High-speed Multi-contrast Optical-resolution Photoacoustic Microscopy


Student thesis: Doctoral Thesis

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Award date25 May 2020


Optical-resolution photoacoustic microscopy (OR-PAM) is a new noninvasive imaging technique that can provide sub-cellular insight of endogenous and exogenous contrasts. Compared with optical imaging and ultrasonic imaging, OR-PAM offers optical-absorption contrast and high spatial resolution in a single modality. Via selecting multiple optical wavelengths, OR-PAM can provide functional and molecular information, such as total hemoglobin concentration (CHb), blood flow (BF), oxygen saturation (sO2), metabolic rate of oxygen, and concentrations of molecular probes. High-speed imaging of these parameters can even enable us to capture abundant dynamic information, such as neural activities, strokes, and many other physiological and pathological dynamics. However, the challenges are, (1) advanced laser technique to generate multiple proper wavelengths and switch among them with a short time; (2) OR-PAM imaging system that can acquire and quantify multi-contrasts at high imaging speed. Therefore, my doctoral research focuses on the development of new high-speed and multi-contrast OR-PAM, including laser sources with multiple wavelengths, sufficient pulse energy, and short wavelength switching time and methods to acquire and quantify multiple functional and molecular contrasts.

The first part of my thesis discusses the development of single-shot multi-wavelength OR-PAM. In this part, based on the stimulated-Raman-scattering (SRS) effect, I develop a high-pulse-repetition-rate multi-wavelength laser system. The specification of the system consists of, (1) single-shot multi-wavelengths (532-nm, 545-nm, 558-nm, 570-nm, 620/640-nm) from one 532-nm laser source for various functional application; (2) enough pulsed energy of each wavelength for PA sensing (≥ 90 nJ); (3) ultrashort switching time among different pulses within sub-microseconds scale for capturing dynamic tissue activities; (4) high laser repetition rate (5 MHz) enabling fast PA imaging. This technique satisfies laser source with multiple wavelengths, sufficient pulse energy for each wavelength, and short switching time among different wavelengths, providing an ideal laser source for fast functional OR-PAM imaging.

The second part of my thesis discusses the simultaneous measurement of in vivo multi-functional parameters via the single-shot multi-wavelength OR-PAM. For total hemoglobin concentration (CHb), it can be normalized and measured by single wavelength which is an isosbestic point for hemoglobin (e.g. 532-nm, 545-nm). For oxygen saturation (sO2), first I use conventional linear spectral unmixing method via 532-nm and 558-nm to measure it in vivo in the mouse brain (skull-removed) and ear. The arteries and veins can be discriminated obviously. However, the sO2 in arteries is underestimated by 19% while the sO2 in veins is overestimated by 7% compared with the normal values due to the saturation effect. Then a nonlinear sO2 measurement method is developed via 532-nm, 545-nm and 558-nm to do calibration. The resulted nonlinear sO2 in arteries and veins can be compensated back to the normal values, especially that the sO2 values in arteries can be improved by 19% compared with the conventional linear values. For blood flow speed (BF), I develop a new photoacoustic flowmetry via 532-nm, 545-nm and 558-nm based on the Grueneisen relaxion effect. This method helps blood flow speed measurement in sub-microseconds scale, which is hundreds of times faster than the traditional photoacoustic flowmetry method. Furthermore, to study other biological molecules besides blood, I develop a 5-wavelength system including 620/640-nm because the hemoglobin absorptivity is weak in such spectral range. Then, I use dyes which are sensitive to 620/640-nm to mark other body tissues distinguished with the blood vessels. In this work, by injecting Evans Blue into the ear of mouse, I can label the lymphatic vessels in the ear and realize the simultaneous single-shot imaging of CHb, sO2, BF, and lymphatic vessels in sub-microseconds. For all functional imaging, it can acquire, (1) the maximum image field of view (FOV) of 1 cm× 1 cm; (2) 20 dB high SNR for capillaries PA sensing; (3) simultaneous measurement of different functional parameters.

The third part of my thesis focuses on the development of high-speed OR-PAM. I first replace the conventional ball-screw stage with a louder speaker. The vibration of louder speaker can be deemed as the raster scanning. It can provide 100 Hz B-scan rate which is 20 times than conventional stage, and 1 Hz imaging speed for C-scan, which enables real-time imaging. However, limited by the power, the maximum field of view is 500 μm × 500 μm, which can be only used for local imaging. To trade off the high imaging speed and large FOV, a voice-coil scanner is used instead. Finally, I can achieve 40 Hz B-scan rate and 7 mm× 5 mm field of view by voice-coil. The whole C-scan imaging time is 50 seconds. Although it is larger than the loudspeaker, it is still much faster than the conventional method when acquiring the same imaging area. It increases the imaging speed 40 times faster than the conventional method when reaching a large imaging area. Combining with the multi-wavelength OR-PAM system, voice-coil can realize the simultaneous multi-functional PA imaging with large imaging area and faster scanning speed.

In conclusion, during my study, I develop new high-speed and multi-contrast OR-PAM, including laser sources with multiple wavelengths, sufficient pulse energy, and short wavelength switching time and methods to acquire and quantify multiple functional and molecular contrasts. These works would positively promote the development of fast functional photoacoustic imaging in the future biomedical applications.

    Research areas

  • optical-resolution photoacoustic microscopy, biomedical application, stimulated Raman scattering, multi-contrast, high-speed imaging, photoacoustic flowmetry, single-shot