Three-Dimensional Optical-Resolution Photoacoustic Microscopy - Biomedical Optical Imaging Technologies: Design and Applications - page 56

waves, which can be detected to map optical absorption [ 2 ]. Thus, the photoacoustic
effect provides an exquisite way to resolve the optical absorption distribution in
biological tissue ultrasonically. Taking advantage of the much weaker ultrasonic
scattering in tissues (1,000 times weaker than optical scattering), researchers have
developed reconstruction-based photoacoustic computed tomography (PACT) [ 3 ]
and focused-scanning-based photoacoustic microscopy (PAM) [ 4 , 5 ] for deep tissue
imaging in the optical quasidiffusive or diffusive regime. When the spatial resolution
in all directions is entirely determined by the photoacoustic wave, the technology
is called acoustic-resolution PAT. Although having achieved great success, the
acoustic-resolution PAT is inadequate for examining the anatomy and function
of biological tissues at the cellular or subcellular level. To fill this gap, we have
developed optical-resolution photoacoustic microscopy (OR-PAM), improving the
lateral resolution of PAT to the cellular [ 6 ] or even subcellular scale [ 7 ]. As a
unique optical absorption microscopy technology and a valuable complement to the
existing technologies, OR-PAM has demonstrated broad biomedical applications
since its invention [ 8 - 14 ]. Taking advantage of the strong optical absorption of
endogenous hemoglobin, OR-PAM enables label-free, noninvasive, and volumetric
microvascular imaging down to single capillaries, providing both anatomical (such
as vessel diameter, connectivity, and tortuosity) and functional (such as hemoglobin
oxygen saturation (sO 2 ) and blood flow velocity) information [ 8 - 11 , 15 ]. With the
aid of exogenous molecular contrast agents, OR-PAM is also capable of molecular
imaging [ 13 ].
This chapter provides a detailed description of the design, operation, and
application of OR-PAM, including the principle, system design, system configura-
tion, system alignment, experimental procedures, laser safety, functional imaging
scheme, recent technical advances, and sample biomedical applications. Future
directions of OR-PAM development are also predicted at the conclusion of this
chapter.

2.2 Principle and System Design

In PAM, volumetric imaging is realized by two-dimensional (2-D) raster scanning
of the dual foci of optical excitation and ultrasonic detection, in combination with
depth-resolved ultrasonic detection. The two foci are configured coaxially and
confocally to maximize the imaging sensitivity. Thus, the lateral resolution of PAM
is determined by the product of the two point spread functions.
In dark-field acoustic-resolution PAM (AR-PAM) [ 5 ], the maturest version of
AR-PAM, a pulsed laser beam passes through a conical lens to form a ring-shaped
illumination and then is weakly focused into biological tissues to overlap the tight
ultrasonic focus [Fig. 2.1 a]. Since the ultrasonic focus is smaller than the optical
focus, the lateral resolution of AR-PAM is determined acoustically as [ 16 ]

0:71 0
NA

r R D ; (2.1)