Sounding out the dynamics: a concise review of high-speed photoacoustic microscopy.

Significance: Photoacoustic microscopy (PAM) offers advantages in high-resolution and high-contrast imaging of biomedical chromophores. The speed of imaging is critical for leveraging these benefits in both preclinical and clinical settings. Ongoing technological innovations have substantially boosted PAM's imaging speed, enabling real-time monitoring of dynamic biological processes. Aim: This concise review synthesizes historical context and current advancements in high-speed PAM, with an emphasis on developments enabled by ultrafast lasers, scanning mechanisms, and advanced imaging processing methods. Approach: We examine cutting-edge innovations across multiple facets of PAM, including light sources, scanning and detection systems, and computational techniques and explore their representative applications in biomedical research. Results: This work delineates the challenges that persist in achieving optimal high-speed PAM performance and forecasts its prospective impact on biomedical imaging. Conclusions: Recognizing the current limitations, breaking through the drawbacks, and adopting the optimal combination of each technology will lead to the realization of ultimate high-speed PAM for both fundamental research and clinical translation.

Efficient label-free in vivo photoacoustic imaging of melanoma cells using a condensed NIR-I spectral window.

In this paper, we propose an efficient label-free in vivo photoacoustic (PA) imaging of melanoma using a condensed near infrared-I (NIR-I) supercontinuum light source. Although NIR-II spectral window is advantageous such as longer penetration depth compared to the NIR-I region, supercontinuum light sources emitting both NIR-I and NIR-II region could lower the efficiency to target melanoma because of low optical power density in the melanoma's absorption spectra. To exploit efficient in vivo PA imaging of melanoma, we demonstrated the light source emitting from visible (532-600 nm) to NIR-I (600-1000 nm) by optimizing stimulated Raman scattering induced supercontinuum generation. The melanoma's structure is successfully differentiated from blood vessels at a high pulse energy of 2.5 µJ and a flexible pulse repetition rate (PRR) of 5-50 kHz. The proposed light source with the microjoules energies and tens of kHz of PRR can potentially accelerate clinical trials such as early diagnosis of melanoma.

High-speed photoacoustic microscopy: A review dedicated on light sources.

In recent years, many methods have been investigated to improve imaging speed in photoacoustic microscopy (PAM). These methods mainly focused upon three critical factors contributing to fast PAM: laser pulse repetition rate, scanning speed, and computing power of the microprocessors. A high laser repetition rate is fundamentally the most crucial factor to increase the PAM speed. In this paper, we review methods adopted for fast PAM systems in detail, specifically with respect to light sources. To the best of our knowledge, ours is the first review article analyzing the fundamental requirements for developing high-speed PAM and their limitations from the perspective of light sources.

Ultra-widefield photoacoustic microscopy with a dual-channel slider-crank laser-scanning apparatus for in vivo biomedical study.

Photoacoustic microscopy (PAM) is an important imaging tool that can noninvasively visualize the anatomical structure of living animals. However, the limited scanning area restricts traditional PAM systems for scanning a large animal. Here, we firstly report a dual-channel PAM system based on a custom-made slider-crank scanner. This novel scanner allows us to stably capture an ultra-widefield scanning area of 24 mm at a high B-scan speed of 32 Hz while maintaining a high signal-to-noise ratio. Our system's spatial resolution is measured at ∼3.4 µm and ∼37 µm for lateral and axial resolution, respectively. Without any contrast agent, a dragonfly wing, a nude mouse ear, an entire rat ear, and a portion of mouse sagittal are successfully imaged. Furthermore, for hemodynamic monitoring, the mimicking circulating tumor cells using magnetic contrast agent is rapidly captured in vitro. The experimental results demonstrated that our device is a promising tool for biological applications.

Quickly Alternating Green and Red Laser Source for Real-time Multispectral Photoacoustic Microscopy.

Multispectral photoacoustic microscopy uses a wavelength-dependent absorption difference as a contrast mechanism to image the target molecule. In this paper, we present a novel multispectral pulsed fiber laser source, which selectively alternates the excitation wavelengths between green and red colors based on the stimulated Raman scattering (SRS) effect for imaging. This laser has a high pulse repetition rate (PRR) of 300 kHz and high pulse energy of more than 200 nJ meeting the real-time requirements of optical-resolution photoacoustic microscopy imaging. By switching the polarization state of the pump light and optical paths of the pump light, the operating wavelengths of the light source can be selectively alternated at the same fast PRR for any two SRS peak wavelengths between 545 and 655 nm. At 545 nm excitation wavelength, molecular photoacoustic signals from both blood vessels and gold nanorods were obtained simultaneously. However, at 655 nm, the photoacoustic signals of gold nanorods were dominant because the absorption of light by the blood vessels decreased drastically in the spectral region over 600 nm. Thus the multispectral photoacoustic system designed using the novel laser source implemented here could simultaneously monitor the time-dependent fast movement of two molecules independently, having different wavelength-dependent absorption properties at a high repetition rate of 0.49 frames per second (fps).

Photoacoustic Imaging-Guided Photothermal Therapy with Tumor-Targeting HA-FeOOH@PPy Nanorods.

Cancer theragnosis agents with both cancer diagnosis and therapy abilities would be the next generation of cancer treatment. Recently, nanomaterials with strong absorption in near-infrared (NIR) region have been explored as promising cancer theragnosis agents for bio-imaging and photothermal therapy (PTT). Herein, we reported the synthesis and application of a novel multifunctional theranostic nanoagent based on hyaluronan (HA)-coated FeOOH@polypyrrole (FeOOH@PPy) nanorods (HA-FeOOH@PPy NRs) for photoacoustic imaging (PAI)-guided PTT. The nanoparticles were intentionally designed with rod-like shape and conjugated with tumor-targeting ligands to enhance the accumulation and achieve the entire tumor distribution of nanoparticles. The prepared HA-FeOOH@PPy NRs showed excellent biocompatible and physiological stabilities in different media. Importantly, HA-FeOOH@PPy NRs exhibited strong NIR absorbance, remarkable photothermal conversion capability, and conversion stability. Furthermore, HA-FeOOH@PPy NRs could act as strong contrast agents to enhance PAI, conducting accurate locating of cancerous tissue, as well as precise guidance for PTT. The in vitro and in vivo photothermal anticancer activity results of the designed nanoparticles evidenced their promising potential in cancer treatment. The tumor-bearing mice completely recovered after 17 days of PTT treatment without obvious side effects. Thus, our work highlights the great potential of using HA-FeOOH@PPy NRs as a theranostic nanoplatform for cancer imaging-guided therapy.

Unfolding displacement measurement method for the aliasing interferometer signal of a wavelength-comb-swept laser.

We demonstrate an unfolding displacement measurement method to overcome the aliasing problem of wavelength-comb-swept laser (WCSL). Compared to the conventional wavelength-swept laser (WSL), the WCSL exhibits an extended coherence length, owing to the narrowing spectral linewidth of the etalon filter. However, the aliasing interference signal induces an unexpected back-bounced phenomenon during displacement measurement because of the discretely distributed comb-like periodic spectra of the etalon filter. By using the dual-reference interferometry method, the back-bounced displacement measurement can be successfully unfolded to extend the measurement range by two times. In addition, we demonstrate a longer-range surface profiling image over 18 mm within the 200 mm of measurement range using a line-field beam of a parallel-swept source-optical coherence tomography system.

In vivo photoacoustic monitoring using 700-nm region Raman source for targeting Prussian blue nanoparticles in mouse tumor model.

Photoacoustic imaging (PAI) is a noninvasive imaging tool to visualize optical absorbing contrast agents. Due to high ultrasonic resolution and superior optical sensitivity, PAI can be used to monitor nanoparticle-mediated cancer therapy. The current study synthesized Food and Drug Administration-approved Prussian blue (PB) in the form of nanoparticles (NPs) with the peak absorption at 712 nm for photoacoustically imaging tumor-bearing mouse models. To monitor PB NPs from the background tissue in vivo, we also developed a new 700-nm-region stimulated Raman scattering (SRS) source (pulse energy up to 200 nJ and repetition rate up to 50 kHz) and implemented optical-resolution photoacoustic microscopy (OR-PAM). The SRS-assisted OR-PAM system was able to monitor PB NPs in the tumor model with micrometer resolution. Due to strong light absorption at 712 nm, the developed SRS light yielded a two-fold higher contrast from PB NPs, in comparison with a 532-nm pumping source. The proposed laser source involved cost-effective and simple system implementation along with high compatibility with the fiber-based OR-PAM system. The study highlights the OR-PAM system in conjunction with the tunable-color SRS light source as a feasible tool to assist NP-mediated cancer therapy.

Real-time functional optical-resolution photoacoustic microscopy using high-speed alternating illumination at 532 and 1064 nm.

Optical-resolution photoacoustic microscopy (OR-PAM), which has been widely used and studied as a noninvasive and in vivo imaging technique, can yield high-resolution and absorption contrast images. Recently, metallic nanoparticles and dyes, such as gold nanoparticles, methylene blue, and indocyanine green, have been used as contrast agents of OR-PAM. This study demonstrates real-time functional OR-PAM images with high-speed alternating illumination at 2 wavelengths. To generate 2 wavelengths, second harmonic generation at 532 nm with an LBO crystal and a pump wavelength of 1064 nm is applied at a pulse repetition rate of 300 kHz. For alternating illumination, an electro-optical modulator is used as an optical switch. Therefore, the A-line rate for the functional image is 150 kHz, which is half of the laser repetition rate. To enable fast signal processing and real-time displays, parallel signal processing using a graphics processing unit (GPU) is performed. OR-PAM images of the distribution of blood vessels and gold nanorods in a BALB/c-nude mouse's ear can be simultaneously obtained with 500 × 500 pixels and real-time display at 0.49 fps.

Optical measurements of paintings and the creation of an artwork database for authenticity.

Paintings have high cultural and commercial value, so that needs to be preserved. Many techniques have been attempted to analyze properties of paintings, including X-ray analysis and optical coherence tomography (OCT) methods, and enable conservation of paintings from forgeries. In this paper, we suggest a simple and accurate optical analysis system to protect them from counterfeit which is comprised of fiber optics reflectance spectroscopy (FORS) and line laser-based topographic analysis. The system is designed to fully cover the whole area of paintings regardless of its size for the accurate analysis. For additional assessments, a line laser-based high resolved OCT was utilized. Some forgeries were created by the experts from the three different styles of genuine paintings for the experiments. After measuring surface properties of paintings, we could observe the results from the genuine works and the forgeries have the distinctive characteristics. The forgeries could be distinguished maximally 76.5% with obtained RGB spectra by FORS and 100% by topographic analysis. Through the several executions, the reliability of the system was confirmed. We could verify that the measurement system is worthwhile for the conservation of the valuable paintings. To store the surface information of the paintings in micron scale, we created a numerical database. Consequently, we secured the databases of three different famous Korean paintings for accurate authenticity.

Parallel Imaging of 3D Surface Profile with Space-Division Multiplexing.

We have developed a modified optical frequency domain imaging (OFDI) system that performs parallel imaging of three-dimensional (3D) surface profiles by using the space division multiplexing (SDM) method with dual-area swept sourced beams. We have also demonstrated that 3D surface information for two different areas could be well obtained in a same time with only one camera by our method. In this study, double field of views (FOVs) of 11.16 mm × 5.92 mm were achieved within 0.5 s. Height range for each FOV was 460 µm and axial and transverse resolutions were 3.6 and 5.52 µm, respectively.