Towards affordable biomedical imaging: Recent advances in low‐cost, high‐resolution optoacoustic microscopy

Abstract

This short review discusses the recent developments in low‐cost, high‐resolution optoacoustic microscopy systems, integrating laser diodes for signal excitation, which are 20–40 times cheaper than the typically employed Q‐switched nanosecond laser sources. The development of laser diode‐based microscopes can substantially improve not only cost efficiency, but also multispectral capabilities, robustness, portability and overall imaging performance of the optoacoustic technique. To this end, we demonstrate relevant implementations in both time and frequency domain, highlighting their representative applications in biomedical research such as microvasculature imaging, oxygen saturation assessments, hybrid and multiview microscopy of model organisms and tissues and Doppler flow speed measurements. Finally, we analyse the benefits and limitations of each approach, identifying the respective application contexts where they achieve optimum performance.

1. INTRODUCTION

During the previous years, optoacoustic (OA) microscopy has been utilised as a powerful imaging method for the in vivo acquisition of valuable anatomical, molecular, functional, and flow dynamic information in biomedical studies with high resolution and contrast specificity. OA microscopy typically uses focused laser beams that travel through tissues, where some photons are absorbed by molecules, and their energy is partially or fully converted into heat. The heat then induces an initial pressure rise due to local thermoelastic expansion, which propagates in the form of an acoustic wave. An ultrasonic transducer (or transducer array) detects this acoustic perturbation to generate images which map the optical energy deposition in the tissue. ¹ As different components exhibit unique absorption spectra, the use of multiple excitation wavelengths may differentiate between various tissue chromophores (e.g., oxy‐ and deoxy‐haemoglobin). By providing optical absorption contrast through the generation of light‐induced ultrasonic waves typically found in the MHz regime, the diagnostic capabilities of OA microscopy have found various applications including the in‐depth imaging of malignant tumours, the assessment of haemoglobin oxygen saturation (sO₂) for the study of metabolic disorders as well as the uncovering of drug release mechanisms. ² OA microscopes typically utilise ultrashort nanosecond pulses emitted by Q‐switched laser sources to generate strong energy gradients, leading to efficient signal generation. However, these laser technologies present several drawbacks as they can be highly expensive, complex, and bulky, while they also provide limited wavelength availability and cannot simultaneously deliver multiple excitation lines needed for multispectral imaging capabilities. Such issues significantly restrict both the accessibility and the application potential of the OA technique, and thus, there is an urgent need for novel implementations improving cost efficiency, multispectral capabilities, robustness, portability and overall imaging performance of such microscopes. In this short review, we present some recent advances in the field of low‐cost and high‐resolution OA microscopy (known also as optical‐resolution photoacoustic microscopy – OR‐PAM), ² covering both technological developments and the novel applications of such systems. To this end, we discriminate between two fundamentally different types of cost‐efficient microscopes implemented in the time and frequency domain respectively. While time‐domain OA microscopes utilise short laser pulses emitted by continuous‐wave (CW) or pulsed laser diodes (LDs) for the excitation of broadband signals detected in time, frequency‐domain counterparts integrate intensity‐modulated CW beams following sinusoidal (or square) waveforms. In this latter case, only the signal's amplitude and phase values are detected through the use of an appropriate demodulation scheme, sacrificing though microscope's axial resolution. We demonstrate that both implementations provide valuable diagnostic insights across a broad range of applications, thereby advancing the development of next‐generation OA microscopes.

2. ADVANCES IN LOW‐COST TIME‐DOMAIN OA MICROSCOPY

A novel low‐cost time‐domain OA microscopy system integrating pulsed‐driven CW LDs has been recently demonstrated for high quality microvasculature and oxygen saturation (sO₂) imaging. ³ The main components of the developed microscope (Figure 1A) included commercial pulse drivers connected to two LDs emitting at 458 and 517 nm respectively, and a ring‐shaped transducer that allowed for signal detection in reflection‐mode. The LDs provided pulses with 6.8 ns duration and energy of ∼34 nJ at a repetition rate of 10 kHz, defining the maximum imaging speed. Standard linear unmixing methods were utilised to generate an sO₂ map (Figure 1B) with adequate lateral resolution (∼6 µm) following the sequential acquisition of OA amplitude images in a mouse ear in vivo using the two available excitation wavelengths. The estimated sO₂ values of arteries and veins were consistent (Figure 1C) with the typical oxygen saturation of arteries (>95%) and veins (65–75%) under normal conditions. A similar excitation technology time‐domain OA microscope operating in transmission mode was shown to offer optimised performance in terms of both lateral resolution and imaging speed compared to previously demonstrated systems. ⁴ To achieve this, the microscope integrated a blue LD emitting at 439 nm when operating in pulsed mode. The pulsed LD provided 25 ns duration pulses of ∼100 nJ at 30 kHz, which were subsequently focused by an objective lens to provide a near‐diffraction‐limited lateral resolution of 4.8 µm. A novel combination of 1D galvo scanning and a motorised stage displacing the sample holder could take advantage of the full repetition rate of the optical source, offering concurrently higher imaging speed and large field of view in the order of a few tens of mm². The system was successfully applied for the in vivo imaging of a mouse ear, delineating the microvasculature networks with signal‐to‐noise‐ratio (SNR) values up to 31 dB for fluences well‐below the ANSI safety limit of 20 mJ/cm² (Figure 1D and E). Due to the high SNR, no signal averaging was required, and thus, a typical scan of 40 mm² took ∼11.5 min to complete. In a different approach, another study employed commercially available, cost‐efficient pulsed LDs emitting 908 nm central wavelength pulses of 100 ns and up to 8.9 µJ in energy with a 10 kHz repetition rate. ⁵ Nevertheless, such laser beams are of low quality and cannot be tightly focused, as required in high‐resolution OA microscopes. The authors developed a new strategy to homogenise and shape the pulsed LD beam using a square‐core multimode optical fibre, which allowed them to attain competitive lateral resolutions while keeping large working distances of ∼1 cm. The improved‐quality beam was utilised for signal excitation in a custom‐made reflection‐mode OA microscope integrating a linear phased array placed at an angle of 23° (as determined by the system's geometry) with respect to the imaging plane. The homogenised focused beam could provide a lateral resolution of up to 25 µm following the mechanical raster scanning of the microscope's head across the specimens. The time‐domain OA system was then employed for the ex vivo imaging of a rabbit ear, providing information about the spatial distribution of blood vessels and melanin‐rich hair follicles (bright spots) with SNR values exceeding 26 dB (Figure 1F). The OA images were compared with respective trans‐illumination photographs (Figure 1G), validating the high sensitivity of the method regarding vasculature visualisation.

FIGURE 1.

Cost‐efficient LD‐based time‐domain OA microscopy. (A) Schematic of a dual‐wavelength, low‐cost OA microscope incorporating a ring‐shaped transducer for sO₂ measurements. Abbreviations: AL, aspheric lens; CL, cylindrical lens. (B) sO₂ map of mouse ear vasculature. (C) Average sO₂ values for the lines indicated in (B). (D, E) Microvasculature of a mouse ear as imaged by a spatial resolution and imaging speed‐optimised CW LD‐based time‐domain OA system. (F) OA image of rabbit ear vessels using a pulsed LD in the near‐infrared. (G) Photograph of the same region as in (F). The figures have been adapted and reproduced with permission from references [3, 4, 5].

Finally, a recent study demonstrated the promising capabilities of a novel OA microscope incorporating three overdriven CW LDs at 445, 520, and 638 nm for fast, simultaneous multi‐wavelength imaging with spatial resolution below ∼2 µm. ⁶ The LDs are overdriven in the sense that they are supplied with current that is considerably higher than the nominal one for very short time intervals, contributing to the generation of high‐power nanosecond laser pulses. The system enabled a precise temporal synchronisation of the excitation sources, providing pulse widths <8 ns, pulse energies between 11–26 nJ, repetition rates of up to 1 MHz, and frame rates beyond 5 Hz across a standard microscopy field of view. The microscope's high spatial and temporal precision was utilised for the monitoring and discrimination of blood cells and melanocytes in vivo, as well as the monitoring of blood oxygenation in response to oxygenation stress challenges. Specifically, B16F10 melanocytes were injected into the blood vessels of a mouse ear to mimic circulating tumour cells. The injection region was subsequently imaged over time using the three wavelengths to estimate the respective OA signal trajectories within a selected region of interest, indicated by a dotted white circle (Figure 2A). While the blue and green wavelengths displayed similar signal patterns, the red wavelength contained two peaks in the trajectory, indicating a single B16F10 cell passing through the interrogation area, thus suggesting a discrimination between red blood cells and melanocytes. In a second experiment, an anaesthetised mouse was subjected to oxygen‐stress conditions by changing the carrier gas of the anaesthesia from 100% O₂ to air with 20% O₂ every 30 s. The normalised ratio of OA signals obtained with blue and green wavelengths was estimated and plotted over time (Figure 2B; black line) together with the responses from the blue and green wavelengths (Figure 2B; blue and green line), as well as the measurements of a standard oximeter (Figure 2B; dotted line), in the vessel region indicated by the dotted ellipses. The measurements revealed an expected sawtooth behaviour of OA signals as a function of time, highlighting the capability of the system for monitoring blood oxygenation with high accuracy.

FIGURE 2.

In vivo monitoring of melanocyte circulation and oxygen stress using an overdriven LD‐based OA microscope. (A) Triple‐wavelength OA signal trajectories over time for a region containing circulating blood cells and melanocytes. (B) Similar data for vessels subjected to time‐variable oxygen stress. The figures have been adapted and reproduced with permission from reference [6].

3. ADVANCES IN FREQUENCY‐DOMAIN OA MICROSCOPY

One of the first attempts towards the development of high‐resolution frequency‐domain OA microscopy systems was performed in 2016 by Langer et al. ⁷ The microscope integrated a LD at 405 nm which was directly modulated between 3 and 10 MHz, as higher frequencies would result in considerable losses of modulation depth. The modulated beam was focused on the sample using a 0.5 numerical aperture objective lens providing a measured lateral resolution of 750 nm. The generated OA waves were detected through a hydrophone, amplified and finally transmitted to a lock‐in amplifier which estimated the amplitude and phase of the signals. The system could also detect the emitted luminescence by employing a second lock‐in amplifier and a photodiode. The image acquisition was performed by raster scanning the specimen over the focused beam and required ∼5 min to complete. The developed microscope was used to image successfully red blood cells (RBC) label‐free, by exploiting the intrinsic OA contrast from haemoglobin (Figure 3A; red colour) and the luminescence emitted by Heinz bodies depicted as bright spots (Figure 3B; green colour). A combined image (Figure 3C) was also presented and directly compared with a brightfield view (Figure 3D).

FIGURE 3.

Label‐free imaging of RBC. (A) Frequency‐domain OA and (B) luminescence images. (C) Composite image combining (A) and (B). (D) Respective brightfield view. The figures have been adapted and reproduced from reference [7].

A recent study demonstrated a novel hybrid imaging instrument integrating frequency‐domain OA and traditional confocal fluorescence microscopy for the acquisition of complementary diagnostic information from the eggs of the emerging model organism Parhyale hawaiensis. ⁸ The developed low‐cost system (Figure 4A) integrated a dual‐wavelength excitation scheme, following the intensity modulation of the respective CW beams at 9.5 MHz, using an external acousto‐optic modulator providing enhanced modulation depths. The chosen modulation frequency optimised signal generation by achieving a balance among: (a) rapid optical intensity modulation, (b) sufficient optical modulation depth, and (c) reduced ultrasonic attenuation. The instrument was capable of exciting and concurrently detecting OA and fluorescence signals through a piezoelectric transducer and a photomultiplier tube respectively, with a sufficient lateral resolution of approximately 1.5 µm. OA signal's amplitude and phase estimation was performed by integrating a cost‐efficient I/Q demodulator. Hybrid images of fluorescently labelled Parhyale embryos at cleavage (Figure 4B) and mid‐germband (Figure 4C) stages were recorded using an excitation wavelength at 532 nm. In both cases the emitted SYTO® 24 fluorescence signals (green colour) could clearly reveal the nuclei of the blastomeres with high contrast specificity, whereas the corresponding label‐free OA images (magenta colour) delineated the spatial distribution of yolk mass, the blastomeres’ membranes, as well as the rigid eggshell surrounding the embryos. The application potential of a hybrid frequency‐domain OA and confocal fluorescence microscopy system was further enhanced through the integration of multiview imaging capabilities, significantly expanding the amount of anatomical, structural and functional features that can be recorded in intact organisms and tissues. ⁹ In specific, the multiview capability involved the acquisition of images from different angles, providing a comprehensive and detailed representation of the investigated specimen. A first demonstration of this approach was performed on zebrafish larvae, in an attempt to provide label‐free diagnostic information at various views of interest. Specifically, an agarose‐embedded specimen was rotated with respect to the imaging plane at 90° angle steps for the recording of four sequential hybrid images depicting the head of the fish (Figure 4D–G). Strong frequency‐domain OA signals (magenta colour) were generated in regions of high melanin accumulation such as the eyes and several superficial melanocytes, whereas complementary autofluorescence emissions (green colour) revealed various structures including the trabeculae of the cranial area (TCA) and the upper jaws (UJ) (Figure 4D), the craniofacial sides and the eyes’ lenses (EL) (Figure 4E and G), as well as the lower jaws (LJ) (Figure 4F). Despite its relatively low spatiotemporal resolution (∼5 µm/3 min per view), the multiview microscope has the potential to offer the missing axial resolution and 3D reconstruction capabilities in the case of the frequency‐domain OA modality, by taking advantage of common back‐projection algorithms or other image processing methods.

FIGURE 4.

Hybrid and hybrid multiview frequency‐domain OA imaging. (A) Schematic representation of a hybrid frequency‐domain OA and confocal fluorescence microscope. Abbreviations: FMM, flip mount mirror; L (1–6), lenses; AOM, acousto‐optic modulator; FG, function generator; AP, aperture; DM, dichroic mirror; GMS, galvanometric mirror scanner; M, broadband mirror; OL, objective lens; E, embryo; XYZ, microscope's positioning and focusing system; SH, sample holder; UT, ultrasonic transducer; A, RF amplifier; RFF, bandpass RF filter; I/Q, demodulator; DAQ, data acquisition card; PMT, photomultiplier tube; F, longpass optical filter; PH, pinhole; PC, computer. (B) Hybrid frequency‐domain OA (magenta) and confocal fluorescence (green) image of a Parhyale embryo at the cleavage stage. (C) Similar image of a Parhyale embryo at mid‐germband stage. (D–G) Hybrid images of the head of a zebrafish larva recorded by rotating the specimen at 0°, 90°, 180°, and 270° respectively. All scale bars are equal to 100 µm. Please refer to the main text for explanations of abbreviations. The figures have been adapted and reproduced with permission from references [8, 9].

All previously presented studies involved frequency‐domain OA systems that integrate single‐frequency modulation schemes aiming to record optical absorption contrast images in biological specimens. Nevertheless, the development of similar technology microscopes operating at various discrete frequencies, ¹⁰ provided the opportunity to analyse and examine thoroughly the contribution of each frequency component in the formation of the final image, as happens in the case of broadband excitation through pulsed sources. By imaging a zebrafish eye (Figure 5A), the spatial distribution of low (L), mid (M) and high (H) frequency OA signals could be visualised for an excitation bandwidth between 10 and 50 MHz, revealing the respective amplitude contributions in the final colour‐coded image of frequency‐space representation (FSR). The study demonstrated that different modulation frequencies provide distinct spatial features of the examined specimen according to the OA signal's interference conditions occurring across the transducer's finite surface. The frequency‐domain OA modality was additionally combined with multiphoton microscopy to offer complementary data in mouse ear tissues. In specific, the recorded multimodal image (Figure 5B) depicted the collagen layer through Second Harmonic Generation (SHG; blue colour), the hair follicles through Third Harmonic Generation (THG; green colour), blood vessels and injected melanoma cells (MC) using frequency‐domain OA excitation in the visible (red colour) and finally MC through frequency‐domain OA excitation in the near‐infrared (yellow colour). The multimodal image was directly compared to brightfield (BF) microscopy (Figure 5C) to highlight the superiority of the method in terms of contrast sensitivity and specificity. Finally, the OA Doppler effect was exploited for microcirculatory blood flow measurements in mouse ears in vivo. For this experiment, the transducer was placed off‐axis at 55° with respect to the horizontal plane to detect the frequency shift of OA signals due to the blood flow, following excitation with a 42 MHz intensity‐modulated beam. A colour‐coded image of microvasculature (Figure 5D) shows the detected positive (cyan hot) and negative (orange hot) frequency shifts, also indicating the blood flow directions. The exact flow speed values were subsequently estimated through previous calibration measurements in the order of a few tenths of mm/sec.

FIGURE 5.

Frequency‐domain OA microscopy operating at multiple discrete frequencies. (A) Zebrafish eye imaged using low (L), mid (M), and high (H) OA frequencies within an excitation bandwidth of 10–50 MHz. FSR is generated by merging the previous images. (B) Multimodal image including SHG (blue colour), THG (green colour), and dual‐wavelength frequency‐domain OA (visible: red colour; near‐infrared: yellow colour) contrast. Scale bar is 50 µm. (C) Respective brightfield image of the region shown in (B). (D) OA Doppler shift map depicting the blood flow in mouse ear vessels (white arrows). The figures have been adapted and reproduced with permission from reference [10].

4. CONCLUSIONS

The recent technological advances in LD‐based OA microscopy have paved the way for the emergence of cost‐efficient and high‐performance imaging systems which can be employed in a wide range of demanding biomedical applications. The cost of LD sources (a few hundred USD) is typically 20–40 times lower compared to that of the Q‐switched lasers (several thousand USD) integrated in conventional OA systems, substantially improving the accessibility of the method. Thus, a two‐wavelength LD‐based OA system could be developed at an estimated cost of ∼5–6 kUSD, making it at least 5 times more affordable than a comparable conventional microscope integrating Q‐switched lasers. In principle, low‐cost time‐domain OA microscopes offer axial resolution, high SNRs, and minimum imaging artefacts; however, they may be sometimes limited by either the provided pulse energy or the repetition rate. Furthermore, they require high‐speed data acquisition equipment which can increase the total budget for the microscope's development. Hence, the cost reduction in time‐domain approaches arises primarily from the lower price of LDs compared to Q‐switched laser sources, becoming even more pronounced as the number of excitation lines increases in multispectral OA microscopes. On the other hand, frequency‐domain OA microscopes can integrate inexpensive data acquisition cards (<100 USD), and may potentially provide improved temporal resolutions due to the orders of magnitude higher duty cycle of intensity‐modulated sources compared to time‐domain approaches (MHz versus kHz). In this case, an external acousto‐optic modulator is usually required (∼1000 USD), a function generator (∼150 USD), as well as an I/Q demodulator ^{8 , 9 , 10} and a noise‐rejection filter (∼500 USD) for efficient signal detection, avoiding the use of much more expensive lock‐in amplifiers ⁷ (∼10,000 USD). Furthermore, frequency‐domain approaches demonstrate a superior performance in developmental studies as the applied optical power is relatively low (∼10 mW), enabling thus prolonged observations. Finally, these microscopes can be inherently combined with a confocal fluorescence modality and provide Doppler flow measurements in a straightforward manner. Their main drawbacks include the lack of axial resolution and lower SNR, rendering them less ideal for some applications. In conclusion, the emerging new‐generation OA microscopes are anticipated to reshape the biomedical diagnostics ecosystem by providing valuable information at low cost, substantially complementing state‐of‐the‐art fluorescence‐based imaging methods.

Kalitsounakis, P. , Zacharakis, G. , & Tserevelakis, G. J. (2025). Towards affordable biomedical imaging: Recent advances in low‐cost, high‐resolution optoacoustic microscopy. Journal of Microscopy, 298, 3–9. 10.1111/jmi.13378