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Photoacoustic Microscopy Method Delivers Higher Sensitivity

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A multispectral, super-low-dose photoacoustic microscopy (SLD-PAM) system developed by City University of Hong Kong (CUHK) achieves significantly higher sensitivity than traditional optical resolution photoacoustic imaging.

By providing an exceptionally high level of sensitivity, SLD-PAM could help broaden the use of photoacoustic microscopy in biomedical applications. In the future, it could translate to clinical settings; for example, it could be used for ophthalmic exams where a low-power laser is preferred for the patient’s safety and comfort. Long-term monitoring of pharmacokinetics or blood flow also requires low-dose imaging to alleviate perturbation to tissue function.
(a): SLD-PAM system and (b): sensitivity improvement from the probe and filter. Courtesy of Zhang, Y. et al., https://onlinelibrary.wiley.com/doi/10.1002/advs.202302486.
(a) SLD-PAM system and (b) sensitivity improvement from the probe and filter. Courtesy of Zhang, Y. et al., https://onlinelibrary.wiley.com/doi/10.1002/advs.202302486.

“High sensitivity is important for high-quality imaging,” said CUHK professor Lidai Wang. “It helps detect chromophores — molecules that confer color on materials by absorbing particular wavelengths of visible light — that do not strongly absorb light.”

Additionally, since a low-power light source is sufficient to operate SLD-PAM, it reduces the chances of photobleaching, phototoxicity, or perturbation to delicate tissues.

To build SLD-PAM, the researchers optimized the photoacoustic probe design of a technique they developed previously and implemented a spectral-spatial filter.

The researchers customized a high-numerical-aperture acoustic lens to optimize the optical and acoustic beam combiner and improve the alignment between the optical and acoustic foci. They combined these improvements with an innovative, 4D, spectral-spatial filter algorithm based on discrete wavelet transform. The algorithm enabled SLD-PAM to filter coherent photoacoustic signals in all 3D space and all-optical wavelengths, greatly enhancing the microscope’s ability to recover weak signals.

These improvements increased the sensitivity of the microscope by a factor of between 6 and 33, compared to the researchers’ previous technique.
Comparison of the in vivo results of (a): Traditional-PAM, (b): SLD-PAM at super-low pulse energy with a green-light source, and (c): oxygen saturation image acquired by SLD-PAM via dual-wavelength spectrum unmixing. Courtesy of Zhang, Y. et al., https://onlinelibrary.wiley.com/doi/10.1002/advs.202302486.
Comparison of the in vivo results of (a) traditional-PAM, (b) SLD-PAM at super-low pulse energy with a green-light source, and (c) oxygen saturation image acquired by SLD-PAM via dual-wavelength spectrum unmixing. Courtesy of Zhang, Y. et al., https://onlinelibrary.wiley.com/doi/10.1002/advs.202302486.


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SLD-PAM uses a low-cost, multiwavelength, pulsed laser that provides 11 wavelengths ranging from green to red light. The laser operates at a repetition frequency up to megahertz, and the spectral switching time is in submicroseconds.

The researchers tested SLD-PAM through in vivo animal imaging at super-low pulse energy with green-light and red-light sources. SLD-PAM demonstrated high-quality anatomical and functional imaging.

Using tens of times less pulse energy than the lowest reported in vivo results, SLD-PAM visualized microvessels and quantified oxygen saturation with approximately 1% of the maximum permissible exposure. This significantly reduced potential phototoxicity or perturbation to normal tissue function. The successful use of SLD-PAM to image fragile eye and brain tissue indicates that the technique has potential for use in clinical settings.

In molecular imaging, the much-reduced laser energy required by SLD-PAM led to a reduction in photobleaching of about 85%. SLD-PAM achieved high-quality molecular imaging using 80% fewer contrast agents, potentially reducing biotoxicity and metabolic burden.
Image comparison of the in vivo results of (a): Low-dose PAM and (b): SLD-PAM, both at super-low pulse energy with a red-light source, and (c): oxygen saturation image acquired by SLD-PAM via dual-wavelength spectrum unmixing. Courtesy of Zhang, Y. et al., https://onlinelibrary.wiley.com/doi/10.1002/advs.202302486.
Comparison of the in vivo results of (a) low-dose PAM and (b) SLD-PAM, both at super-low pulse energy with a red-light source, and (c) oxygen saturation image acquired by SLD-PAM via dual-wavelength spectrum unmixing. Courtesy of Zhang, Y. et al., https://onlinelibrary.wiley.com/doi/10.1002/advs.202302486.

SLD-PAM can be used with a broad range of low-absorbing nano-agents, small molecules, and genetically encoded biomarkers, as well as low-power light sources in a wide spectrum. The system’s cost is relatively low, making it affordable for research laboratories and clinics.

“SLD-PAM enables noninvasive imaging of biological tissue with minimal damage to the subjects, offering a powerful and promising tool for anatomical, functional, and molecular imaging,” Wang said. “We believe that SLD-PAM can help advance the applications of photoacoustic imaging, enable numerous new biomedical applications, and pave a new avenue for clinical translation.”

Wang and his team plan to test a broader range of small molecules and genetically encoded biomarkers in biological imaging using the SLD-PAM system. They also intend to adopt more types of low-power light sources in a wider spectrum to develop wearable or portable optical resolution photoacoustic microscopy systems.

The research was published in Advanced Science (www.doi.org/10.1002/advs.202302486).

Published: September 2023
Glossary
ophthalmology
Ophthalmology is a branch of medicine that focuses on the anatomy, physiology, and diseases of the eyes and visual system. Ophthalmologists are medical doctors who specialize in the diagnosis, treatment, and prevention of eye disorders and diseases. They are trained to provide comprehensive eye care, including medical, surgical, and optical interventions. Key areas within ophthalmology include: General eye care: Ophthalmologists perform routine eye examinations to assess visual acuity,...
photobleaching
Photobleaching is a phenomenon in which the fluorescence of a fluorophore (a fluorescent molecule or dye) is permanently reduced or eliminated upon prolonged exposure to light. This process occurs due to the photochemical destruction or alteration of the fluorophore molecules, rendering them non-fluorescent. Key points about photobleaching: Mechanism: Photobleaching is typically a result of chemical reactions induced by the absorbed photons. The excessive light exposure causes the...
phototoxicity
Phototoxicity refers to the harmful effects caused by exposure to light, particularly intense or ultraviolet (UV) light, on living cells or organisms. This phenomenon is often associated with the interaction of light with certain substances, known as photosensitizers, which can lead to cellular damage or death. Phototoxic reactions can occur in various biological systems, including cells, tissues, and organisms. Key points about phototoxicity include: Photosensitizers: Photosensitizers are...
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