Wide Spectrum Optical Resolution Photoacoustic Microsopy
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 the 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 image of a target. PAM offers 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.
Wide Spectrum OR-PAM is a technique that further improves the quality of the OR-PAM images and allows for multiple contrast imaging, which is a huge improvement over traditional OR-PAM.
Wide Spectrum OR-PAM System Diagram
Figure 1 shows a system diagram for a Wide Spectrum OR-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 Wide Spectrum OR-PAM, fixed optical paths are made to allow for different contrast imaging within the same system. The grey box on the left side represents the nano second pulsed wide spectrum laser. Dichroic mirrors (DM1-7) are used to allow for certain ranges of wavelengths to pass through. Double-cylindrical lenses 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.
Advantages
Resolution
Lateral Resolution in OR-PAM is defined by the following relationship: , where is the optical wavelength and 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.
Multiple Contrast Imaging
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.
| Absorbers | Wavelength (nm) | Spectrum |
|---|---|---|
| Ex. DNA & RNA | 180-400 | Ultraviolet (UV) |
| Ex. Melanin | 400-700 | Visible Light |
| Ex. Glucose | 700-1400 | Near-Infrared |
The table above shows widely imaged absorbers versus 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, Wide Spectrum OR-PAM has greater advantages over single wavelength lasers.
Image Reconstruction
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 some results of the Wide Spectrum OR-PAM system. One image shows a slice of a mouse brain (at 260nm) and the other two show the cells of a green frog (at 532nm). This system is considered a super-resolution system since the resolution is in the sub-micron level.
Figure 3 is a way to visualize the Maximum Intensity Projection method. This method is a great way to visualize the vessels and the vasculature of a target.
Imaging Depth
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]
Current Challenges
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
- 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 systems in general. The higher the laser rep rate, the faster the system will be able to acquire the data.
Scanning Speed
- 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]
References
- ↑ Moothanchery, M.; Bi, R.; Kim, J. Y.; Jeon, S.; Kim, C.; Olivo, M. (2018). "Optical resolution photoacoustic microscopy based on multimode fibers". Biomedical Optics Express. 9 (3): 1190–1197. doi:10.1364/BOE.9.001190. PMC 5846522. PMID 29541512.
- ↑ Wang, L. V.; Yao, J. (2016). "A Practical Guide to Photoacoustic Tomography in the Life Sciences". Nature Methods. 13 (8): 627–638. doi:10.1038/nmeth.3925. PMC 4980387. PMID 27467726.
- ↑ "Using Eppendorf BioSpectrometer® Fluorescence for Nucleic Acid Concentration Measurements". 19 November 2015.
- ↑ "Absorption spectrum of melanin".
- ↑ "Optical-Resolution Photoacoustic Microscopy: Auscultation of Biological Systems at the Cellular Level" (PDF).
- ↑ "Pulse Repetition Rate".
- ↑ DiSpirito A III; Li, D.; Vu, T.; Chen, M.; Zhang, D.; Luo, J.; Horstmeyer, R.; Yao, J. (2021). "Reconstructing Undersampled Photoacoustic Microscopy Images using Deep Learning". IEEE Transactions on Medical Imaging. 40 (2): 562–570. doi:10.1109/TMI.2020.3031541. PMC 7858223 Check
|pmc=value (help). PMID 33064648 Check|pmid=value (help).
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