Small near-infrared photochromic protein for photoacoustic multi-contrast imaging and detection of protein interactions in vivo

Abstract

Photoacoustic (PA) computed tomography (PACT) benefits from genetically encoded probes with photochromic behavior, which dramatically increase detection sensitivity and specificity through photoswitching and differential imaging. Starting with a DrBphP bacterial phytochrome, we have engineered a near-infrared photochromic probe, DrBphP-PCM, which is superior to the full-length RpBphP1 phytochrome previously used in differential PACT. DrBphP-PCM has a smaller size, better folding, and higher photoswitching contrast. We have imaged both DrBphP-PCM and RpBphP1 simultaneously on the basis of their unique signal decay characteristics, using a reversibly switchable single-impulse panoramic PACT (RS-SIP-PACT) with a single wavelength excitation. The simple structural organization of DrBphP-PCM allows engineering a bimolecular PA complementation reporter, a split version of DrBphP-PCM, termed DrSplit. DrSplit enables PA detection of protein-protein interactions in deep-seated mouse tumors and livers, achieving 125-µm spatial resolution and 530-cell sensitivity in vivo. The combination of RS-SIP-PACT with DrBphP-PCM and DrSplit holds great potential for noninvasive multi-contrast deep-tissue functional imaging.

Fig. 1

Spectral and photoacoustic characterization of the DrBphP-PCM. a Molar extinction spectra of oxy-hemoglobin (HbO₂), deoxy-hemoglobin (Hb), Pfr (ON), and Pr (OFF) state of DrBphP-PCM and RpBphP1. b Schematic of the whole-body photoacoustic computed tomography (PACT) system with a ring-shaped illumination pattern. BC beam combiner, CL conical lens, DAQ data acquisition unit, ED engineering diffuser, M mirror, OC optical condenser, P prism, PC personal computer, pre-A pre-amplifier, USTA ultrasonic transducer array. L1, a Ti:Sapphire laser fired at 780 nm is used for PA imaging and switching off BphPs. L2, the optical parametric oscillator (OPO) laser, fired at 630 nm, switches BphPs ON. c Time sequence of photoswitching and imaging of BphPs (pop., population). d Absorbance of DrBphP-PCM at 780 nm, switched OFF with 780 nm light illumination and then switched ON with 630 nm illumination. The photoswitching period was 180 s for both wavelengths. e PA images of transparent silicone tubes filled with proteins in clear media. Left column: ON state PA image of BphPs and hemoglobin, middle column: frequency lock-in reconstructed (LIR) PA image of BphPs and hemoglobin; right column, decay constant encoded image showing a reliable separation of DrBphP-PCM, RpBphP1, and non-switchable hemoglobin. Scale bar, 500 µm. f PA signal changes upon 780 nm light illumination and their fits, where PA signals from either DrBphP-PCM or RpBphP1 exponentially decrease during photoswitching, but with different decay constants. However, the blood signal remains at the original level. Thus, the difference in decay constants enables a good separation of hemoglobin, DrBphP-PCM, and RpBphP1. g The switching ratio of BphPs and hemoglobin, defined as the ratio between the ON and OFF states of the PA amplitude, in both clear medium (0 mm in depth) and scattering medium (12 mm in depth); error bars are s.e.m. (n = 40), calculated based on the pixel values from regions of interest

Fig. 2

Photoacoustic characterization of the BphPs in cultured cells. a LIR image of bovine blood, U87 cells expressing either RpBphP1 or DrBphP-PCM. Scale bar, 2 mm. b PA signal decays and their fits from bovine blood, U87 cells expressing either RpBphP1 or DrBphP-PCM during 780 nm light illumination. c Decay constant encoded image showing different photoswitching rates of U87 cells expressing either RpBphP1 or DrBphP-PCM and the non-switchable bovine blood. Scale bar, 2 mm. d The computed decay constants of the three types of cells; error bars are s.e.m. (n = 40), calculated based on the pixel values from regions of interest. e The contrast-to-noise ratio (CNR) of LIR signals from bovine blood and from U87 cells expressing either RpBphP1 or DrBphP-PCM in a clear medium (0 mm in depth) and a scattering medium (15 mm in depth). f PA signal decays and their fits for the two types of cells—HEK-293 cells expressing both DrBphP-PCM and RpBphP1, and U87 cells expressing only DrBphP-PCM—under different illumination fluences. g LIR images (top row) of U87 cells (left) and HEK-293 cells (right) and the images of computed coefficients of b (middle row) and c (bottom row) under different illumination fluences. The signal decays can be modeled in the form of $g\left(t\right)=a+b\cdot e^{\left(\frac{-t}{T_1}\right)}+c\cdot e^{\left(\frac{-t}{T_2}\right)}$, where T₁ > T₂. The signals from HEK-293 cells were fitted with two similar coefficients b ≈ c ≈ 0.5, while the signals from U87 cells were fitted with very different coefficients b ≈ 1, c ≈ 0. Scale bar, 2 mm. h The computed coefficients k, defined as $k=\frac{\max \left\{b,c\right\}}{\min \left\{b,c\right\}}$, under different light fluences, showing a reliable separation of the two types of cells in f, g. Independent of the light fluence, the coefficient k for HEK-293 cells is ~1, and the coefficient k for U87 cells is much larger (>10); error bars are s.e.m. (n = 120), calculated based on the pixel values from regions of interest

Fig. 3

Multi-contrast PA imaging of BphPs in the mouse brain in vivo. a Conventional PA image of the tumor-bearing mouse brain cortex vasculature (ON state). Approximately 1 × 10⁶ U87 cells expressing DrBphP-PCM were injected into the left front of the brain, ~1 × 10⁶ U87 cells expressing RpBphP1 were injected into the right rear of the brain. The tumors are invisible in the ON state images due to the overwhelming background signals from blood. Scale bar, 2 mm. b LIR image overlaid on the mouse brain cortex vasculature, highlighting the two tumors of U87 cells expressing either RpBphP1 or DrBphP-PCM. The overlay image shows the BphP signals in color and the background blood signals in gray. c PA signals from two tumors were modulated at the same frequency by the illumination but with different signal decay constants. d Temporal frequency spectra of the PA signals from brain tumors and the cortical arteries, showing both the harmonics of the illumination modulation frequency and the heartbeat frequency from the arteries. e PA signal decays and their fits for the two tumors expressing either DrBphP-PCM or RpBphP1. f The computed decay constants of the two tumors; error bars are s.e.m. (n = 160), calculated based on the pixel values from regions of interest. g Decay constant encoded image illustrating good separation of the two tumors. Scale bar, 2 mm. h Decay constant encoded image overlaid on the mouse brain cortical vasculature, showing reliable separation of two tumors

Fig. 4

In vivo lock-in reconstruction of BphPs in kidneys and their decay analysis. a LIR image overlaid on a conventional PACT cross-sectional image by 780 nm illumination, highlighting the two tumors of U87 cells expressing either RpBphP1 (right side) or DrBphP-PCM (left side) on the two kidneys. The overlay image shows the BphPs signal in color and the background blood signal in gray. b Decay constant encoded image overlaid on a conventional PACT cross-sectional image made by 780 nm illumination. The LIR image was used to form a binary mask, and the decay constant computation was implemented in the masked regions. c Representative H&E histological images of the two isolated kidneys, showing the tumors (bordered by green lines) corresponding to a and b. Scale bar, 1 mm. d The computed decay constants of the two tumors. e PA signal decays and their fits in the tumor regions. f Temporal frequency spectra of the PA signals in the kidney tumors and the internal organs, showing both the harmonics of the illumination modulation frequency and the harmonics of the respiratory frequency. g The LIR method provides approximately two–threefold better CNR of tumor cells expressing either DrBphP-PCM or RpBphP1 than the differential imaging method; error bars are s.e.m. (n = 80), calculated based on the pixel values from regions of interest. Scale bar, 5 mm

Fig. 5

In vivo separation of two types of cells at depths. The PA excitation wavelength was 780 nm. a LIR image overlaid on a conventional PACT cross-sectional image by 780 nm illumination, highlighting the two tumors of HEK-293 cells expressing both DrBphP-PCM and RpBphP1 (left lobe) or U87 cells expressing DrBphP-PCM (right lobe) inside the liver (n = 3). The overlay image shows the BphP signals in color and the background blood signals in gray. b Coefficient b encoded image overlaid on a conventional PACT cross-sectional image. The computed coefficient, b, is shown in color, and the background anatomy is shown in gray. The LIR image in a was used to form a binary mask, and the decay analysis was implemented in the masked regions. c Coefficient c encoded image overlaid on a conventional PACT cross-sectional image. The computed coefficient, c, is shown in color, and the background anatomy is shown in gray. d Normalized coefficient k encoded image overlaid on a conventional PACT cross-sectional image. Because the HEK-293 tumors contain two different photochromic proteins and U87 tumors contain only one photochromic protein, the normalized coefficient k of HEK-293 tumors is much smaller than that of U87 tumor, showing a reliable separation of the two tumors. The LIR image was used to form a binary mask, and the decay constant computation was implemented in the masked regions. e PA signal decays and their fits in the tumor regions. f The computed coefficients of b, c, and k from the tumor regions, where k, showing the largest difference, can be used to separate the two types of tumors. Independent of the light fluence, the coefficient k for HEK-293 tumors is ~1, and the coefficient k for U87 tumors is much larger (>8); error bars are s.e.m. (n = 140), calculated based on the pixel values from regions of interest. Scale bar, 5 mm

Fig. 6

Development of the bimolecular photoacoustic complementation (BiPC) reporter DrSplit. a DrBphP-PCM consists of three domains, PAS, GAF, and PHY. The biliverdin (BV) chromophore is covalently bound with conservative cysteine from the PAS domain and secured to a chromophore-binding pocket in the GAF domain. DrBphP-PCM was genetically split into two parts, the PAS domain and GAF-PHY domain, together named DrSplit. In this case, BV does not bind with any part of DrSplit. Genetically fusing one protein of interest (protein A) to one part of DrSplit and another protein of interest (protein B) to another part of DrSplit makes possible the monitoring of protein–protein interactions (PPIs) between protein A and protein B. b We used a model rapamycin-induced PPI between the FRB and FKBP proteins for evaluation of DrSplit. FRB was fused to the PAS domain and FKBP was fused to the GAF-PHY domains. Upon addition of rapamycin to the DrSplit, DrBphP-PCM was re-functionalized

Fig. 7

PA characterization of DrSplit in mammalian cells. a ON state PA image of MTLn3 cells expressing DrSplit (left) and MTLn3 cells expressing DrSplit in the presence of rapamycin (right). Scale bar, 2 mm. b OFF state PA image of MTLn3 cells expressing DrSplit (left) and MTLn3 cells expressing DrSplit in the presence of rapamycin (right). c LIR PA image of MTLn3 cells expressing DrSplit (left) and MTLn3 cells expressing DrSplit in the presence of rapamycin (right). The induction with rapamycin reconstitutes the functional DrBphP-PCM, which responds to the periodical light modulation. d Repeated fluorescence changes of the lysate of HeLa cells expressing DrBphP-PCM detected at 720 nm during recurrent illumination cycles with 780/20 nm and 636/20 nm. e Repeated fluorescence changes of the lysate of HeLa cells expressing DrSplit in the presence (black line) or absence (blue line) of rapamycin, detected at 720 nm during recurrent illumination cycles with 780/20 nm and 636/20 nm. f MTLn3 cells expressing DrSplit and MTLn3 cells expressing DrSplit in the presence of rapamycin and blood (dilute 10×) show similar CNRs in the ON state PA image; while the LIR image shows an outstanding CNR for MTLn3 cells expressing DrSplit in the presence of rapamycin. g LIR image of blood and MTLn3 cells expressing DrSplit in the presence of rapamycin in a clear medium (0 mm in depth) and a scattering medium (15 mm in depth). Scale bar, 2 mm. h The switching ratio of blood and MTLn3 cells expressing DrSplit in the presence of rapamycin in both a clear medium (0 mm in depth) and a scattering medium (15 mm in depth); error bars are s.e.m. (n = 40), calculated based on the pixel values from regions of interest. Rapa is short for rapamycin

Fig. 8

Longitudinal imaging of PPIs in a tumor and monitoring of tumor metastases in a mouse liver. Approximately 1 × 10⁶ MTLn3 cells expressing DrSplit were injected into the mouse liver. The mice (n = 4) were imaged at multiple time points after tumor cell injection, and rapamycin was injected via the tail vein ~40–44 h before each PA imaging. a–d PA images of the mouse on a day 5, b day 15, c day 24, and d day 33 after injection of tumor cells, where the white arrows indicate the secondary tumor. LIR images are overlaid on the anatomical images. The overlay image shows the DrSplit signal in color and the background blood signal in gray. Scale bar, 5 mm. e Tumor growth curve, in-plane tumor area vs. time (quantified from LIR images). Error bars represent s.e.m. for results from four animals. f A representative H&E histological image of a harvested left lobe of a tumorous liver, showing the tumor metastasis, where the primary tumor and secondary tumor are bordered by green and yellow lines, respectively. Scale bar, 1 mm. The close-up H&E image shows the secondary tumor, which can be clearly differentiated from normal tissue. Scale bar, 100 µm

Fig. 9

In vivo monitoring of PPIs in a mouse liver after hydrodynamic transfection. a PA image of the mouse liver 24 h after hydrodynamic transfection, without induction with rapamycin. The LIR image was overlaid on the anatomical images, but no photoswitching signal was detected. Scale bar, 5 mm. b PA image of the mouse liver 42 h after rapamycin injection, with the LIR image overlaid on the anatomical image. c Ex vivo PA image (ON state) of the liver excised from the hydrodynamic-transfected mouse. Scale bar, 2 mm. d Ex vivo PA image (OFF state) of the excised mouse liver. e LIR image of the mouse liver excised from the hydrodynamic-transfected mouse. f Overlay of the LIR image and the anatomical image of the excised mouse liver. g CNR comparison of the mouse liver region in the LIR image with and without rapamycin injection. Rapamycin was injected through the tail vein 24 h after hydrodynamic liver transfection; Error bars represent s.e.m. for results from four animals. Rapa is short for rapamycin