Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins.
ABSTRACT: Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted fluorescent proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths, and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal, and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8?mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo.
Project description:Near-infrared fluorescent proteins (FPs) are in high demand for in vivo imaging. We developed four spectrally distinct near-infrared FPs--iRFP670, iRFP682, iRFP702 and iRFP720--from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging with standard approaches in living cells and mice. The four new iRFPs and the previously engineered iRFP713 allow multicolor imaging with spectral unmixing in living mice.
Project description:Near-infrared fluorescent proteins, iRFPs, are recently developed genetically encoded fluorescent probes for deep-tissue in vivo imaging. Their functions depend on the corresponding fluorescence efficiencies and electronic excited state properties. Here we report the electronic excited state deactivation dynamics of the most red-shifted iRFPs: iRFP702, iRFP713 and iRFP720. Complementary measurements by ultrafast broadband fluorescence and absorption spectroscopy show that single exponential decays of the excited state with 600~700 ps dominate in all three iRFPs, while photoinduced isomerization was completely inhibited. Significant kinetic isotope effects (KIE) were observed with a factor of ~1.8 in D2O, and are interpreted in terms of an excited-state proton transfer (ESPT) process that deactivates the excited state in competition with fluorescence and chromophore mobility. On this basis, new approaches for rational molecular engineering may be applied to iRFPs to improve their fluorescence.
Project description:At present, clinicians routinely apply ultrasound endoscopy in a variety of interventional procedures that provide treatment solutions for diseased organs. Ultrasound endoscopy not only produces high-resolution images, but also is safe for clinical use and broadly applicable. However, for soft tissue imaging, its mechanical wave-based image contrast fundamentally limits its ability to provide physiologically specific functional information. By contrast, photoacoustic endoscopy possesses a unique combination of functional optical contrast and high spatial resolution at clinically relevant depths, ideal for imaging soft tissues. With these attributes, photoacoustic endoscopy can overcome the current limitations of ultrasound endoscopy. Moreover, the benefits of photoacoustic imaging do not come at the expense of existing ultrasound functions; photoacoustic endoscopy systems are inherently compatible with ultrasound imaging, thereby enabling multimodality imaging with complementary contrast. Here we present simultaneous photoacoustic and ultrasonic dual-mode endoscopy and show its ability to image internal organs in vivo, thus illustrating its potential clinical application.
Project description:Spectrally resolved photoacoustic imaging is promising for label-free imaging in optically scattering materials. However, this technique often requires acquisition of a separate image at each wavelength of interest. This reduces imaging speeds and causes errors if the sample changes in time between images acquired at different wavelengths. We demonstrate a solution to this problem by using dual-comb spectroscopy for photoacoustic measurements. This approach enables a photoacoustic measurement at thousands of wavelengths simultaneously. In this technique, two optical-frequency combs are interfered on a sample and the resulting pressure wave is measured with an ultrasound transducer. This acoustic signal is processed in the frequency-domain to obtain an optical absorption spectrum. For a proof-of-concept demonstration, we measure photoacoustic signals from polymer films. The absorption spectra obtained from these measurements agree with those measured using a spectrophotometer. Improving the signal-to-noise ratio of the dual-comb photoacoustic spectrometer could enable high-speed spectrally resolved photoacoustic imaging.
Project description:Optical-resolution photoacoustic microscopy (OR-PAM) is an imaging modality with superb penetration depth and excellent absorption contrast. Here we demonstrate, for the first time, that this technique can advance quantitative analysis of conventional chromogenic histochemistry. Because OR-PAM can quantify the absorption contrast at different wavelengths, it is feasible to spectrally resolve the specific biomolecules involved in a staining color. Furthermore, the tomographic capability of OR-PAM allows for noninvasive volumetric imaging of a thick sample without microtoming it. By immunostaining the sample with different chromogenic agents, we further demonstrated the ability of OR-PAM to resolve different types of cells in a coculture sample with imaging depths up to 1?mm. Taken together, the integration of OR-PAM with (immuno)histochemistry offers a simple and versatile technique with broad applications in cell biology, pathology, tissue engineering, and related biomedical studies.
Project description:Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial phytochromes but were not systematically compared in neurons. To fluoresce, NIR FPs utilize an enzymatic derivative of heme, the linear tetrapyrrole biliverdin, as a chromophore whose level in neurons is poorly studied. Here, we evaluated NIR FPs of the iRFP protein family, which were reported to be the brightest in non-neuronal mammalian cells, in primary neuronal culture, in brain slices of mouse and monkey, and in mouse brain in vivo. We applied several fluorescence imaging modes, such as wide-field and confocal one-photon and two-photon microscopy, to compare photochemical and biophysical properties of various iRFPs. The iRFP682 and iRFP670 proteins exhibited the highest brightness and photostability under one-photon and two-photon excitation modes, respectively. All studied iRFPs exhibited efficient binding of the endogenous biliverdin chromophore in cultured neurons and in the mammalian brain and can be readily applied to neuroimaging.
Project description:Photoacoustic tomography is a rapidly growing imaging modality that can provide images of high spatial resolution and high contrast at depths up to 5 cm. We report here the design, synthesis, and evaluation of an activatable probe that shows great promise for enabling detection of the cleaved probe in the presence of high levels of nonactivated, uncleaved probe, a difficult task to attain in absorbance-based modality. Before the cleavage by its target, proteolytic enzyme MMP-2, the probe, an activatable cell-penetrating peptide, Ceeee[Ahx]PLGLAGrrrrrK, labeled with two chromophores, BHQ3 and Alexa750, shows photoacoustic signals of similar intensity at the two wavelengths corresponding to the absorption maxima of the chromophores, 675 and 750 nm. Subtraction of the images taken at these two wavelengths makes the probe effectively photoacoustically silent, as the signals at these two wavelengths essentially cancel out. After the cleavage, the dye associated with the cell-penetrating part of the probe, BHQ3, accumulates in the cells, while the other dye diffuses away, resulting in photoacoustic signal seen at only one of the wavelengths, 675 nm. Subtraction of the photoacoustic images at two wavelengths reveals the location of the cleaved (activated) probe. In the search for the chromophores that are best suited for photoacoustic imaging, we have investigated the photoacoustic signals of five chromophores absorbing in the near-infrared region. We have found that the photoacoustic signal did not correlate with the absorbance and fluorescence of the molecules, as the highest photoacoustic signal arose from the least absorbing quenchers, BHQ3 and QXL 680.
Project description:To noninvasively map sentinel lymph nodes (SLNs) and lymphatic vessels in rats in vivo by using dual-modality nonionizing imaging-volumetric spectroscopic photoacoustic imaging, which measures optical absorption, and planar fluorescence imaging, which measures fluorescent emission-of indocyanine green (ICG).Institutional animal care and use committee approval was obtained. Healthy Sprague-Dawley rats weighing 250-420 g (age range, 60-120 days) were imaged by using volumetric photoacoustic imaging (n = 5) and planar fluorescence imaging (n = 3) before and after injection of 1 mmol/L ICG. Student paired t tests based on a logarithmic scale were performed to evaluate the change in photoacoustic signal enhancement of SLNs and lymphatic vessels before and after ICG injection. The spatial resolutions of both imaging systems were compared at various imaging depths (2-8 mm) by layering additional biologic tissues on top of the rats in vivo. Spectroscopic photoacoustic imaging was applied to identify ICG-dyed SLNs.In all five rats examined with photoacoustic imaging, SLNs were clearly visible, with a mean signal enhancement of 5.9 arbitrary units (AU) + or - 1.8 (standard error of the mean) (P < .002) at 0.2 hour after injection, while lymphatic vessels were seen in four of the five rats, with a signal enhancement of 4.3 AU + or - 0.6 (P = .001). In all three rats examined with fluorescence imaging, SLNs and lymphatic vessels were seen. The average full width at half maximum (FWHM) of the SLNs in the photoacoustic images at three imaging depths (2, 6, and 8 mm) was 2.0 mm + or - 0.2 (standard deviation), comparable to the size of a dissected lymph node as measured with a caliper. However, the FWHM of the SLNs in fluorescence images widened from 8 to 22 mm as the imaging depth increased, owing to strong light scattering. SLNs were identified spectroscopically in photoacoustic images.These two modalities, when used together with ICG, have the potential to help map SLNs in axillary staging and to help evaluate tumor metastasis in patients with breast cancer.
Project description:Photoacoustic tomography (PAT) is a non-ionizing imaging modality capable of acquiring high contrast and resolution images of optical absorption at depths greater than traditional optical imaging techniques. Practical considerations with instrumentation and geometry limit the number of available acoustic sensors and their "view" of the imaging target, which result in image reconstruction artifacts degrading image quality. Iterative reconstruction methods can be used to reduce artifacts but are computationally expensive. In this work, we propose a novel deep learning approach termed pixel-wise deep learning (Pixel-DL) that first employs pixel-wise interpolation governed by the physics of photoacoustic wave propagation and then uses a convolution neural network to reconstruct an image. Simulated photoacoustic data from synthetic, mouse-brain, lung, and fundus vasculature phantoms were used for training and testing. Results demonstrated that Pixel-DL achieved comparable or better performance to iterative methods and consistently outperformed other CNN-based approaches for correcting artifacts. Pixel-DL is a computationally efficient approach that enables for real-time PAT rendering and improved image reconstruction quality for limited-view and sparse PAT.
Project description:Molecular photoacoustic imaging has shown great potential in medical applications; its sensitivity is normally in pico-to-micro-molar range, dependent on exogenous imaging agents. However, tissue can produce strong background signals, which mask the signals from the imaging agents, resulting in orders of magnitude sensitivity reduction. As such, an elaborate spectral scan is often required to spectrally un-mix the unwanted background signals. Here we show a new single-wavelength photoacoustic dynamic contrast-enhanced imaging technique by employing a stimuli-responsive contrast agent. Our technique can eliminate intrinsic background noises without significant hardware or computational resources. We show that this new contrast agent can generate up to 30 times stronger photoacoustic signals than the concentration-matched inorganic nanoparticle counterparts. By dynamically modulating signals from the contrast agents with an external near-infrared optical stimulus, we can further suppress the background signals leading to an additional increase of more than five-fold in imaging contrast in vivo.