Optical-resolution photoacoustic microscopy can visualize wavelength-dependent optical absorption at the cellular level. However, this technique suffers from a limited depth of field 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, which can extend the depth of field to around a 28-fold Rayleigh length via customized diffractive optical elements. These diffractive optical elements generate a needle-shaped beam with a well-maintained beam diameter, a uniform axial intensity distribution and negligible sidelobes. The advantage of using needle-shaped beam photoacoustic microscopy is demonstrated via 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. This approach provides new perspectives for slide-free intraoperative pathological imaging and in vivo organ-level imaging.
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The data that support the findings of this study are available within the paper and its Supplementary Information. The raw data are too large to be publicly shared, yet they are available for research purposes from the corresponding authors upon reasonable request.
The code that supports the plots and images within this paper is available from the corresponding author upon reasonable request.
Glaser, A. K. et al. Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat. Biomed. Eng. 1, 0084 (2017).
Liu, S. & Hua, H. Extended depth-of-field microscopic imaging with a variable focus microscope objective. Opt. Express 19, 353–362 (2011).
Li, B., Qin, H., Yang, S. & Xing, D. In vivo fast variable focus photoacoustic microscopy using an electrically tunable lens. Opt. Express 22, 20130–20137 (2014).
Xiao, S., Tseng, H., Gritton, H., Han, X. & Mertz, J. Video-rate volumetric neuronal imaging using 3D targeted illumination. Sci. Rep. 8, 7921 (2018).
Shain, W. J., Vickers, N. A., Goldberg, B. B., Bifano, T. & Mertz, J. Extended depth-of-field microscopy with a high-speed deformable mirror. Opt. Lett. 42, 995–998 (2017).
Patel, K. B. et al. High-speed light-sheet microscopy for the in-situ acquisition of volumetric histological images of living tissue. Nat. Biomed. Eng. 6, 569–583 (2022).
Descloux, A. et al. Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy. Nat. Photon. 12, 165–172 (2018).
Geissbuehler, S. et al. Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging. Nat. Commun. 5, 5830 (2014).
Abrahamsson, S. et al. Fast multicolor 3D imaging using aberration-corrected multifocus microscopy. Nat. Methods 10, 60–63 (2013).
Oudjedi, L. et al. Astigmatic multifocus microscopy enables deep 3D super-resolved imaging. Biomed. Opt. Express 7, 2163–2173 (2016).
Zheng, G., Horstmeyer, R. & Yang, C. Wide-field, high-resolution Fourier ptychographic microscopy. Nat. Photon. 7, 739–745 (2013).
Planchon, T. A. et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–423 (2011).
Gao, L., Shao, L., Chen, B.-C. & Betzig, E. 3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy. Nat. Protoc. 9, 1083–1101 (2014).
Jia, S., Vaughan, J. C. & Zhuang, X. Isotropic three-dimensional super-resolution imaging with a self-bending point spread function. Nat. Photon. 8, 302–306 (2014).
Hu, Y., Chen, Z., Xiang, L. & Xing, D. Extended depth-of-field all-optical photoacoustic microscopy with a dual non-diffracting Bessel beam. Opt. Lett. 44, 1634–1637 (2019).
Yang, J., Gong, L., Shen, Y. & Wang, L. V. Synthetic Bessel light needle for extended depth-of-field microscopy. Appl. Phys. Lett. 113, 181104 (2018).
Thériault, G., Koninck, Y. D. & McCarthy, N. Extended depth of field microscopy for rapid volumetric two-photon imaging. Opt. Express 21, 10095–10104 (2013).
Thériault, G., Cottet, M., Castonguay, A., McCarthy, N. & De Koninck, Y. Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging. Front. Cell. Neurosci. https://doi.org/10.3389/fncel.2014.00139 (2014).
Snoeyink, C. Imaging performance of Bessel beam microscopy. Opt. Lett. 38, 2550–2553 (2013).
Wu, Y. et al. Three-dimensional virtual refocusing of fluorescence microscopy images using deep learning. Nat. Methods 16, 1323–1331 (2019).
Jin, L. et al. Deep learning extended depth-of-field microscope for fast and slide-free histology. Proc. Natl Acad. Sci. USA 117, 33051–33060 (2020).
Zhou, Y., Sun, N. & Hu, S. Deep learning-powered Bessel-beam multi-parametric photoacoustic microscopy. IEEE Trans. Med. Imaging https://doi.org/10.1109/TMI.2022.3188739 (2022).
Wang, L. V. & Hu, S. Photoacoustic tomography: in vivo imaging from organelles to organs. Science 335, 1458–1462 (2012).
Wang, L. V. & Yao, J. A practical guide to photoacoustic tomography in the life sciences. Nat. Methods 13, 627–638 (2016).
Wong, T. T. W. et al. Fast label-free multilayered histology-like imaging of human breast cancer by photoacoustic microscopy. Sci. Adv. 3, e1602168 (2017).
Shi, J. et al. High-resolution, high-contrast mid-infrared imaging of fresh biological samples with ultraviolet-localized photoacoustic microscopy. Nat. Photon. 13, 609–615 (2019).
Wong, T. T. W. et al. Label-free automated three-dimensional imaging of whole organs by microtomy-assisted photoacoustic microscopy. Nat. Commun. 8, 1386 (2017).
Zhang, C., Zhang, Y. S., Yao, D.-K., Xia, Y. & Wang, L. V. Label-free photoacoustic microscopy of cytochromes. J. Biomed. Opt. 18, 020504 (2013).
Yao, J. et al. High-speed label-free functional photoacoustic microscopy of mouse brain in action. Nat. Methods 12, 407–410 (2015).
Cao, R. et al. Functional and oxygen-metabolic photoacoustic microscopy of the awake mouse brain. Neuroimage 150, 77–87 (2017).
Cao, R. et al. Photoacoustic microscopy reveals the hemodynamic basis of sphingosine 1-phosphate-induced neuroprotection against ischemic stroke. Theranostics 8, 6111–6120 (2018).
Zhou, Y., Xing, W., Maslov, K. I., Cornelius, L. A. & Wang, L. V. Handheld photoacoustic microscopy to detect melanoma depth in vivo. Opt. Lett. 39, 4731–4734 (2014).
He, Y. et al. Label-free imaging of lipid-rich biological tissues by mid-infrared photoacoustic microscopy. J. Biomed. Opt. 25, 106506 (2020).
Buma, T., Conley, N. C. & Choi, S. W. Multispectral photoacoustic microscopy of lipids using a pulsed supercontinuum laser. Biomed. Opt. Express 9, 276–288 (2017).
Maslov, K., Zhang, H. F., Hu, S. & Wang, L. V. Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries. Opt. Lett. 33, 929–931 (2008).
Hu, S., Maslov, K. & Wang, L. V. Second-generation optical-resolution photoacoustic microscopy with improved sensitivity and speed. Opt. Lett. 36, 1134–1136 (2011).
Park, B. et al. Reflection-mode switchable subwavelength Bessel-beam and Gaussian-beam photoacoustic microscopy in vivo. J. Biophotonics 12, e201800215 (2019).
Jiang, B., Yang, X. & Luo, Q. Reflection-mode Bessel-beam photoacoustic microscopy for in vivo imaging of cerebral capillaries. Opt. Express 24, 20167–20176 (2016).
Shi, J., Wang, L., Noordam, C. & Wang, L. V. Bessel-beam Grueneisen relaxation photoacoustic microscopy with extended depth of field. J. Biomed. Opt. 20, 116002 (2015).
Xu, Z. et al. Cortex-wide multiparametric photoacoustic microscopy based on real-time contour scanning. Neurophotonics 6, 035012 (2019).
Ning, B. et al. Ultrasound-aided multi-parametric photoacoustic microscopy of the mouse brain. Sci. Rep. 5, 18775 (2015).
Yang, X., Jiang, B., Song, X., Wei, J. & Luo, Q. Fast axial-scanning photoacoustic microscopy using tunable acoustic gradient lens. Opt. Express 25, 7349–7357 (2017).
Liu, S. et al. GPU-accelerated two dimensional synthetic aperture focusing for photoacoustic microscopy. APL Photonics 3, 026101 (2018).
Jeon, S., Park, J., Managuli, R. & Kim, C. A novel 2-D synthetic aperture focusing technique for acoustic-resolution photoacoustic microscopy. IEEE Trans. Med. Imaging 38, 250–260 (2019).
Amjadian, M., Mostafavi, S. M., Chen, J., Wang, L. & Luo, Z. Super-resolution photoacoustic microscopy via modified phase compounding. IEEE Trans. Med. Imaging https://doi.org/10.1109/TMI.2022.3184711 (2022).
Amjadian, M. et al. Super-resolution photoacoustic microscopy using structured-illumination. IEEE Trans. Med. Imaging 40, 2197–2207 (2021).
Yang, J. et al. Motionless volumetric photoacoustic microscopy with spatially invariant resolution. Nat. Commun. 8, 780 (2017).
L.V.W. was sponsored by the United States National Institutes of Health grants R01 EB028277, U01 NS099717 (BRAIN Initiative) and R35 CA220436 (Outstanding Investigator Award). A.d.l.Z. was supported by the National Institutes of Health grants DP50D012179 and K23CA211793, the United States National Science Foundation (NSF 1438340) and the United States Air Force (FA9550–15–1–0007).
L.V.W. has a financial interest in MicroPhotoAcoustics, CalPACT and Union Photoacoustic Technologies, although they did not support this work. The remaining authors declare no competing interests.
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Cao, R., Zhao, J., Li, L. et al. Optical-resolution photoacoustic microscopy with a needle-shaped beam. Nat. Photon. 17, 89–95 (2023). https://doi.org/10.1038/s41566-022-01112-w
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