Tiny Photoswitchable Protein Opens a New Window on Deep-Tissue Molecular Imaging

For more than a decade, photoacoustic tomography has promised something extraordinary: the ability to peer centimeters into living tissue with the molecular sensitivity of optics and the spatial precision of ultrasound. The technique works by firing short laser pulses into the body and listening for the ultrasonic waves that tissues emit as they absorb light and thermally expand. Blood vessels light up naturally because hemoglobin is a voracious absorber, but that same abundance has long been the method’s Achilles heel. Molecular probes designed to report on specific genes or proteins produce signals so faint that they drown in the sea of endogenous absorption, particularly in blood-rich organs like the liver. A study published in Nature Methods now reports a solution that is as elegant as it is small: an engineered, photoswitchable protein of just 18.8 kilodaltons that lets researchers subtract away the background and reveal molecular events hidden deep inside living animals.

The new probe, named rsmiRFPnano, belongs to a family of photoreceptors called cyanobacteriochromes, or CBCRs, found in cyanobacteria. These proteins normally require only a single GAF domain to bind their light-sensing chromophore, making them far more compact than the bacteriophytochromes that previously dominated near-infrared photoacoustic imaging. Bacteriophytochrome-based probes, even in their truncated forms, exceed 55 kilodaltons and demand the full three-domain PAS-GAF-PHY assembly to bind biliverdin IXalpha, the heme-breakdown product that mammalian cells supply naturally. That bulk has constrained how these probes can be fused to other proteins or incorporated into reporters. The research team, led by Vladislav Verkhusha of the University of Helsinki and Albert Einstein College of Medicine together with Junjie Yao of Duke University, reasoned that a verdin-specific CBCR lineage, whose members naturally accept biliverdin-like chromophores and absorb at red-shifted wavelengths, could be coaxed into a mammalian-compatible photoswitch a third of the size.

The starting material was the third GAF domain of a CBCR called JSC1_58120 from the cyanobacterium Leptolyngbya sp. JSC1. In its native form, the domain showed promising photochemistry in bacteria, flipping reversibly between a near-infrared-absorbing ground state with an absorbance peak at 715 nanometers and a far-red-absoring photoproduct peaking at 650 nanometers. But in mammalian cells it performed poorly, a fatal flaw for any genetically encoded in vivo probe. Mutations in the biliverdin-binding pocket tend to disrupt the delicate photocycle, so the team took a different tack: iterative saturation mutagenesis aimed at structural regions outside the pocket, targeting paired residues in the N- and C-terminal helices that face each other in the folded structure. Four sequential rounds of evolution, screened first in bacteria and then validated in transfected mammalian cells, yielded rsmiRFPnano, a 163-residue protein carrying seven substitutions that collectively tripled mammalian-cell fluorescence relative to the parent while preserving, and even slightly enhancing, the light-absorption machinery.

The numbers behind the engineered protein are striking. rsmiRFPnano exhibits molar extinction coefficients of 85,000 and 88,000 per molar per centimeter in its two photoswitchable states, among the highest values reported for genetically encoded near-infrared probes. Its pKa values of roughly 4.2 in both states mean spectral properties remain stable across the physiological pH range, and native gel electrophoresis confirmed it behaves as a monomer with no detectable aggregation. In HEK293T cells, flow cytometry showed a 3.5-fold fluorescence increase over the parental protein, and the probe accumulated with kinetics comparable to enhanced green fluorescent protein, reaching half-maximal signal in about 27 hours with an intracellular half-life of roughly 18 hours. These are the characteristics of a probe built to survive and function inside living cells, not just in a cuvette.

The real magic, however, lies in how the probe exploits its photoswitching. Under alternating illumination with 635 and 725 nanometer light, rsmiRFPnano cycles between an ON state that absorbs strongly in the near-infrared and an OFF state that absorbs in the far-red. By acquiring photoacoustic images in both states and subtracting the OFF image from the ON image, the technique known as differential photoacoustic tomography isolates only the signals that actually switch, suppressing the static background from hemoglobin and water. In phantom experiments, purified rsmiRFPnano at 17 micromolar concentration embedded in whole blood produced contrast-to-noise ratios essentially identical to those measured in buffer, demonstrating that the differential contrast is virtually independent of the surrounding optical environment. The team estimated a noise-equivalent detection sensitivity of about 0.33 micromolar against a whole-blood background, a level of performance that transforms what is detectable inside a living animal.

To prove the probe could report on biology rather than just its own presence, the researchers built transcriptional reporters for two master regulators of inflammation, NF-kappaB and AP-1. Each construct placed tandem response elements upstream of a minimal promoter driving rsmiRFPnano expression, so that pathway activation directly translated into probe production. In cultured cells, the reporters showed minimal basal leakiness and strong, dose-dependent induction upon stimulation with tumor necrosis factor or phorbol ester. The team then delivered the reporters to mouse livers via hydrodynamic transfection and induced systemic acute inflammation with lipopolysaccharide. Differential photoacoustic imaging tracked the molecular response longitudinally over five days, revealing that the NF-kappaB reporter peaked rapidly at 12 hours post-treatment while the AP-1 reporter rose more gradually to a maximum at 48 hours, a temporal distinction consistent with known signaling dynamics in liver injury. Control animals receiving saline instead of the inflammatory stimulus showed peak signals fivefold to sevenfold weaker, confirming that the readouts reflected genuine inflammation rather than transfection stress.

The imaging platform went further by integrating super-resolution ultrasound localization microscopy on the same system. Microbubble tracking resolved the hepatic microvasculature in three dimensions and co-registered it with the molecular photoacoustic maps, revealing significant decreases in liver blood-flow speed and blood volume at 48 hours after lipopolysaccharide treatment. An integrated inflammation index, combining the spatial extent and intensity of reporter activation, rose in parallel with the hemodynamic decline. To validate the in vivo findings independently, the collaborators performed multiplexed spatial proteomics using CODEX imaging with a 50-antibody panel on liver sections collected at baseline and at 24 and 72 hours. Single-cell segmentation and clustering showed immune cell infiltration peaking at 24 hours, with macrophages and CD45-positive cells surging early, while inflamed hepatocytes expressing the MHC class I molecule H-2Db dominated by 72 hours. The cellular choreography mapped ex vivo matched the temporal patterns the living-imaging approach had captured noninvasively.

Perhaps the most forward-looking application came from fusing rsmiRFPnano into nanobody scaffolds. Nanobodies, the 15-kilodalton single-domain antibody fragments derived from camelid heavy chains, bind intracellular antigens with high specificity, but unbound nanobodies accumulate and generate background that obscures the signal of interest. The team’s solution, called rsNIR-Fbs, inserts rsmiRFPnano into a loop in the nanobody’s third framework region, distal from the antigen-binding surface. The design makes the fusion protein unstable unless it binds its target: unbound probes are rapidly degraded by the proteasome, while antigen-bound probes are stabilized and generate photoacoustic signal. AlphaFold3 modeling confirmed the insertion preserves the canonical VHH beta-sandwich fold and the antigen-binding loops. The researchers built rsNIR-Fbs against enhanced green fluorescent protein, HIV-1 gp41, and the SARS-CoV-2 spike receptor-binding domain, and in each case fluorescence appeared only when the cognate antigen was coexpressed.

In vivo proof of concept followed in mice whose livers expressed green fluorescent protein via hydrodynamic transfection while the anti-GFP rsNIR-Fb was delivered systemically through an adeno-associated virus. Differential photoacoustic imaging combined with ultrasound localization microscopy revealed strong, spatially confined signals in the liver, co-registered with the microvasculature, and nothing in control organs. When the antigen plasmid was omitted, virtually no differential signal appeared, demonstrating that the photoacoustic output was governed by antigen presence rather than probe delivery or nonspecific accumulation. The result establishes a new class of conditional probes in which the imaging signal itself is gated by molecular recognition inside cells, within organs whose dense blood supply would ordinarily defeat molecular specificity.

The implications extend well beyond inflammation models. Because rsmiRFPnano is threefold smaller than the most compact bacteriophytochrome-derived photoacoustic probes, it offers a flexible scaffold for reporters of virtually any transcriptional pathway, from oncogenic signaling to neuronal plasticity, and the rsNIR-Fb strategy opens photoacoustic immuno-imaging to endogenous intracellular targets including signaling intermediates, viral antigens, and cancer-associated proteins. The authors note that further improvements in biliverdin binding and switching contrast should push detection limits lower still. What the study ultimately delivers is a convergence of protein engineering, acoustic detection, and spatial omics validation: a genetically encodable molecular flashlight, small enough to fit inside a nanobody, bright enough to be heard through centimeters of blood-filled tissue, and switchable enough to be distinguished from everything the body does on its own. For a field that has long struggled to make molecular signals audible above the noise of biology, that is a genuinely resonant advance.

Subject of Research: Engineering of a compact cyanobacteriochrome-derived photoswitchable protein for photoacoustic molecular imaging of inflammation and intracellular antigens

Article Title: Near-infrared photochromic miniprotein enables photoacoustic molecular imaging of acute inflammation and intracellular antigens

Article References: Near-infrared photochromic miniprotein enables photoacoustic molecular imaging of acute inflammation and intracellular antigens. (n.d.). https://doi.org/10.1038/s41592-026-03255-y

Image Credits: AI Generated

DOI: 10.1038/s41592-026-03255-y

Keywords: photoacoustic tomography, rsmiRFPnano, cyanobacteriochrome, near-infrared imaging, NF-kappaB, AP-1, inflammation, nanobody, biliverdin, molecular imaging, protein engineering, spatial omics