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Photoacoustic imaging (PA) is an emerging medical imaging technology. PA imaging uses the Photoacoustic effect and Ultrasound Detection to image a target. In PA imaging a target is excited by a laser pulse, then the target absorbs that energy and in PA effect, this energy is converted into thermal energy resulting in local thermal expansion. Because of the thermal expansion, the target emits Ultrasound waves that can be detected using an ultrasound transducer. From the idea of PA imaging, Photoacoustic microscopy (PAM) was developed to have a more detailed and high-quality images of a target. PAM offers a high-resolution images of biological molecules and the vasculature of different organs^[1]. Acoustic and Optical resolution PAM, AR-PAM and OR-PAM, respectively are two of the widely used applications of PAM.

Hyperspectral PAM is a technique that further improve the quality of the OR-PAM images and allow for multiple contrast imaging which is a huge improvement over traditional OR-PAM.

Figure 1 shows a system diagram for a Hyperspectral PAM. In this system, the light can follow different optical paths and thus providing different wavelengths that will reach the target. Instead of having a single wavelength laser, in Hyperspectral PAM, fixed optical paths are made to allow for different contrast imaging within the same system. Grey box on the left side represents the nano second pulsed wide spectrum laser. Dichroic mirrors (DM1-7) are used to allow for certain range of wavelengths to pass through. Double-cylindrical lens are used to reshape the laser beam from a rectangular beam to a circular beam. Finally, the Objective Lens (OL) is used to focus the light and improve the resolution of the final image of the target.

Lateral Resolution in OR-PAM is defined by the following relationship: ${\displaystyle LateralResolution=0.51*{\frac {\lambda }{NA}}}$, where ${\displaystyle \lambda }$ is the optical wavelength and ${\displaystyle NA}$ is the Numerical Aperture of the optical objective lens. From the given relationship, by increasing the Numerical Aperture of the objective lens and/or having a shorter excitation wavelength, the lateral resolution can be improved.^[2]. The resolution of OR-PAM systems can reach the sub-micron levels.

In the system described above, a wide spectrum laser is used (200nm - 2000nm) and an objective lens that has a wide spectrum as well that covers the wavelength spectrum of the laser. Having a wide spectrum laser as well as a wide spectrum objective lens is what makes the system suitable for multiple contrast imaging.

The table above shows widely imaged absorbers vs. their respective wavelengths. In the Ultraviolet spectrum, the major absorbers are DNA and RNA which are important molecules since they store and read the genetic information.^[3]. In the Visible Light spectrum, Melanin is an important molecule to image^[4], since it is found in Melanoma (Skin Cancer). The ability to image Melanin is critical in the biomedical imaging field since it can lead to the early detection of skin cancer. Since different biological molecules absorb light at different wavelengths, Hyperspectral PAM has greater advantages over single wavelength lasers.

The image reconstruction of OR-PAM is based on a method called Maximum Intensity Projection (MIP). MIP projects the maximum intensity of voxels.

Figure 2 shows a slice of a mouse brain (at 260nm) with a resolution of 625 nm. This system is considered a super-resolution system since the resolution is in the sub-micron level. The left picture of figure 3 shows an image of a mouse brain at 532 nm; The right image is a mouse ear image at 1210 nm.

Figure 4 is a way to visualize the Maximum Intensity Projection method. This method is great way to visualize the vessels and the vasculature of a target.

The maximum imaging depth of OR-PAM is around ~1mm and this is due to the scattering of the photons while penetrating the target and reaching the optical diffusion limit^[5].

Despite having a super-resolution imaging system, there are many trade-offs to this advantage. The process of acquiring an image using OR-PAM systems is a long process. This is due to different factors:

• Laser Repetition Rate: This is the number of pulses emitted from the laser source per second and has units of Hertz (Hz)^[6]. Usually, the rep rate is 10Hz. In the System shown above, the rep rate is 100Hz. However, this is still a current challenge and a limitation for OR-PAM system in general. The higher the laser rep rate, the faster the system will be able to acquire the data.

• Scanning Step Size: The scanning step size is the scanning step size of the motor that moves the sample that is being imaged. According to the Nyquist–Shannon sampling theorem and to avoid under-sampling, the scanning step size is limited by the theoretical spatial resolution; The scanning step size must be less than half the theoretical spatial resolution of the system.^[7]