Photoacoustic microscopy is an imaging method based on the photoacoustic effect and is a subset of photoacoustic tomography. Photoacoustic microscopy takes advantage of the local temperature rise that occurs as a result of light absorption in tissue. Using a nanosecond pulsed laser beam, tissues undergo thermoelastic expansion, resulting in the release of a wide-band acoustic wave that can be detected using a high-frequency ultrasound transducer. Since ultrasonic scattering in tissue is weaker than optical scattering, photoacoustic microscopy is capable of achieving high-resolution images at greater depths than conventional microscopy methods. Furthermore, photoacoustic microscopy is especially useful in the field of biomedical imaging due to its scalability. By adjusting the optical and acoustic foci, lateral resolution may be optimized for the desired imaging depth.
Photoacoustic signal The goal of photoacoustic microscopy is to find the local pressure rise p 0 {\displaystyle p_{0}} , which can be used to calculate the absorption coefficient μ a {\displaystyle \mu _{a}} according to the formula:
p 0 = Γ η t h μ a F , {\displaystyle p_{0}=\Gamma \eta _{th}\mu _{a}F,}
where η t h {\displaystyle \eta _{th}} is the percentage of light converted to heat, F {\displaystyle F} is the local optical fluence (J/cm2), and the dimensionless Gruneisen parameter Γ {\displaystyle \Gamma } is defined as:
Γ = β κ ρ C V , {\displaystyle \Gamma ={\frac {\beta }{\kappa \rho C_{V}}},}
where β {\displaystyle \beta } is the thermal coefficient of volume expansion (K−1), κ {\displaystyle \kappa } is the isothermal compressibility (Pa−1), and ρ {\displaystyle \rho } is the density (kg/m3). Following the initial pressure rise, a photoacoustic wave propagates at the speed of sound within the medium and can be detected with an ultrasound transducer.
Image reconstruction One of the major benefits of photoacoustic microscopy is the simplicity of image reconstruction. A laser pulse excites tissue in the axial direction and the resulting photoacoustic waves are detected by an ultrasound transducer. The transducer then converts the mechanical energy into a voltage signal that can be read by an analog-to-digital converter for post-processing. A one-dimensional image, known as an A-line, is formed as a result of each laser pulse. Hilbert transform of an A-line reveals depth-encoded information. A 3D photoacoustic image can then be formed by combining multiple A-lines produced by 2D raster scanning.
Synthetic Aperture Image Reconstruction Altering delays of the elements on an ultrasound transducer allows one to focus ultrasound waves similar to passing through an acoustic lens. This delay-and-sum method enables one to find the signal at each focal point. However, the lateral resolution is limited by the presence of side lobes, which appear at polar angles and are dependent on the width of each element.
Contrast In photoacoustic imaging modalities, including photoacoustic microscopy, contrast is based on photon excitation and is thus determined by the optical properties of the tissue. When an electron absorbs a photon, it moves to a higher energy state. Upon returning to a lower energy level, the electron undergoes either radiative or nonradiative relaxation. During radiative relaxation, the electron releases energy in the form of a photon. On the other hand, an electron undergoing nonradiative relaxation releases energy as heat. The heat then induces a pressure rise that propagates as a photoacoustic wave. Due to the fact that almost all molecules are capable of nonradiative relaxation, photoacoustic microscopy has the potential to image a wide range of endogenous and exogenous agents. By contrast, fewer molecules are capable of radiative relaxation, thus limiting fluorescence microscopy techniques such as one-photon and two-photon microscopy. Current research in photoacoustic microscopy takes advantage of both endogenous and exogenous contrast agents to gain functional information about the body, from blood saturation levels to cancer proliferation rate.
Endogenous Contrast Agents
Endogenous contrast agents, molecules naturally occurring within the body, are useful in photoacoustic microscopy due to the fact that they may be imaged non-invasively. Endogenous agents are also non-toxic and do not affect the properties of the tissue being studied. In particular, endogenous absorbers can be classified based on their absorbing wavelengths.
Ultraviolet Absorbers Within the ultraviolet light range (λ = 180 to 400 nm), the primary absorber in the body is DNA and RNA. By using ultraviolet photoacoustic microscopy, DNA and RNA can be imaged in the cell nuclei without the use of fluorescence labeling. Since cancer is associated with DNA replication failure, UV photoacoustic microscopy has the potential to be used for early cancer detection.
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