Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Optical-resolution photoacoustic microscopy with a needle-shaped beam


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.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Principle of NB-PAM with a customized DOE.
Fig. 2: Characterization of UV-NB-PAM in comparison with that of conventional UV-GB-PAM.
Fig. 3: Volumetric imaging of carbon particles using UV-GB-PAM and UV-NB-PAM with a 200 × 1.2 μm NB.
Fig. 4: Depth-resolved imaging of carbon fibres for VIS-GB-PAM and for VIS-NB-PAM with the 1,000 × 2.3 μm NB.
Fig. 5: Label-free UV-GB-PAM and UV-NB-PAM with the 200 × 1.2 μm NB for slide-free fresh mouse lung and brain samples.
Fig. 6: In vivo label-free depth-encoded VIS-NB-PAM with the 1,000 × 2.3 μm NB and VIS-GB-PAM of brain vasculature with and without a skull.

Data availability

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.

Code availability

The code that supports the plots and images within this paper is available from the corresponding author upon reasonable request.


  1. Glaser, A. K. et al. Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat. Biomed. Eng. 1, 0084 (2017).

    Google Scholar 

  2. Liu, S. & Hua, H. Extended depth-of-field microscopic imaging with a variable focus microscope objective. Opt. Express 19, 353–362 (2011).

    ADS  Google Scholar 

  3. 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).

    ADS  Google Scholar 

  4. Xiao, S., Tseng, H., Gritton, H., Han, X. & Mertz, J. Video-rate volumetric neuronal imaging using 3D targeted illumination. Sci. Rep. 8, 7921 (2018).

    ADS  Google Scholar 

  5. 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).

    ADS  Google Scholar 

  6. 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).

    Google Scholar 

  7. 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).

    ADS  Google Scholar 

  8. Geissbuehler, S. et al. Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging. Nat. Commun. 5, 5830 (2014).

    ADS  Google Scholar 

  9. Abrahamsson, S. et al. Fast multicolor 3D imaging using aberration-corrected multifocus microscopy. Nat. Methods 10, 60–63 (2013).

    Google Scholar 

  10. Oudjedi, L. et al. Astigmatic multifocus microscopy enables deep 3D super-resolved imaging. Biomed. Opt. Express 7, 2163–2173 (2016).

    Google Scholar 

  11. Zheng, G., Horstmeyer, R. & Yang, C. Wide-field, high-resolution Fourier ptychographic microscopy. Nat. Photon. 7, 739–745 (2013).

    ADS  Google Scholar 

  12. Planchon, T. A. et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–423 (2011).

    Google Scholar 

  13. 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).

    Google Scholar 

  14. 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).

    ADS  Google Scholar 

  15. 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).

    ADS  Google Scholar 

  16. 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).

    ADS  Google Scholar 

  17. Thériault, G., Koninck, Y. D. & McCarthy, N. Extended depth of field microscopy for rapid volumetric two-photon imaging. Opt. Express 21, 10095–10104 (2013).

    ADS  Google Scholar 

  18. 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. (2014).

  19. Snoeyink, C. Imaging performance of Bessel beam microscopy. Opt. Lett. 38, 2550–2553 (2013).

    ADS  Google Scholar 

  20. Wu, Y. et al. Three-dimensional virtual refocusing of fluorescence microscopy images using deep learning. Nat. Methods 16, 1323–1331 (2019).

    Google Scholar 

  21. 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).

    ADS  Google Scholar 

  22. Zhou, Y., Sun, N. & Hu, S. Deep learning-powered Bessel-beam multi-parametric photoacoustic microscopy. IEEE Trans. Med. Imaging (2022).

  23. Wang, L. V. & Hu, S. Photoacoustic tomography: in vivo imaging from organelles to organs. Science 335, 1458–1462 (2012).

    ADS  Google Scholar 

  24. Wang, L. V. & Yao, J. A practical guide to photoacoustic tomography in the life sciences. Nat. Methods 13, 627–638 (2016).

    Google Scholar 

  25. 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).

    ADS  Google Scholar 

  26. 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).

    ADS  Google Scholar 

  27. 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).

    ADS  Google Scholar 

  28. 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).

    ADS  Google Scholar 

  29. Yao, J. et al. High-speed label-free functional photoacoustic microscopy of mouse brain in action. Nat. Methods 12, 407–410 (2015).

    Google Scholar 

  30. Cao, R. et al. Functional and oxygen-metabolic photoacoustic microscopy of the awake mouse brain. Neuroimage 150, 77–87 (2017).

    Google Scholar 

  31. Cao, R. et al. Photoacoustic microscopy reveals the hemodynamic basis of sphingosine 1-phosphate-induced neuroprotection against ischemic stroke. Theranostics 8, 6111–6120 (2018).

    Google Scholar 

  32. 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).

    ADS  Google Scholar 

  33. He, Y. et al. Label-free imaging of lipid-rich biological tissues by mid-infrared photoacoustic microscopy. J. Biomed. Opt. 25, 106506 (2020).

    ADS  Google Scholar 

  34. 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).

    Google Scholar 

  35. 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).

    ADS  Google Scholar 

  36. Hu, S., Maslov, K. & Wang, L. V. Second-generation optical-resolution photoacoustic microscopy with improved sensitivity and speed. Opt. Lett. 36, 1134–1136 (2011).

    ADS  Google Scholar 

  37. Park, B. et al. Reflection-mode switchable subwavelength Bessel-beam and Gaussian-beam photoacoustic microscopy in vivo. J. Biophotonics 12, e201800215 (2019).

    Google Scholar 

  38. 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).

    ADS  Google Scholar 

  39. 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).

    ADS  Google Scholar 

  40. Xu, Z. et al. Cortex-wide multiparametric photoacoustic microscopy based on real-time contour scanning. Neurophotonics 6, 035012 (2019).

    Google Scholar 

  41. Ning, B. et al. Ultrasound-aided multi-parametric photoacoustic microscopy of the mouse brain. Sci. Rep. 5, 18775 (2015).

    ADS  Google Scholar 

  42. 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).

    ADS  Google Scholar 

  43. Liu, S. et al. GPU-accelerated two dimensional synthetic aperture focusing for photoacoustic microscopy. APL Photonics 3, 026101 (2018).

    ADS  Google Scholar 

  44. 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).

    Google Scholar 

  45. Amjadian, M., Mostafavi, S. M., Chen, J., Wang, L. & Luo, Z. Super-resolution photoacoustic microscopy via modified phase compounding. IEEE Trans. Med. Imaging (2022).

  46. Amjadian, M. et al. Super-resolution photoacoustic microscopy using structured-illumination. IEEE Trans. Med. Imaging 40, 2197–2207 (2021).

    Google Scholar 

  47. Yang, J. et al. Motionless volumetric photoacoustic microscopy with spatially invariant resolution. Nat. Commun. 8, 780 (2017).

    ADS  Google Scholar 

Download references


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).

Author information

Authors and Affiliations



R.C. and L.V.W. designed the experiment. R.C., L.L. and Y.Z. built the PAM system. J.Z. designed and fabricated the DOEs. L.D. contributed to the mask preparation and wafer dividing. L.J. and Q.Z. manufactured the ultrasonic transducer. R.C. prepared the sample and animals and performed the imaging experiment. R.C., S.D. and Y.L. contributed to image processing. L.V.W. and A.d.l.Z. supervised the project. All authors were involved in discussions and manuscript preparation.

Corresponding authors

Correspondence to Adam de la Zerda or Lihong V. Wang.

Ethics declarations

Competing interests

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.

Peer review

Peer review information

Nature Photonics thanks Ruiqing Ni and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Note, Figs. 1–9 and Tables 1–3.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Cao, R., Zhao, J., Li, L. et al. Optical-resolution photoacoustic microscopy with a needle-shaped beam. Nat. Photon. 17, 89–95 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing