Photoacoustic imaging schematic

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.^[1] 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.^[2]

Photoacoustic signal

The goal of photoacoustic microscopy is to find the local pressure rise ${\displaystyle p_{0}}$, which can be used to calculate the absorption coefficient ${\displaystyle \mu _{a}}$ according to the formula:

${\displaystyle p_{0}=\Gamma \eta _{th}\mu _{a}F,}$

where ${\displaystyle \eta _{th}}$ is the percentage of light converted to heat, ${\displaystyle F}$ is the local optical fluence (J/cm²), and the dimensionless Gruneisen parameter ${\displaystyle \Gamma }$ is defined as:

${\displaystyle \Gamma ={\frac {\beta }{\kappa \rho C_{V}}},}$

where ${\displaystyle \beta }$ is the thermal coefficient of volume expansion (K⁻¹), ${\displaystyle \kappa }$ is the isothermal compressibility (Pa⁻¹), and ${\displaystyle \rho }$ is the density (kg/m³).^[3]

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.^[3]

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.^[3] 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

Absorption profile of oxy- and deoxyhemoglobin

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.^[2]

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.^[5]

Visible Light Absorbers

Visible light absorbers (λ = 400 to 700 nm) include oxyhemoglobin, deoxyhemoglobin, melanin, and cytochrome c. Visible light photoacoustic microscopy is particularly useful in determining hemoglobin concentration and oxygen saturation due to the difference in absorption profiles of oxyhemoglobin and deoxyhemoglobin. Real-time analysis can then be used to determine blood flow speed and oxygen metabolism rate.^[3] In addition, photoacoustic microscopy is capable of early melanoma detection due to the high concentration of melanin found in skin cancer cells.

Near-Infrared Absorbers

Near-Infrared absorbers (λ = 700 to 1400 nm) include water, lipids, and glucose. Photoacoustic determination of blood glucose levels can be used for treating diabetes, while studying lipid concentrations within blood vessels is important for monitoring the progression of atherosclerosis.^[2] It is still feasible to quantify and compare deoxyhemoglobin and hemoglobin concentrations at this wavelength, trading deeper tissue penetration for lower absorption.^[6]

Resolution

Mouse ear vasculature imaged using OR-PAM at 532 nm

Mouse ear vasculature imaged using AR-PAM at 532 nm

Photoacoustic micrograph of methanol fixed human red blood cells using 405 nm.

Photoacoustic microscopy achieves greater penetration than conventional microscopy due to ultrasonic detection. As a result, axial resolution is defined acoustically and is determined by the formula:

${\displaystyle R_{axial}=0.88{\frac {\nu _{A}}{\Delta f_{A}}},}$

where ${\displaystyle \nu _{A}}$ is the speed of sound in the medium and ${\displaystyle \Delta f_{A}}$ is the photoacoustic signal bandwidth. The axial resolution of the system can be improved by using a wider bandwidth ultrasound transducer as long as the bandwidth matches that of the photoacoustic signal. The lateral resolution of photoacoustic microscopy depends on the optical and acoustic foci of the system. Optical-resolution photoacoustic microscopy (OR-PAM) uses a tighter optical focus than acoustic focus, while acoustic-resolution photoacoustic microscopy (AR-PAM) uses a tighter acoustic focus than optical focus.^[9][10]

Dark-field Confocal Photoacoustic microscopy

Depiction of PAM raster scanning path

By ignoring ballistic light, dark-field confocal photoacoustic microscopy reduces surface signal. This method uses a dark-field pulsed laser and high-NA ultrasonic detection, with the fiber output end coaxially aligned with the focused ultrasound transducer. Filtration of ballistic light relies on the altered shape of the excitation laser beam instead of an opaque disk, as used in conventional dark-field microscopy. The general reconstruction technique is used to convert the photoacoustic signal into one A-line, and B-line images are produced by raster scanning.^[4]

Biomedical applications

Photoacoustic microscopy has a wide range of applications in the biomedical field. Due to its ability to image a variety of molecules based on optical wavelength, photoacoustic microscopy can be used to gain functional information about the body noninvasively. Blood flow dynamics and oxygen metabolic rates can be measured and correlated to studies of atherosclerosis or tumor proliferation. Exogenous agents can be used to bind to cancerous tissue, enhancing image contrast and aiding in surgical removal. On the same note, photoacoustic microscopy is useful in early cancer diagnosis due to the difference in optical absorption properties compared to healthy tissue.^[1]

See also

• Photoacoustic imaging
• Deep learning in photoacoustic imaging

References