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Ultrasound detectors use high-frequency sound waves to image objects and measure distances, but the resolution of these readings is limited by the physical dimensions of the detecting element. Point-like broadband ultrasound detection can greatly increase the resolution of ultrasonography and optoacoustic (photoacoustic) imaging1,2, but current ultrasound detectors, such as those used for medical imaging, cannot be miniaturized sufficiently. Piezoelectric transducers lose sensitivity quadratically with size reduction3, and optical microring resonators4 and Fabry-Pérot etalons5 cannot adequately confine light to dimensions smaller than about 50 micrometres. Micromachining methods have been used to generate arrays of capacitive6 and piezoelectric7 transducers, but with bandwidths of only a few megahertz and dimensions exceeding 70 micrometres. Here we use the widely available silicon-on-insulator technology to develop a miniaturized ultrasound detector, with a sensing area of only 220 nanometres by 500 nanometres. The silicon-on-insulator-based optical resonator design provides per-area sensitivity that is 1,000 times higher than that of microring resonators and 100,000,000 times better than that of piezoelectric detectors. Our design also enables an ultrawide detection bandwidth, reaching 230 megahertz at -6 decibels. In addition to making the detectors suitable for manufacture in very dense arrays, we show that the submicrometre sensing area enables super-resolution detection and imaging performance. We demonstrate imaging of features 50 times smaller than the wavelength of ultrasound detected. Our detector enables ultra-miniaturization of ultrasound readings, enabling ultrasound imaging at a resolution comparable to that achieved with optical microscopy, and potentially enabling the development of very dense ultrasound arrays on a silicon chip.
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Optoacoustic (photoacoustic) imaging has seen marked advances in detection and data analysis, but there is less progress in understanding the photophysics of common optoacoustic contrast agents. This gap blocks the development of novel agents and the accurate analysis and interpretation of multispectral optoacoustic images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. Herein, we employ MLS to analyze contrast agents (methylene blue, rhodamine 800, Alexa Fluor 750, IRDye 800CW, and indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720, and smURFP). We found that the optical absorption spectrum does not correlate with the optoacoustic spectrum for the majority of the analytes. We determined that for dyes, the transition underlying an aggregation state has more optoacoustic signal generation efficiency than the monomer transition. For proteins we found a favored optoacoustic relaxation that stems from the neutral or zwitterionic chromophores and unreported photoswitching behavior of tdTomato and HcRed. We then crystalized HcRed in its photoswitch optoacoustic state, confirming structurally the change in isomerization with respect to HcReds' fluorescence state. Finally, on the example of the widely used label tdTomato and the dye indocyanine green, we show the importance of correct photophysical (e.g., spectral and kinetic) information as a prerequisite for spectral-unmixing for in vivo imaging.
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Absorción Fisicoquímica , Colorantes/química , Proteínas Luminiscentes/química , Imagen Molecular , Técnicas Fotoacústicas , Límite de Detección , Modelos Moleculares , Conformación ProteicaRESUMEN
A long-standing objective in neuroscience has been to image distributed neuronal activity in freely behaving animals. Here we introduce NeuBtracker, a tracking microscope for simultaneous imaging of neuronal activity and behavior of freely swimming fluorescent reporter fish. We showcase the value of NeuBtracker for screening neurostimulants with respect to their combined neuronal and behavioral effects and for determining spontaneous and stimulus-induced spatiotemporal patterns of neuronal activation during naturalistic behavior.
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Conducta Animal , Peces/fisiología , Animales , Microscopía/métodos , Neuronas/fisiología , Natación/fisiologíaRESUMEN
Reversibly switchable fluorescent proteins (rsFPs) have had a revolutionizing effect on life science imaging due to their contribution to sub-diffraction-resolution optical microscopy (nanoscopy). Initial studies showed that their use as labels could also be highly beneficial for emerging photo- or optoacoustic imaging. It could be shown that their use in optoacoustics (i) strongly improves the imaging contrast-to-noise ratio due to modulation and locked-in detection, (ii) facilitates fluence calibration, affording precise measurements of physiological parameters, and finally (iii) could boost spatial resolution following similar concepts as used for nanoscopy. However, rsFPs show different photophysical behavior in optoacoustics than in optical microscopy because optoacoustics requires pulsed illumination and depends on signal generation via nonradiative energy decay channels. This implies that rsFPs optimized for fluorescence imaging may not be ideal for optoacoustics. Here, we analyze the photophysical behavior of a broad range of rsFPs with optoacoustics and analyze how the experimental factors central to optoacoustic imaging influence the different types of rsFPs. Finally, we discuss how knowledge of the switching behavior can be exploited for various optoacoustic imaging approaches using sophisticated temporal unmixing schemes.
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Proteínas Luminiscentes/química , Técnicas Fotoacústicas/métodos , Fluorescencia , Microscopía Fluorescente/métodosRESUMEN
We show that nanoscopy based on the principle called RESOLFT (reversible saturable optical fluorescence transitions) or nonlinear structured illumination can be effectively parallelized using two incoherently superimposed orthogonal standing light waves. The intensity minima of the resulting pattern act as 'doughnuts', providing isotropic resolution in the focal plane and making pattern rotation redundant. We super-resolved living cells in 120 µm × 100 µm-sized fields of view in <1 s using 116,000 such doughnuts.
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Microscopía Fluorescente/métodos , Nanotecnología/métodos , Animales , Línea Celular , Femenino , Humanos , Masculino , Microscopía Fluorescente/instrumentación , Neuronas/ultraestructura , Ratas , Ratas WistarRESUMEN
Up to now, all demonstrations of reversible saturable optical fluorescence transitions (RESOLFT) superresolution microscopy of living cells have relied on the use of reversibly switchable fluorescent proteins (RSFP) emitting in the green spectral range. Here we show RESOLFT imaging with rsCherryRev1.4, a new red-emitting RSFP enabling a spatial resolution up to four times higher than the diffraction barrier. By co-expressing green and red RSFPs in living cells we demonstrate two-color RESOLFT imaging both for single ("donut") beam scanning and for parallelized versions of RESOLFT nanoscopy where an array of >23,000 "donut-like" minima are scanned simultaneously.
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Color , Proteínas Fluorescentes Verdes/análisis , Proteínas Luminiscentes/análisis , Microscopía Fluorescente , Nanotecnología/métodos , Células Cultivadas , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteína Fluorescente RojaRESUMEN
Conventional histology, as well as immunohistochemistry or immunofluorescence, enables the study of morphological and phenotypical changes during tissue inflammation with single-cell accuracy. However, although highly specific, such techniques require multiple time-consuming steps to apply exogenous labels, which might result in morphological deviations from native tissue structures. Unlike these techniques, mid-infrared (mid-IR) microspectroscopy is a label-free optical imaging method that retrieves endogenous biomolecular contrast without altering the native composition of the samples. Nevertheless, due to the strong optical absorption of water in biological tissues, conventional mid-IR microspectroscopy has been limited to dried thin (5-10 µm) tissue preparations and, thus, it also requires time-consuming steps-comparable to conventional imaging techniques. Here, as a step towards label-free analytical histology of unprocessed tissues, we applied mid-IR optoacoustic microscopy (MiROM) to retrieve intrinsic molecular contrast by vibrational excitation and, simultaneously, to overcome water-tissue opacity of conventional mid-IR imaging in thick (mm range) tissues. In this proof-of-concept study, we demonstrated application of MiROM for the fast, label-free, non-destructive assessment of the hallmarks of inflammation in excised white adipose tissue; i.e., formation of crown-like structures and changes in adipocyte morphology.
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Short-wave infrared (SWIR) fluorescence could become the new gold standard in optical imaging for biomedical applications due to important advantages such as lack of autofluorescence, weak photon absorption by blood and tissues, and reduced photon scattering coefficient. Therefore, contrary to the visible and NIR regions, tissues become translucent in the SWIR region. Nevertheless, the lack of bright and biocompatible probes is a key challenge that must be overcome to unlock the full potential of SWIR fluorescence. Although rare-earth-based core-shell nanocrystals appeared as promising SWIR probes, they suffer from limited photoluminescence quantum yield (PLQY). The lack of control over the atomic scale organization of such complex materials is one of the main barriers limiting their optical performance. Here, the growth of either homogeneous (α-NaYF4) or heterogeneous (CaF2) shell domains on optically-active α-NaYF4:Yb:Er (with and without Ce3+ co-doping) core nanocrystals is reported. The atomic scale organization can be controlled by preventing cation intermixing only in heterogeneous core-shell nanocrystals with a dramatic impact on the PLQY. The latter reached 50% at 60 mW/cm2; one of the highest reported PLQY values for sub-15 nm nanocrystals. The most efficient nanocrystals were utilized for in vivo imaging above 1450 nm.
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Introduction: The intrinsic autofluorescence of biological tissues interferes with the detection of fluorophores administered for fluorescence guidance, an emerging auxiliary technique in oncological surgery. Yet, autofluorescence of the human brain and its neoplasia is sparsely examined. This study aims to assess autofluorescence of the brain and its neoplasia on a microscopic level by stimulated Raman histology (SRH) combined with two-photon fluorescence. Methods: With this experimentally established label-free microscopy technique unprocessed tissue can be imaged and analyzed within minutes and the process is easily incorporated in the surgical workflow. In a prospective observational study, we analyzed 397 SRH and corresponding autofluorescence images of 162 samples from 81 consecutive patients that underwent brain tumor surgery. Small tissue samples were squashed on a slide for imaging. SRH and fluorescence images were acquired with a dual wavelength laser (790 nm and 1020 nm) for excitation. In these images tumor and non-tumor regions were identified by a convolutional neural network that reliably differentiates between tumor, healthy brain tissue and low quality SRH images. The identified areas were used to define regions.of- interests (ROIs) and the mean fluorescence intensity was measured. Results: In healthy brain tissue, we found an increased mean autofluorescence signal in the gray (11.86, SD 2.61, n=29) compared to the white matter (5.99, SD 5.14, n=11, p<0.01) and in the cerebrum (11.83, SD 3.29, n=33) versus the cerebellum (2.82, SD 0.93, n=7, p<0.001), respectively. The signal of carcinoma metastases, meningiomas, gliomas and pituitary adenomas was significantly lower (each p<0.05) compared to the autofluorescence in the cerebrum and dura, and significantly higher (each p<0.05) compared to the cerebellum. Melanoma metastases were found to have a higher fluorescent signal (p<0.01) compared to cerebrum and cerebellum. Discussion: In conclusion we found that autofluorescence in the brain varies depending on the tissue type and localization and differs significantly among various brain tumors. This needs to be considered for interpreting photon signal during fluorescence-guided brain tumor surgery.
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Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.
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Técnicas Fotoacústicas , Animales , Línea Celular , Ratones , Microscopía Fluorescente/métodos , Técnicas Fotoacústicas/métodosRESUMEN
Purpose: The Bragg peak located at the end of the ion beam range is one of the main advantages of ion beam therapy compared to X-Ray radiotherapy. However, verifying the exact position of the Bragg peak within the patient online is a major challenge. The goal of this work was to achieve submillimeter proton beam range verification for pulsed proton beams of an energy of up to 220 MeV using ionoacoustics for a clinically relevant dose deposition of typically 2 Gy per fraction by i) using optimal proton beam characteristics for ionoacoustic signal generation and ii) improved signal detection by correlating the signal with simulated filter templates. Methods: A water tank was irradiated with a preclinical 20 MeV proton beam using different pulse durations ranging from 50 ns up to 1 µs in order to maximise the signal-to-noise ratio (SNR) of ionoacoustic signals. The ionoacoustic signals were measured using a piezo-electric ultrasound transducer in the MHz frequency range. The signals were filtered using a cross correlation-based signal processing algorithm utilizing simulated templates, which enhances the SNR of the recorded signals. The range of the protons is evaluated by extracting the time of flight (ToF) of the ionoacoustic signals and compared to simulations from a Monte Carlo dose engine (FLUKA). Results: Optimised SNR of 28.0 ± 10.6 is obtained at a beam current of 4.5 µA and a pulse duration of 130 ns at a total peak dose deposition of 0.5 Gy. Evaluated ranges coincide with Monte Carlo simulations better than 0.1 mm at an absolute range of 4.21 mm. Higher beam energies require longer proton pulse durations for optimised signal generation. Using the correlation-based post-processing filter a SNR of 17.8 ± 5.5 is obtained for 220 MeV protons at a total peak dose deposition of 1.3 Gy. For this clinically relevant dose deposition and proton beam energy, submillimeter range verification was achieved at an absolute range of 303 mm in water. Conclusion: Optimal proton pulse durations ensure an ideal trade-off between maximising the ionoacoustic amplitude and minimising dose deposition. In combination with a correlation-based post-processing evaluation algorithm, a reasonable SNR can be achieved at low dose levels putting clinical applications for online proton or ion beam range verification into reach.
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Despite its importance in regulating cellular or tissue function, electrical conductivity can only be visualized in tissue indirectly as voltage potentials using fluorescent techniques, or directly with radio waves. These either requires invasive procedures like genetic modification or suffers from limited resolution. Here, we introduce radio-frequency thermoacoustic mesoscopy (RThAM) for the noninvasive imaging of conductivity by exploiting the direct absorption of near-field ultrashort radio-frequency pulses to stimulate the emission of broadband ultrasound waves. Detection of ultrasound rather than radio waves enables micrometer-scale resolutions, over several millimeters of tissue depth. We confirm an imaging resolution of <30 µm in phantoms and demonstrate microscopic imaging of conductivity correlating to physical structures in 1- and 512-cell zebrafish embryos, as well as larvae. These results support RThAM as a promising method for high-resolution, label-free assessment of conductivity in tissues.
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Magneto-plasmonic nanocomposites can possess properties inherent to both individual components (iron oxide and gold nanoparticles) and are reported to demonstrate high potential in targeted drug delivery and therapy. Herein, we report on Fe3O4/Au magneto-plasmonic nanocomposites (MPNC) synthesized with the use of amino acid tryptophan via chemical and photochemical reduction of Au ions in the presence of nanosized magnetite. The magnetic field (MF) induced aggregation was accompanied by an increase in the absorption in the near-infrared (NIR) spectral region, which was demonstrated to provide an enhanced photothermal (PT) effect under NIR laser irradiation (at 808 nm). A possibility for therapeutic application of the MPNC was illustrated using cancer cells in vitro. Cultured HeLa cells were treated by MPNC in the presence of MF and without it, following laser irradiation and imaging using confocal laser scanning microscopy. After scanning laser irradiation of the MPNC/MF treated cells, a formation and rise of photothermally-induced microbubbles on the cell surfaces was observed, leading to a damage of the cell membrane and cell destruction. We conclude that the synthesized magneto-plasmonic Fe3O4/Au nanosystems exhibit magnetic field-induced reversible aggregation accompanied by an increase in NIR absorption, allowing for an opportunity to magnetophoretically control and locally enhance a NIR light-induced thermal effect, which holds high promise for the application in photothermal therapy.
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An approach to study bimolecular interactions in model lipid bilayers and biological membranes is introduced, exploiting the influence of membrane-associated electron spin resonance labels on the triplet state kinetics of membrane-bound fluorophores. Singlet-triplet state transitions within the dye Lissamine Rhodamine B (LRB) were studied, when free in aqueous solutions, with LRB bound to a lipid in a liposome, and in the presence of different local concentrations of the electron spin resonance label TEMPO. By monitoring the triplet state kinetics via variations in the fluorescence signal, in this study using fluorescence correlation spectroscopy, a strong fluorescence signal can be combined with the ability to monitor low-frequency molecular interactions, at timescales much longer than the fluorescence lifetimes. Both in solution and in membranes, the measured relative changes in the singlet-triplet transitions rates were found to well reflect the expected collisional frequencies between the LRB and TEMPO molecules. These collisional rates could also be monitored at local TEMPO concentrations where practically no quenching of the excited state of the fluorophores can be detected. The proposed strategy is broadly applicable, in terms of possible read-out means, types of molecular interactions that can be followed, and in what environments these interactions can be measured.
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Colorantes Fluorescentes/química , Membrana Dobles de Lípidos/química , Rodaminas/química , Espectrometría de Fluorescencia/métodos , Óxidos N-Cíclicos , Difusión , Cinética , Liposomas/química , SolucionesRESUMEN
For a spatially restricted excitation volume, the effective modulation of the excitation in time is influenced by the passage times of the molecules through the excitation volume. By applying an additional time-modulated excitation, the buildup of photoinduced reversible dark states in fluorescent molecules can be made to vary significantly with their passage times through the excitation volume. The variations in the dark state populations are reflected by the time-averaged fluorescence intensity, which thus can be used to characterize the mobilities of the molecules. The concept was experimentally verified by measuring the fluorescence response of freely diffusing cyanine fluorophores (Cy5), undergoing trans-cis isomerization when subject to time-modulated excitation in a focused laser beam. From the fluorescence response, and by applying a simple photodynamic model, the transition times of the Cy5 molecules could be well reproduced when applying different laminar flow speeds through the detection volume. The presented approach puts no constraints on sample concentration, no requirements for high time resolution or sensitivity in the detection, nor requires a high fluorescence brightness of the characterized molecules. This can make the concept useful for a broad range of biomolecular mobility studies.
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Difusión , Fluorescencia , Carbocianinas , Colorantes Fluorescentes , Cinética , EstereoisomerismoRESUMEN
Electrostatic interactions between dielectric surfaces and different fluorophores used in ultrasensitive fluorescence microscopy are investigated using objective-based Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS). The interfacial dynamics of cationic rhodamine 123 and rhodamine 6G, anionic/dianionic fluorescein, zwitterionic rhodamine 110 and neutral ATTO 488 are monitored at various ionic strengths at physiological pH. As analyzed by means of the amplitude and time-evolution of the autocorrelation function, the fluorescent molecules experience electrostatic attraction or repulsion at the glass surface depending on their charges. Influences of the electrostatic interactions are also monitored through the triplet-state population and triplet relaxation time, including the amount of detected fluorescence or the count-rate-per-molecule parameter. These TIR-FCS results provide an increased understanding of how fluorophores are influenced by the microenvironment of a glass surface, and show a promising approach for characterizing electrostatic interactions at interfaces.
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Colorantes Fluorescentes/química , Espectrometría de Fluorescencia , Fluoresceína/química , Fluoresceínas/química , Vidrio/química , Concentración de Iones de Hidrógeno , Concentración Osmolar , Rodamina 123/química , Rodaminas/química , Electricidad Estática , Propiedades de SuperficieRESUMEN
We develop mid-infrared optoacoustic microscopy (MiROM) for label-free, bond-selective, live-cell metabolic imaging, enabling spatiotemporal monitoring of carbohydrates, lipids and proteins in cells and tissues. Using acoustic detection of optical absorption, MiROM converts mid-infrared sensing into a positive-contrast imaging modality with negligible photodamage and high sensitivity. We use MiROM to observe changes in intrinsic carbohydrate distribution from a diffusive spatial pattern to tight co-localization with lipid droplets during adipogenesis.
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Aumento de la Imagen/métodos , Gotas Lipídicas/metabolismo , Técnicas Fotoacústicas/métodos , Células 3T3-L1 , Adipogénesis , Animales , Metabolismo de los Hidratos de Carbono , Células HeLa , Humanos , Ratones , Microscopía , Programas Informáticos , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The triplet-state kinetics of several fluorescent dyes used in ultrasensitive fluorescence microscopy are investigated using total internal reflection fluorescence correlation spectroscopy (TIR-FCS). A theoretical outline of the correlation analysis and the physical aspects of evanescent excitation and fluorescence emission at dielectric interfaces are given. From this analysis, the rates of intersystem crossing and triplet decay are deduced for fluorescein, ATTO 488, rhodamine 110, rhodamine 123, and rhodamine 6G in aqueous buffer solutions. All investigated dyes show slightly higher triplet rates at the dielectric interface compared to bulk solution measurements. We attribute this enhancement to possible modifications of the dyes' photophysical properties near a dielectric interface. In the case of rhodamine 6G, the impact of changes in the dye concentration, ionic strength of the solvent, and potassium iodide concentration are also investigated. This leads to a better understanding of the influences of dye-dye, dye-solvent, and dye-surface interactions on the increased triplet intersystem crossing and triplet decay rates. The study shows that analysis of triplet-state kinetics by TIR-FCS not only results in a better understanding of how the photophysical properties of the dyes are affected by the presence of an interface, but also provides a means for probing the microenvironment near dielectric interfaces.
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Colorantes Fluorescentes/química , Impedancia Eléctrica , Fluoresceína/química , Cinética , Rodaminas/química , Espectrometría de FluorescenciaRESUMEN
Macrophages are one of the most functionally-diverse cell types with roles in innate immunity, homeostasis and disease making them attractive targets for diagnostics and therapy. Photo- or optoacoustics could provide non-invasive, deep tissue imaging with high resolution and allow to visualize the spatiotemporal distribution of macrophages in vivo. However, present macrophage labels focus on synthetic nanomaterials, frequently limiting their ability to combine both host cell viability and functionality with strong signal generation. Here, we present a homogentisic acid-derived pigment (HDP) for biocompatible intracellular labeling of macrophages with strong optoacoustic contrast efficient enough to resolve single cells against a strong blood background. We study pigment formation during macrophage differentiation and activation, and utilize this labeling method to track migration of pro-inflammatory macrophages in vivo with whole-body imaging. We expand the sparse palette of macrophage labels for in vivo optoacoustic imaging and facilitate research on macrophage functionality and behavior.
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Ácido Homogentísico/química , Microscopía Intravital/métodos , Activación de Macrófagos , Macrófagos/citología , Técnicas Fotoacústicas/métodos , Pigmentos Biológicos/química , Coloración y Etiquetado/métodos , Animales , Materiales Biocompatibles , Diferenciación Celular , Citocinas/metabolismo , Oro , Células HEK293 , Células HeLa , Humanos , L-Lactato Deshidrogenasa/metabolismo , Macrófagos/metabolismo , Melaninas , Ratones , Nanopartículas , NanotubosRESUMEN
Photo- or optoacoustics (OA) imaging is increasingly being used as a non-invasive imaging method that can simultaneously reveal structure and function in deep tissue. However, the most frequent transgenic OA labels are current fluorescent proteins that are not optimized for OA imaging. Thus, they lack OA signal strength, and their absorption maxima are positioned at short wavelengths, thus giving small penetration depths and strong background signals. Here, we apply insights from our recent determination of the structure of the fluorescent phycobiliprotein smURFP to mutate a range of residues to promote the nonradiative decay pathway that generates the OA signal. We identified hydrophobic and aromatic substitutions within the chromophore-binding pocket that substantially increase the intensity of the OA signal and red-shift the absorption. Our results demonstrate the feasibility of structure-based mutagenesis to repurpose fluorescent probes for OA imaging, and they may provide structure-function insights for de novo engineering of transgenic OA probes.