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.
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, neurology5, 6, ophthalmology7, 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.
1. System configuration
2. System alignment
3. A sample experimental procedure-in vivo OR-PAM of mouse ear vasculature
4. Functional OR-PAM of total concentration and oxygen saturation of hemoglobin
Besides sO2, HbT can be calculated by adding [HbR] and [HbO2] together, or it can be directly measured at isosbestic wavelengths of the molar extinction coefficient spectra of hemoglobin (e.g., 530 nm, 545 nm, 570 nm, and 584 nm)14, where HbR and HbO2 have equal molar extinction coefficients.
5. Representative Results:
Shown in Figure 1 is the vascular anatomy and sO2 in a living nude mouse ear imaged by dual-wavelength (561 and 570 nm) OR-PAM. The typical image acquisition time for dual-wavelength sO2 measurements of such a region of interest (image size: 10 mm x 10 mm; step size: 2.5 μm x 5 μm) is ~80 min.
Figure 1. In vivo optical-resolution photoacoustic microscopy. MAP images of (A) the total hemoglobin concentration showing the vascular anatomy (acquired at 570 nm) and (B) the hemoglobin oxygen saturation (acquired at 561 nm and 570 nm) in a nude mouse ear. (C) Close-up of the boxed region in panel (A). The scale bar in panel (A) applies to both (A) and (B).
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.
The authors have nothing to disclose.
The authors appreciate Dr. Lynnea Brumbaugh’s close reading of the manuscript. This work was sponsored by National Institutes of Health Grants R01 EB000712, R01 EB008085, R01 CA134539, U54 CA136398, and 5P60 DK02057933. Prof. Lihong V. Wang has a financial interest in Microphotoacoustics, Inc. and Endra, Inc., which, however, did not support this work.
Home-made acoustic-optical beam combiner: