral unmixing, avoiding wavelength‐dependent errors and computational noises. With reduced laser power, SLD‐PAM can reduce photobleaching by ≈85%. It is also demonstrated that SLD‐PAM achieves similar molecular imaging quality using 80% fewer contrast agents. Therefore, SLD‐PAM enables the use of a broader range of low‐absorbing nano‐agents, small molecules, and genetically encoded biomarkers, as well as more types of low‐power light sources in wide spectra. It is believed that SLD‐PAM offers a powerful tool for anatomical, functional, and molecular imaging.

. Representative multi-wavelength lasers used in the SLD-PAM system. A) Dualwavelength of 532 nm and 558 nm laser source for vascular morphology and oxygen saturation (sO2) imaging. B) Single wavelength of 620 nm laser source for the exogenous molecular imaging (Evans blue). C) Single wavelength of 675 nm laser source for the deoxyhemoglobin (Deoxy-Hb) imaging. BPF, bandpass filter; CP, coupler; DM, dichroic mirror; HWP, half-wave plate; MR, mirror; NDF, neutral density filter; PBS, polarizing beam splitter; PD, photodiode; PM-SMF, polarization-maintaining single-mode fiber; SRS, stimulated Raman scattering.            Movie S1. Comparison of vascular imaging at 1-nJ laser pulse energy acquired by LD-PAM and SLD-PAM. The tracked green solid circle records the corresponding A-line signal at different positions.

Supplementary Text S1. Multi-wavelength low-cost laser systems
For the dual-wavelength optical path, the output laser beam from a pump laser (VPFL-G-20, Spectra-Physics) is split into two paths by a polarization beam splitter (PBS, PBS051, Thorlabs, Inc.), and the energy ratio of the two paths is adjusted with a half-wave plate (HWP, GCL-060633, Daheng Optics). One path transmits in free space, and the other one is coupled into a 60-m polarization-maintaining single-mode fiber (PM-SMF, HB450-SC, Fibercore Limited). Via stimulated Raman scattering (SRS), the 532-nm pulse in the 60-m fiber generates a 558-nm pulse with a 300-ns delay. An HWP is placed before the 60-m PM-SMF to adjust the polarization of the 532-nm light to maximize the SRS efficiency. The 532-nm beam is combined with the delayed 558-nm beam using a dichroic mirror (DM, T550lpxr-UF1, Chroma Technology), and the dualwavelength is coupled into a 2-m PM-SMF (P1-460Y-FC, Thorlabs).
For the 620-nm and 675-nm lasers, the pump laser beam is coupled into a 20-m PM-SMF (HB450-SC, Fibercore Limited), and the HWP is placed before the 20-m PM-SMF to adjust the polarization of the pump laser to adjust the SRS efficiency. Two different optical filters are used to pass the 620 nm (#67-083 dichroic filter, Edmund Optics) and 675 nm (ET675/20m bandwidth filter, Chroma) wavelengths. The filtered laser beam is coupled into a 2-m PM-SMF for photoacoustic imaging.
Finally, ̂4 is re-assigned to their original positions of both volumetric data. Sub-volume data from different 4D groups or within the same group may be overlapped. Weighting factors are used to generate new averaged volumetric data as The molar extinction coefficient matrix is written as

Supplementary Text S4. Spatial resolution and imaging depth measurement
The lateral resolution of our system was measured by scanning the stainless-steel blade across the sharp edges. We fitted the amplitude of the photoacoustic signal across the sharp edge to obtain edge spread functions (ESF). Then the ESF function was derived to calculate the line spread functions (LSF). Finally, the full width at half maximum (FWHM) of the LSF was calculated for quantizing the lateral resolution. The axial resolution was measured by scanning a 10-μm-diameter tungsten filament. Then the A-line signals were extracted and the FWHM was calculated after a Hilbert transformation for quantizing the axial resolution.
To measure maximal imaging depth, we obliquely inserted a segment of a tungsten filament with a diameter of 250 μm into a fresh chicken breast tissue. The maximal imaging depth is determined by the 6-dB signal-to-noise ratio (SNR) compared with the background noise.