Three-dimensional Optical-resolution Photoacoustic Microscopy

Summary

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Abstract

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable.

The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)¹, where the optical irradiation is focused to the diffraction limit to achieve cellular¹ or even subcellular² level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy³ or with optical-scattering-based OCT⁴ (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra.

Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology^{5, 6}, ophthalmology^{7, 8}, vascular biology⁹, and dermatology¹⁰. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Protocol

1. System configuration

1. Optical irradiation
   1. Optical irradiation source: a diode-pumped solid-state pulsed laser (INNOSLAB, Edgewave) and a dye laser (CBR-D, Sirah).
   2. The output laser beam (pulse width: 7 ns) is focused by a condenser lens (LA1131, Thorlabs) to pass through a 50-μm pinhole (P50C, Thorlabs).
   3. The pinhole is positioned slightly away from the focus of the condenser lens to match the pinhole diameter with the fundamental-mode beam diameter for effective spatial filtering.
   4. The filtered beam is attenuated by a neutral-density filter (NDC-50C-2M, Thorlabs) and then coupled into a single-mode opt....

Discussion

In this video, we provide a detailed instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, and typical experimental procedures. Label-free, noninvasive OR-PAM has enabled studies of microvascular functioning and metabolism on a single capillary basis and thereby holds the potential to expand our understanding of microcirculation-related physiology and pathology. Microphotoacoustics is currently manufacturing this OR-PAM system.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Home-made acoustic-optical beam combiner:
right-angle prism	Edmund Scientific	NT32-545
rhomboid prism	Edmund Scientific	NT49-419
silicone oil	Clearco Products	1000cSt
OR-PAM system	Microphotoacoustics