The integration of near-infrared genetically encoded reporters (NIR-GERs) with photoacoustic (PA) imaging offers a promising approach for visualizing deep-tissue-specific functions (e.g., metabolism, neural activity) at high resolution. However, one critical limitation lies in the PA response intensity of the probes. Directed evolution methods enable iterative optimization of probe performance by expressing mutant NIR-GER genes in Escherichia coli (E. coli) colonies, yet challenges persist in directly quantifying PA signals at the individual colony level, coupled with inefficiencies and low precision in screening. Existing PA microscopy-based screening platforms rely on mechanical scanning with single-element transducers, requiring several minutes per sample. This prolonged imaging duration exacerbates photobleaching and colony dissolution while failing to correct quantification errors caused by colony morphology and illumination heterogeneity, thereby hindering the development of novel PA probes.

To address the limitations of slow speed, low throughput, and insufficient accuracy in NIR-GER screening, the research team developed Self-Calibrated Photo-Acoustic Screening (SCAPAS). This technology employs a 360° ring-shaped ultrasonic transducer array and a dual-wavelength switchable illumination system to achieve single-pulse parallel imaging of multiple colonies. By integrating a self-calibration strategy with co-expressed reference proteins, SCAPAS eliminates systematic and sample-induced quantification biases while significantly improving throughput. Experimental results demonstrate that SCAPAS completes whole-sample detection within 5 seconds, with a per-colony screening time of ~50 ms, achieving a 36-fold efficiency improvement over conventional PA microscopy. Validation tests using two NIR-GERs (iRFP713 and SNIFP) revealed a quantification accuracy of ~2.8% and precision of ~6.47%. These advancements position SCAPAS as a robust tool for developing high-performance PA molecular probes and advancing molecular imaging. Relevant research results were recently published in Photonics Research, Volume 13, Issue 4, 2025. [Xuanhao Wang, Yan Luo, Fudong Xue, Lijuan Ma, Yang Xiao, Dikui Zhou, Junhui Shi, Mingshu Zhang, Pingyong Xu, Cheng Ma, "Fast parallel quantification for near-infrared genetically encoded reporters with self-calibrated photoacoustic screening," Photonics Res. 13, 941 (2025)]

As illustrated in Fig. 1(a), the SCAPAS system comprises a pump laser and optical parametric oscillator (OPO) to generate 532 nm and tunable near-infrared pulsed illumination. A rotating engineered diffuser produces uniform light distribution matching colony dimensions while suppressing speckle noise. PA signals from colonies are captured in parallel by a 256-element ring-shaped piezoelectric transducer array, whose acoustic focus coincides with the sample plane, enabling full-sample imaging without mechanical scanning. For self-calibration quantification [Fig. 1(b)], each target reporter (T-GER) is co-expressed with a reference reporter (R-GER) exhibiting non-overlapping absorption spectra. Dual-wavelength imaging at T-GER and R-GER excitation wavelengths allows normalization of T-GER signals against R-GER signals, thereby correcting errors induced by colony morphology and spatial response variations. In validation tests [Fig. 1(c)], direct readouts from single-wavelength imaging failed to distinguish probe types, whereas self-calibrated results clearly differentiated iRFP713 and SNIFP, aligning closely with ground truth. Statistical analysis [Fig. 1(d)] confirmed SCAPAS's quantification accuracy (~2.8%) and precision (~6.47%), demonstrating its dual advantages of speed and accuracy in probe screening.

The unique advantage of PA imaging lies in its capacity for centimeter-depth visualization of optical absorption. While endogenous hemoglobin provides strong contrast for vascular imaging, it also introduces overwhelming background signals, obscuring dynamic physiological processes (e.g., drug targeting, neural activity) in deep tissues. Thus, developing NIR-GERs with enhanced PA responses remains an urgent need for practical applications. Unlike fluorescence imaging, which benefits from mature probe screening protocols, PA imaging—as an emerging modality—lacks analogous methodologies. The proposed self-calibrated quantification approach fills this gap, offering a novel framework for rapid and precise PA probe development.

Looking ahead, this technology may extend beyond conventional directed evolution. For instance, the discovery of green fluorescent protein (GFP) in Aequorea victoria jellyfish highlights the potential for uncovering novel PA probes in unexplored biological systems. The team plans to advance SCAPAS through automated system design, large-scale experimental validation, and exploration of undiscovered PA probes. Integration with deep learning and sequence-generation algorithms may further accelerate iterative cycles in directed evolution. These efforts aim to unlock new possibilities for high-resolution, deep-tissue molecular imaging and broaden the horizons of PA probe discovery.

Fig. 1 (a) Schematic diagram of the Self-Calibrated Photo-Acoustic Screening (SCAPAS) system. (b) Workflow for co-expressed sample preparation and implementation of the self-calibration method. (c) Comparison of PA response intensity calculations for two PA probes (iRFP713 and SNIFP) using the self-calibrated quantification method versus the traditional direct readout approach. (d) Statistical analysis of quantification results.