Optical-resolution photoacoustic microscopy with a needle-shaped beam

Abstract

Optical-resolution photoacoustic microscopy (OR-PAM) can visualize wavelength-dependent optical absorption at the cellular level. However, OR-PAM suffers from a limited depth of field (DOF) due to the tight focus of the optical excitation beam, making it challenging to acquire high-resolution images of samples with uneven surfaces or high-quality volumetric images without z-scanning. To overcome this limitation, we propose needle-shaped beam photoacoustic microscopy (NB-PAM), which can extend the DOF to up to ~28-fold Rayleigh lengths via customized diffractive optical elements (DOEs). The DOE generate a needle beam with a well-maintained beam diameter, a uniform axial intensity distribution, and negligible sidelobes. The advantage of using NB-PAM is demonstrated by both histology-like imaging of fresh slide-free organs using a 266 nm laser and in vivo mouse brain vasculature imaging using a 532 nm laser. The approach provides new perspectives for slide-free intraoperative pathological imaging and in-vivo organ-level imaging.

Figure 1.. Principle of NB-PAM with a customized DOE.

a DOE phase pattern for NB composed of multiple phases of M foci. b Principle of a DOE combining M foci to form the desired NB. c YZ-profile of a Gaussian beam with a focal spot size of 1.2 μm at 266 nm and XY-profile at different z positions. Scale bars, 10 μm. d YZ-profile of the needle-shaped beam generated by DOE for 200 μm×1.2 μm NB at 266 nm and XY-profile at different z positions. Scale bars, 10 μm. e Experimental setup of NB-PAM system. BS, beam sampler; PH, pinhole; CL, correction lens; UT, ultrasonic transducer; DAQ, data acquisition. f Principle of conventional GB-PAM. g Principle of NB-PAM. In f and g, simulated YZ-projection images of uniformly distributed microspheres with a diameter of 7 μm show the difference between the GB-PAM with 0.16 numerical aperture (NA) and the NB-PAM with 1000 μm×2.3 μm DOE.

Figure 2.. Characterization of UV-NB-PAM in comparison with that of conventional UV-GB-PAM.

a Images of a 1951 USAF resolution target at different axial positions acquired by UV-GB-PAM with 0.16 NA and UV-NB-PAM with 200 μm×1.2 μm NB. The focal plane of UV-GB-PAM is at $\mathrm{z}=0\mathrm{\mu }\mathrm{m}.$ b, c Close-ups of images acquired by conventional UV-GB-PAM at $\mathrm{z}=0\mathrm{\mu }\mathrm{m}$ and $\mathrm{z}=+105\mathrm{\mu }\mathrm{m}$, respectively. d, e Close-ups of images acquired by UV-NB-PAM at $\mathrm{z}=0\mathrm{\mu }\mathrm{m}$ and $\mathrm{z}=+105\mathrm{\mu }\mathrm{m}$, respectively. f Profile of element 6 from group 7 measured by conventional UV-GB-PAM and UV-NB-PAM at $\mathrm{z}=0\mathrm{\mu }\mathrm{m}$. g Lateral FWHM resolutions measured by imaging a sharp edge and quantified by edge spread functions and derived line spread functions (inset).

Figure 3.. Volumetric imaging of carbon particles by UV-NB-PAM with 200 μm×1.2 μm NB and UV-GB-PAM.

XY-MAP images of carbon particles acquired by a UV-GB-PAM and b UV-NB-PAM. Scale bars, 250 μm. c Virtually sectioned XY-MAP images at different depths by UV-GB-PAM and UV-NB-PAM after time-dependent gain compensation. Scale bars, 50 μm.

Figure 4.. Depth-resolved imaging of carbon fibers by VIS-GB-PAM and VIS-NB-PAM with 1000 μm×2.3 μm NB.

a VIS-GB-PAM and b VIS-NB-PAM of ~6 μm carbon fibers randomly distributed in an agarose block. Scale bar, 1 mm. The comparison of c-f Close-up VIS-GB-PAM images and g-j close-up VIS-GB-PAM images demonstrates the improved DOF Scale bars, 250 μm.

Figure 5.. Label-free UV-GB-PAM and UV-NB-PAM with 200 μm×1.2 μm NB of slide-free fresh mouse lung and mouse brain.

a UV-GB-PAM and b UV-NB-PAM of a fresh mouse lung embedded in agarose block. Scale bars, 250 μm. Close-up images of the area indicated by yellow boxes in a and b show the difference between UV-GB-PAM and UV-NB-PAM. Scale bars, 50 μm. c UV-GB-PAM and d UV-NB-PAM of a fresh mouse brain embedded in agarose block. Scale bars, $500\mu \mathrm{m}$. Close-up images of representative areas in UV-GB-PAM show the compromised image quality due to out-of-focus locations. Close-up images in UV-NB-PAM show the well-maintained resolution in corresponding areas. Scale bars, 100 μm.

Figure 6.. In vivo label-free depth-encoded VIS-NB-PAM with 1000 μm×2.3 μm NB and VIS-GB-PAM of brain vasculature with and without a skull.

a VIS-GB-PAM and b VIS-NB-PAM of a mouse brain without a skull show the depth-encoded brain vasculature. c VIS-GB-PAM and d VIS-NB-PAM of a mouse with an intact skull show the depth-encoded brain vasculature. Both mouse brain vasculature images by VIS-NB-PAM show more blood vessels in the edge areas than the corresponding conventional VIS-GB-PAM. Scale bars, 1 mm.