Current state of stimulated Brillouin scattering microscopy for the life sciences.

Stimulated Brillouin scattering (SBS) microscopy is a nonlinear all-optical imaging method that provides mechanical contrast based on the interaction of laser radiation and acoustical vibrational modes. Featuring high mechanical specificity and sensitivity, three-dimensional sectioning, and practical imaging times, SBS microscopy with (quasi) continuous wave excitation is rapidly advancing as a promising imaging tool for label-free visualization of viscoelastic information of materials and living biological systems. In this article, we introduce the theory of SBS microscopy and review the current state-of-the-art as well as recent innovations, including different approaches to system designs and data analysis. In particular, various performance parameters of SBS microscopy and its applications in the life sciences are described and discussed. Future perspectives for SBS microscopy are also presented.

Three-dimensional single particle tracking using 4π self-interference of temporally phase-shifted fluorescence.

Single particle tracking in three dimensions is an indispensable tool for studying dynamic processes in various disciplines, including material sciences, physics, and biology, but often shows anisotropic three-dimensional spatial localization precision, which restricts the tracking precision, and/or a limited number of particles that can be tracked simultaneously over extended volumes. Here we developed an interferometric, three-dimensional fluorescence single particle tracking method based on conventional widefield excitation and temporal phase-shift interference of the emitted, high-aperture-angle, fluorescence wavefronts in a greatly simplified, free-running, triangle interferometer that enables tracking of multiple particles at the same time with <10-nm spatial localization precision in all three dimensions over extended volumes (~35 × 35 × 2 µm3) at video rate (25 Hz). We applied our method to characterize the microenvironment of living cells and up to ~40 µm deep in soft materials.

High-sensitivity and high-specificity biomechanical imaging by stimulated Brillouin scattering microscopy.

Label-free, non-contact imaging with mechanical contrast and optical sectioning is a substantial challenge in microscopy. Spontaneous Brillouin scattering microscopy meets this challenge, but encounters a trade-off between acquisition speed and the specificity for biomechanical constituents with overlapping Brillouin bands. Stimulated Brillouin scattering microscopy overcomes this trade-off and enables the cross-sectional imaging of live Caenorhabditis elegans at the organ and subcellular levels, with both elasticity and viscosity contrasts at high specificity and with practical recording times.

Publisher Correction: High-sensitivity and high-specificity biomechanical imaging by stimulated Brillouin scattering microscopy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Recent progress and current opinions in Brillouin microscopy for life science applications.

Many important biological functions and processes are reflected in cell and tissue mechanical properties such as elasticity and viscosity. However, current techniques used for measuring these properties have major limitations, such as that they can often not measure inside intact cells and/or require physical contact-which cells can react to and change. Brillouin light scattering offers the ability to measure mechanical properties in a non-contact and label-free manner inside of objects with high spatial resolution using light, and hence has emerged as an attractive method during the past decade. This new approach, coined "Brillouin microscopy," which integrates highly interdisciplinary concepts from physics, engineering, and mechanobiology, has led to a vibrant new community that has organized itself via a European funded (COST Action) network. Here we share our current assessment and opinion of the field, as emerged from a recent dedicated workshop. In particular, we discuss the prospects towards improved and more bio-compatible instrumentation, novel strategies to infer more accurate and quantitative mechanical measurements, as well as our current view on the biomechanical interpretation of the Brillouin spectra.

In vivo noninvasive visualization of retinal perfusion dysfunction in murine cerebral malaria by camera-phone laser speckle imaging.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection associated with impaired cerebral blood flow. Visualization of the eye vasculature, which is embryologically derived from that of the brain, is used clinically to diagnose the syndrome. Here, we introduce camera-phone laser speckle imaging as a new tool for in vivo, noncontact two-dimensional mapping of blood flow dynamics in the experimental cerebral malaria (ECM) murine model of Plasmodium berghei ANKA. In a longitudinal study, we show that the camera-phone imager can detect an overall decrease in the retinal blood-flow-speed (BFS) as ECM develops in P. berghei ANKA infected mice, with no similar change observed in uninfected control mice or mice infected with a non-ECM inducing strain (P. berghei NK65). Furthermore, by analyzing relative alterations in the BFS of individual retinal vessels during the progression of ECM, we illustrate the strength of our imager in identifying different BFS-change heterogeneities in the retinas of ECM and uninfected mice. The technique creates new possibilities for objective investigations into the diagnosis and pathogenesis of CM noninvasively through the eye. The camera-phone laser speckle imager along with measured spatial blood perfusion maps of the retina of a mouse infected with P. berghei ANKA-a fatal ECM model-on different days during the progression of the infection (top, day 3 after infection; middle, day 5 after infection; and bottom, day 7 after infection).

Maximum-likelihood analysis of axial displacement in fluorescence phase-shifting interferometry.

Fluorescence phase-shifting interferometry (FPSI) is an optical technique that coherently combines the phase-shifted 4π steradian emission wavefronts of a single fluorescent emitter to obtain multiple interferograms from which the emitter axial displacement can be retrieved with high precision. Here, we study the axial displacement sensitivity in 4-step FPSI within the framework of maximum-likelihood (ML) phase estimation. Using Monte-Carlo simulations, we show that regardless of the method used to preprocess the measured interferograms, the variance of the ML estimate of the axial displacement approaches the Cramér-Rao lower bound and is closely limited from above by the variance of the classical 4-step phase shifting estimator. The difference between these lower and upper bounds depends on the interferogram visibility and signal-to-noise-ratio (SNR), with a percentage change of up to 29% that yields an absolute change of a few sub-nanometers to some nanometers for SNRs larger than ~5. Our results suggest that for these levels of SNR, the use of the computationally simpler classical 4-step phase shifting estimation can be adequate to accurately determine axial displacements in FPSI, as we also experimentally verified. FPSI interferograms with lower SNR but good visibility can benefit from the use of the ML estimator, provided that the spatiotemporal phase stability of the FPSI system is high.

High-speed Continuous-wave Stimulated Brillouin Scattering Spectrometer for Material Analysis.

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as aqueous solutions and biomaterials, with fast acquisition times. Here, we discuss the assembly and operation of a Brillouin spectrometer that uses stimulated Brillouin scattering (SBS) to measure stimulated Brillouin gain (SBG) spectra of water and lipid emulsion-based tissue-like samples in transmission mode with <10 MHz spectral-resolution and <35 MHz Brillouin-shift measurement precision at <100 ms. The spectrometer consists of two nearly counter-propagating continuous-wave (CW) narrow-linewidth lasers at 780 nm whose frequency detuning is scanned through the material Brillouin shift. By using an ultra-narrowband hot rubidium-85 vapor notch filter and a phase-sensitive detector, the signal-to-noise-ratio of the SBG signal is significantly enhanced compared to that obtained with existing CW-SBS spectrometers. This improvement enables measurement of SBG spectra with up to 100-fold faster acquisition times, thereby facilitating high spectral-resolution and high-precision Brillouin analysis of soft materials at high speed.

Background-free Brillouin spectroscopy in scattering media at 780 nm via stimulated Brillouin scattering.

We demonstrate the effectiveness of stimulated Brillouin scattering for background-free Brillouin spectroscopy in scattering media within the biological spectral window. Using two nearly counter-propagating continuous-wave diode laser beams at 780 nm, we acquired transmission stimulated Brillouin point spectra in 10 mm and 500 µm thick Intralipid tissue phantoms with ∼100 µm and ∼16 µm diameter focal points, respectively. Stimulated gain spectra with high signal-to-noise ratio (8.7-30.7 dB) and frequency accuracy (6-72 MHz) were obtained at 20 MHz/10 ms and 20 MHz/100 ms through 0.24-3.36 mean-free paths of tissue phantoms. Our results suggest that stimulated Brillouin gain can be useful for imaging of Brillouin resonances in submillimeter-thick scattering samples.

Static laser speckle contrast analysis for noninvasive burn diagnosis using a camera-phone imager.

Laser speckle contrast analysis (LASCA) is an established optical technique for accurate widefield visualization of relative blood perfusion when no or minimal scattering from static tissue elements is present, as demonstrated, for example, in LASCA imaging of the exposed cortex. However, when LASCA is applied to diagnosis of burn wounds, light is backscattered from both moving blood and static burn scatterers, and thus the spatial speckle contrast includes both perfusion and nonperfusion components and cannot be straightforwardly associated to blood flow. We extract from speckle contrast images of burn wounds the nonperfusion (static) component and discover that it conveys useful information on the ratio of static-to-dynamic scattering composition of the wound, enabling identification of burns of different depth in a porcine model in vivo within the first 48 h postburn. Our findings suggest that relative changes in the static-to-dynamic scattering composition of burns can dominate relative changes in blood flow for burns of different severity. Unlike conventional LASCA systems that employ scientific or industrial-grade cameras, our LASCA system is realized here using a camera phone, showing the potential to enable LASCA-based burn diagnosis with a simple imager.

Quantitative reflection phase mesoscopy by remote coherence tuning of phase-shift interference patterns.

Conventional low-magnification phase-contrast microscopy is an invaluable, yet a qualitative, imaging tool for the interrogation of transparent objects over a mesoscopic millimeter-scale field-of-view in physical and biological settings. Here, we demonstrate that introducing a compact, unbalanced phase-shifting Michelson interferometer into a standard reflected brightfield microscope equipped with low-power infinity-corrected objectives and white light illumination forms a phase mesoscope that retrieves remotely and quantitatively the reflection phase distribution of thin, transparent, and weakly scattering samples with high temporal (1.38 nm) and spatial (0.87 nm) axial-displacement sensitivity and micrometer lateral resolution (2.3 µm) across a mesoscopic field-of-view (2.25 × 1.19 mm(2)). Using the system, we evaluate the etch-depth uniformity of a large-area nanometer-thick glass grating and show quantitative mesoscopic maps of the optical thickness of human cancer cells without any area scanning. Furthermore, we provide proof-of-principle of the utility of the system for the quantitative monitoring of fluid dynamics within a wide region.

Laser speckle spatiotemporal variance analysis for noninvasive widefield measurements of blood pulsation and pulse rate on a camera-phone.

Photoplethysmography is a well-established technique for the noninvasive measurement of blood pulsation. However, photoplethysmographic devices typically need to be in contact with the surface of the tissue and provide data from a single contact point. Extensions of conventional photoplethysmography to measurements over a wide field-of-view exist, but require advanced signal processing due to the low signal-to-noise-ratio of the photoplethysmograms. Here, we present a noncontact method based on temporal sampling of time-integrated speckle using a camera-phone for noninvasive, widefield measurements of physiological parameters across the human fingertip including blood pulsation and resting heart-rate frequency. The results show that precise estimation of these parameters with high spatial resolution is enabled by measuring the local temporal variation of speckle patterns of backscattered light from subcutaneous skin, thereby opening up the possibility for accurate high resolution blood pulsation imaging on a camera-phone. Camera-phone laser speckle imager along with measured relative blood perfusion maps of a fingertip showing skin perfusion response to a pulse pressure applied to the upper arm. The figure is for illustration only; the imager was stabilized on a stand throughout the experiments.

Three-dimensional nano-localization of single fluorescent emitters.

We present a combination of self-interference microscopy with lateral super-resolution microscopy and introduce a novel approach for localizing a single nano-emitter to within a few nanometers in all three dimensions over a large axial range. We demonstrate nanometer displacements of quantum dots placed on top of polymer bilayers that undergo swelling when changing from an air to a water environment, achieving standard deviations below 10 nm for axial and lateral localization.

Fluorescence interferometry: principles and applications in biology.

The use of fluorescence radiation is of fundamental importance for tackling measurement problems in the life sciences, with recent demonstrations of probing biological systems at the nanoscale. Usually, fluorescent light-based tools and techniques use the intensity of light waves, which is easily measured by detectors. However, the phase of a fluorescence wave contains subtle, but no less important, information about the wave; yet, it has been largely unexplored. Here, we introduce the concept of fluorescence interferometry to allow the measurement of phase information of fluorescent light waves. In principle, fluorescence interferometry can be considered a unique form of optical low-coherence interferometry that uses fluorophores as a light source of low temporal coherence. Fluorescence interferometry opens up new avenues for developing new fluorescent light-based imaging, sensing, ranging, and profiling methods that to some extent resemble interferometric techniques based on white light sources. We propose two experimental realizations of fluorescence interferometry that detect the interference pattern cast by the fluorescence fields. This article discusses their measurement capabilities and limitations and compares them with those offered by optical low-coherence interferometric schemes. We also describe applications of fluorescence interferometry to imaging, ranging, and profiling tasks and present experimental evidences of wide-field cross-sectional imaging with high resolution and large range of depth, as well as quantitative profiling with nanometer-level precision. Finally, we point out future research directions in fluorescence interferometry, such as fluorescence tomography of whole organisms and the extension to molecular interferometry by means of quantum dots and bioluminescence.

Speckle reduction in optical coherence tomography images using digital filtering.

Speckle noise is a ubiquitous artifact that limits the interpretation of optical coherence tomography images. Here we apply various speckle-reduction digital filters to optical coherence tomography images and compare their performance. Our results indicate that shift-invariant, nonorthogonal wavelet-transform-based filters together with enhanced Lee and adaptive Wiener filters can significantly reduce speckle and increase the signal-to-noise ratio, while preserving strong edges. The speckle reduction capabilities of these filters are also compared with speckle reduction from incoherent angular compounding. Our results suggest that by using these digital filters, the number of individual angles required to attain a certain level of speckle reduction can be decreased.

Differential near-field scanning optical microscopy.

We theoretically and experimentally illustrate a new apertured near-field scanning optical microscopy (NSOM) technique, termed differential NSOM (DNSOM). It involves scanning a relatively large (e.g., 0.3-2 mum wide) rectangular aperture (or a detector) in the near-field of an object and recording detected power as a function of the scanning position. The image reconstruction is achieved by taking a two-dimensional derivative of the recorded power map. Unlike conventional apertured NSOM, the size of the rectangular aperture/detector does not determine the resolution in DNSOM; instead, the resolution is practically determined by the sharpness of the corners of the rectangular aperture/detector. Principles of DNSOM can also be extended to other aperture/detector geometries such as triangles and parallelograms.

Measurement of fibrous cap thickness in atherosclerotic plaques by spatiotemporal analysis of laser speckle images.

Necrotic-core fibroatheromas (NCFA) with thin, mechanically weak fibrous caps overlying lipid cores comprise the majority of plaques that rupture and cause acute myocardial infarction. Laser speckle imaging (LSI) has been recently demonstrated to enable atherosclerotic plaque characterization with high accuracy. We investigate spatio-temporal analysis of LSI data, in conjunction with diffusion theory and Monte Carlo modeling of light transport, to estimate fibrous cap thickness in NCFAs. Time-varying laser speckle images of 20 NCFAs are selected for analysis. Spatio-temporal intensity fluctuations are analyzed by exponential fitting of the windowed normalized cross-correlation of sequential laser speckle patterns to obtain the speckle decorrelation time constant, tau(rho), as a function of distance rho from the source entry location. The distance, rho', at which tau(rho) dropped to 65% of its maximum value is recorded. Diffusion theory and Monte Carlo models are utilized to estimate the maximum photon penetration depth, zmax(rho'), for a distance equal to rho', measured from LSI. Measurements of zmax(rho') correlate well with histological measurements of fibrous cap thickness (R=0.78, p<0.0001), and paired t-tests show no significant difference between the groups (p=0.4). These results demonstrate that spatio-temporal LSI may allow the estimation of fibrous cap thickness in NCFAs, which is an important predictor of plaque stability.

Noise-reduction capabilities of a raman-mediated wavelength converter.

We describe ultrawideband Raman-mediated wavelength conversion. The nonlinear conversion transfer function is calculated analytically and simulated numerically in the cw regime, and the predicted performance is confirmed experimentally. Data conversion from long- to short-wavelength bands with signal reshaping and significant noise reduction are demonstrated experimentally at 10 Gbits/s and modeled by numerical simulations. Q factors and extinction ratios that are both larger than 10 dB are possible over an effective conversion bandwidth of 35 nm.