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Multiscale photoacoustic tomography using reversibly switchable bacterial phytochrome as a near-infrared photochromic probe.

Yao J, Kaberniuk AA, Li L, Shcherbakova DM, Zhang R, Wang L, Li G, Verkhusha VV, Wang LV - Nat. Methods (2015)

Bottom Line: BphP1 binds a heme-derived biliverdin chromophore and is reversibly photoconvertible between red and near-infrared light-absorption states.We combined single-wavelength PAT with efficient BphP1 photoswitching, which enabled differential imaging with substantially decreased background signals, enhanced detection sensitivity, increased penetration depth and improved spatial resolution.This technology is promising for biomedical studies at several scales.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, Missouri, USA.

ABSTRACT
Photoacoustic tomography (PAT) of genetically encoded probes allows for imaging of targeted biological processes deep in tissues with high spatial resolution; however, high background signals from blood can limit the achievable detection sensitivity. Here we describe a reversibly switchable nonfluorescent bacterial phytochrome for use in multiscale photoacoustic imaging, BphP1, with the most red-shifted absorption among genetically encoded probes. BphP1 binds a heme-derived biliverdin chromophore and is reversibly photoconvertible between red and near-infrared light-absorption states. We combined single-wavelength PAT with efficient BphP1 photoswitching, which enabled differential imaging with substantially decreased background signals, enhanced detection sensitivity, increased penetration depth and improved spatial resolution. We monitored tumor growth and metastasis with ∼ 100-μm resolution at depths approaching 10 mm using photoacoustic computed tomography, and we imaged individual cancer cells with a suboptical-diffraction resolution of ∼ 140 nm using photoacoustic microscopy. This technology is promising for biomedical studies at several scales.

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Optical and photoacoustic characterization of the non-fluorescent bacterial phytochrome BphP1. (a) Molar extinction spectra of oxy-hemoglobin (HbO2), deoxy-hemoglobin (HbR), Pfr (ON) and Pr (OFF) state BphP1. (b) Schematic of the whole-body photoacoustic computed tomography (PACT) system with a ring-shaped illumination pattern. The Ti:Sapphire laser at 780 nm is used for PA imaging and switching off BphP1. The optical parametric oscillator (OPO) laser at 630 nm is used for switching on BphP1. (c) PA signal amplitudes of 30 µM purified ON state rsTagRFP, iRFP720, and ON state BphP1 in clear media, acquired at 567 nm, 715 nm and 780 nm. HbO2 concentration was 2.3 mM for the measurement at 715 nm and 780 nm, and was diluted to 23 µM for the measurement at 567 nm. All the PA signal amplitudes were normalized by that of HbO2 acquired at 780 nm. Error bars, s.d. (d) PA images of transparent plastic tubes filled with proteins in clear media (left column) and with addition of 10 mm thick scattering media (right column). (e) PA signal of purified proteins acquired with increasing imaging depth up to 10 mm in scattering media. (f) Noise-equivalent detectable concentrations of purified proteins at different depths, acquired at their respective absorbing wavelengths. Error bars, s.d.
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Figure 1: Optical and photoacoustic characterization of the non-fluorescent bacterial phytochrome BphP1. (a) Molar extinction spectra of oxy-hemoglobin (HbO2), deoxy-hemoglobin (HbR), Pfr (ON) and Pr (OFF) state BphP1. (b) Schematic of the whole-body photoacoustic computed tomography (PACT) system with a ring-shaped illumination pattern. The Ti:Sapphire laser at 780 nm is used for PA imaging and switching off BphP1. The optical parametric oscillator (OPO) laser at 630 nm is used for switching on BphP1. (c) PA signal amplitudes of 30 µM purified ON state rsTagRFP, iRFP720, and ON state BphP1 in clear media, acquired at 567 nm, 715 nm and 780 nm. HbO2 concentration was 2.3 mM for the measurement at 715 nm and 780 nm, and was diluted to 23 µM for the measurement at 567 nm. All the PA signal amplitudes were normalized by that of HbO2 acquired at 780 nm. Error bars, s.d. (d) PA images of transparent plastic tubes filled with proteins in clear media (left column) and with addition of 10 mm thick scattering media (right column). (e) PA signal of purified proteins acquired with increasing imaging depth up to 10 mm in scattering media. (f) Noise-equivalent detectable concentrations of purified proteins at different depths, acquired at their respective absorbing wavelengths. Error bars, s.d.

Mentions: BphP1 has a natural photochromic behavior: it adopts a Pfr state as the ground state, and undergoes the Pfr→Pr photoconversion upon 730–790 nm light illumination and the Pr→Pfr photoconversion upon 630–690 nm light illumination. From here on, we choose the Pfr state of BphP1 as the ON state, and the Pr state as the OFF state, and used 780 nm light for Pfr→Pr photoconversion and 630 nm light for Pr→Pfr photoconversion. The molar extinction coefficients of the ON state BphP1 at 780 nm and of the OFF-state at 630 nm are respectively ~70-fold and ~40-fold higher than that of oxy-hemoglobin (HbO2) (Fig. 1a, Table 1). We compared BphP1 with the so far reported most red-shifted NIR fluorescent protein (FP), iRFP720, engineered from another BphP20. While the peak absorption of iRFP720 at 705 nm is comparable to that of the ON state BphP1 at 780 nm, iRFP720 is not photoswitchable (Supplementary Fig. 2a, Table 1). We also compared BphP1 with the so far reported most red-shifted photoswitchable FP, rsTagRFP, which can be photoswitched by altering light illumination between 440 nm and 570 nm21, 22. BphP1 was clearly advantageous over rsTagRFP for deep-tissue imaging because of its 2-fold higher extinction coefficient and ~200 nm red-shifted absorption (Supplementary Fig. 2a, Table 1).


Multiscale photoacoustic tomography using reversibly switchable bacterial phytochrome as a near-infrared photochromic probe.

Yao J, Kaberniuk AA, Li L, Shcherbakova DM, Zhang R, Wang L, Li G, Verkhusha VV, Wang LV - Nat. Methods (2015)

Optical and photoacoustic characterization of the non-fluorescent bacterial phytochrome BphP1. (a) Molar extinction spectra of oxy-hemoglobin (HbO2), deoxy-hemoglobin (HbR), Pfr (ON) and Pr (OFF) state BphP1. (b) Schematic of the whole-body photoacoustic computed tomography (PACT) system with a ring-shaped illumination pattern. The Ti:Sapphire laser at 780 nm is used for PA imaging and switching off BphP1. The optical parametric oscillator (OPO) laser at 630 nm is used for switching on BphP1. (c) PA signal amplitudes of 30 µM purified ON state rsTagRFP, iRFP720, and ON state BphP1 in clear media, acquired at 567 nm, 715 nm and 780 nm. HbO2 concentration was 2.3 mM for the measurement at 715 nm and 780 nm, and was diluted to 23 µM for the measurement at 567 nm. All the PA signal amplitudes were normalized by that of HbO2 acquired at 780 nm. Error bars, s.d. (d) PA images of transparent plastic tubes filled with proteins in clear media (left column) and with addition of 10 mm thick scattering media (right column). (e) PA signal of purified proteins acquired with increasing imaging depth up to 10 mm in scattering media. (f) Noise-equivalent detectable concentrations of purified proteins at different depths, acquired at their respective absorbing wavelengths. Error bars, s.d.
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Related In: Results  -  Collection

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Figure 1: Optical and photoacoustic characterization of the non-fluorescent bacterial phytochrome BphP1. (a) Molar extinction spectra of oxy-hemoglobin (HbO2), deoxy-hemoglobin (HbR), Pfr (ON) and Pr (OFF) state BphP1. (b) Schematic of the whole-body photoacoustic computed tomography (PACT) system with a ring-shaped illumination pattern. The Ti:Sapphire laser at 780 nm is used for PA imaging and switching off BphP1. The optical parametric oscillator (OPO) laser at 630 nm is used for switching on BphP1. (c) PA signal amplitudes of 30 µM purified ON state rsTagRFP, iRFP720, and ON state BphP1 in clear media, acquired at 567 nm, 715 nm and 780 nm. HbO2 concentration was 2.3 mM for the measurement at 715 nm and 780 nm, and was diluted to 23 µM for the measurement at 567 nm. All the PA signal amplitudes were normalized by that of HbO2 acquired at 780 nm. Error bars, s.d. (d) PA images of transparent plastic tubes filled with proteins in clear media (left column) and with addition of 10 mm thick scattering media (right column). (e) PA signal of purified proteins acquired with increasing imaging depth up to 10 mm in scattering media. (f) Noise-equivalent detectable concentrations of purified proteins at different depths, acquired at their respective absorbing wavelengths. Error bars, s.d.
Mentions: BphP1 has a natural photochromic behavior: it adopts a Pfr state as the ground state, and undergoes the Pfr→Pr photoconversion upon 730–790 nm light illumination and the Pr→Pfr photoconversion upon 630–690 nm light illumination. From here on, we choose the Pfr state of BphP1 as the ON state, and the Pr state as the OFF state, and used 780 nm light for Pfr→Pr photoconversion and 630 nm light for Pr→Pfr photoconversion. The molar extinction coefficients of the ON state BphP1 at 780 nm and of the OFF-state at 630 nm are respectively ~70-fold and ~40-fold higher than that of oxy-hemoglobin (HbO2) (Fig. 1a, Table 1). We compared BphP1 with the so far reported most red-shifted NIR fluorescent protein (FP), iRFP720, engineered from another BphP20. While the peak absorption of iRFP720 at 705 nm is comparable to that of the ON state BphP1 at 780 nm, iRFP720 is not photoswitchable (Supplementary Fig. 2a, Table 1). We also compared BphP1 with the so far reported most red-shifted photoswitchable FP, rsTagRFP, which can be photoswitched by altering light illumination between 440 nm and 570 nm21, 22. BphP1 was clearly advantageous over rsTagRFP for deep-tissue imaging because of its 2-fold higher extinction coefficient and ~200 nm red-shifted absorption (Supplementary Fig. 2a, Table 1).

Bottom Line: BphP1 binds a heme-derived biliverdin chromophore and is reversibly photoconvertible between red and near-infrared light-absorption states.We combined single-wavelength PAT with efficient BphP1 photoswitching, which enabled differential imaging with substantially decreased background signals, enhanced detection sensitivity, increased penetration depth and improved spatial resolution.This technology is promising for biomedical studies at several scales.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, Missouri, USA.

ABSTRACT
Photoacoustic tomography (PAT) of genetically encoded probes allows for imaging of targeted biological processes deep in tissues with high spatial resolution; however, high background signals from blood can limit the achievable detection sensitivity. Here we describe a reversibly switchable nonfluorescent bacterial phytochrome for use in multiscale photoacoustic imaging, BphP1, with the most red-shifted absorption among genetically encoded probes. BphP1 binds a heme-derived biliverdin chromophore and is reversibly photoconvertible between red and near-infrared light-absorption states. We combined single-wavelength PAT with efficient BphP1 photoswitching, which enabled differential imaging with substantially decreased background signals, enhanced detection sensitivity, increased penetration depth and improved spatial resolution. We monitored tumor growth and metastasis with ∼ 100-μm resolution at depths approaching 10 mm using photoacoustic computed tomography, and we imaged individual cancer cells with a suboptical-diffraction resolution of ∼ 140 nm using photoacoustic microscopy. This technology is promising for biomedical studies at several scales.

Show MeSH
Related in: MedlinePlus