Searching for genes regulating fat accumulation has both fundamental and medical interests. Genetic screening for those genes in Caenorhabditis elegans, a widely-used model organism, requires in vivo quantification of lipids. We demonstrated RNA interference screening based on quantitative imaging of lipids with label-free stimulated Raman scattering microscopy, which overcomes major limitations of coherent anti-stokes Raman scattering microscopy. Our screening yielded eight novel genetic regulators of fat storage.
Stimulated Raman Scattering microscopy allows label-free chemical imaging and has enabled exciting applications in biology, material science, and medicine. It provides a major advantage in imaging speed over spontaneous Raman scattering and has improved image contrast and spectral fidelity compared to coherent anti-Stokes Raman. Wider adoption of the technique has, however, been hindered by the need for a costly and environmentally sensitive tunable ultra-fast dual-wavelength source. We present the development of an optimized all-fibre laser system based on the optical synchronization of two picosecond power amplifiers. To circumvent the high-frequency laser noise intrinsic to amplified fibre lasers, we have further developed a high-speed noise cancellation system based on voltage-subtraction autobalanced detection. We demonstrate uncompromised imaging performance of our fibre-laser based stimulated Raman scattering microscope with shot-noise limited sensitivity and an imaging speed up to 1 frame/s.
ABL1 tyrosine-kinase inhibitors (TKI) are a front-line therapy for chronic myelogenous leukemia and represent the best known examples of targeted cancer therapeutics. However, the dynamic uptake of low molecular weight TKIs into cells and their intracellular behavior is largely unknown due to the difficulty of observing non-fluorescent small molecules at subcellular resolution. Here we report the direct label-free visualization and quantification of two TKI drugs – imatinib and nilotinib inside living cells using hyperspectral stimulated Raman scattering imaging. Both drugs were enriched over 1000-fold in lysosomes as a result of their lysosomotropic properties. In addition, low solubility appeared to contribute significantly to the surprisingly large accumulation of nilotinib. We further show that the lysosomal trapping of imatinib was reduced by more than 10-fold when using chloroquine simultaneously, suggesting that chloroquine may increase the efficacy of TKIs through lysosome mediated drug-drug interaction besides the commonly proposed autophagy inhibition mechanism.
Raman spectroscopy; Hyperspectral SRS imaging; Tyrosine kinase inhibitor; Lysosomotropism
We report a study of DNA deformations using a coarse-grained mechanical model and quantitatively interpret the allosteric effects in protein-DNA binding affinity. A recent single molecule study (Kim et al. (2013) Science, 339, 816) showed that when a DNA molecule is deformed by specific binding of a protein, the binding affinity of a second protein separated from the first protein is altered. Experimental observations together with molecular dynamics simulations suggested that the origin of the DNA allostery is related to the observed deformation of DNA’s structure, in particular the major groove width. In order to unveil and quantify the underlying mechanism for the observed major groove deformation behavior related to the DNA allostery, here we provide a simple but effective analytical model where DNA deformations upon protein binding are analyzed and spatial correlations of local deformations along the DNA are examined. The deformation of the DNA base orientations, which directly affect the major groove width, is found in both an analytical derivation and coarse-grained Monte Carlo simulations. This deformation oscillates with a period of 10 base pairs with an amplitude decaying exponentially from the binding site with a decay length lD~10 base pairs, as a result of the balance between two competing terms in DNA base stacking energy. This length scale is in agreement with that reported from the single molecule experiment. Our model can be reduced to the worm-like chain form at length scales larger than lP but is able to explain DNA’s mechanical properties on shorter length scales, in particular the DNA allostery of protein-DNA interactions.
Protein-DNA interactions; mechanical deformation; network model; base orientations
Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. Here we describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from non-neoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and H&E microscopy for detection of glioma infiltration (κ=0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.
We present a synchronously pumped fiber optical parametric oscillator for coherent anti-Stokes Raman scattering microscopy. Pulses from a 1 μm Yb-doped fiber laser are amplified and frequency converted to 779–808 nm through normal dispersion four-wave mixing in a photonic crystal fiber. The idler frequency is resonant in the oscillator cavity, and we find that bandpass filtering the feedback is essential for a stable, narrow-bandwidth output. Experimental results agree quite well with numerical simulations of the device. Transform-limited 2 ps pulses with energy up to 4 nJ can be generated at the signal wavelength. The average power is 180 mW, and the relative-intensity noise is much lower than that of a similar parametric amplifier. High-quality coherent Raman images of mouse tissues recorded with this source are presented.
Imaging single fluorescent proteins in living mammalian cells is challenging due to out-of-focus fluorescence excitation by common microscopy schemes. We report the development of a novel fluorescence microscopy method, reflected light sheet microscopy (RLSM), which allows selective plane illumination throughout the nucleus of living mammalian cells, for reducing out-of-focus fluorescence signal. Generation of a thin light sheet parallel to the imaging plane and close to the sample surface is achieved by reflecting an elliptical laser beam incident from the top by 45° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to previous illumination schemes and enables imaging of single fluorescent proteins with up to 100 Hz time resolution. We demonstrate the sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determine the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor (ER), enabling us to resolve different modes of DNA binding of GR. Finally, we demonstrate two-color single molecule imaging by observing the spatio-temporal co-localization of two different protein pairs. The combination of our single molecule measurements and statistical analysis reveals dynamic properties of transcription factors in live mammalian cells.
Allostery is well documented for proteins but less recognized for DNA-protein interactions. Here we report that specific binding of a protein on DNA is substantially stabilized or destabilized by another protein bound nearby. The ternary complex's free energy oscillates as a function of the separation between the two proteins with a periodicity of ~10 base pairs, the helical pitch of B-form DNA, and a decay length of ~15 base pairs. The binding affinity of a protein near a DNA hairpin is similarly dependent on their separation, which—together with molecular dynamics simulations—suggests that deformation of the double-helical structure is the origin of DNA allostery. The physiological relevance of this phenomenon is illustrated by its effect on gene expression in live bacteria and on a transcription factor's affinity near nucleosomes.
Stimulated Raman scattering (SRS) microscopy has opened up a wide range of biochemical imaging applications by probing a particular Raman-active molecule vibrational mode in the specimen. However, the original implementation with picosecond pulse excitation can only realize rapid chemical mapping with a single Raman band. Here we present a novel SRS microscopic technique using a grating-based pulse shaper for excitation and a grating-based spectrograph for detection to achieve simultaneous multicolor SRS imaging with high sensitivity and high acquisition speeds. In particular, we used linear combination of the measured CH2 and CH3 stretching signals to map the distributions of protein and lipid contents simultaneously.
coherent Raman scattering; stimulated Raman scattering; multicolor; grating; pulse shaper; lock-in; polystyrene; poly(methyl methacrylate); mouse skin; protein; lipid
Meiotic recombination creates genetic diversity and ensures segregation of homologous chromosomes. Previous population analyses yielded results averaged among individuals and impacted by evolutionary pressures. Here we sequenced 99 sperm from an Asian male using the newly developed amplification method—Multiple Annealing and Looping-Based Amplification Cycles (MALBAC)—to phase the personal genome and map at high resolution recombination events, which are non-uniformly distributed across the genome in the absence of selection pressure. The paucity of recombination near transcription start sites observed in individual sperm indicates such a phenomenon is intrinsic to the molecular mechanism of meiosis. Interestingly, a decreased crossover frequency in companion with an increase of autosomal aneuploidy is observable on a global per-sperm basis.
Kindred cells can have different genomes because of dynamic changes in DNA. Single cell sequencing is needed to characterize these genomic differences but has been hindered by whole-genome amplification bias, resulting in low genome coverage. Here we report a new amplification method: Multiple Annealing and Looping Based Amplification Cycles (MALBAC) that offer high uniformity across the genome. Sequencing MALBAC amplified DNA achieves 93% genome coverage ≥1x for a single human cell at 25x mean sequencing depth. We detected digitized copy number variations (CNVs) of a single cancer cell. By sequencing three kindred cells, we were able to call individual single nucleotide variations (SNVs) with no false positives observed. We directly measured the genome-wide mutation rate of a cancer cell line and found that purine-pyrimidine exchanges occurred unusually frequently among the newly acquired SNVs.
We report a label-free assay for simultaneous optical manipulation and tracking of endogenous lipid droplets as actively transported cargoes in a living mammalian cell with sub-millisecond time resolution. Using an EM-CCD camera as a highly sensitive quadrant detector, we can detect steps of dynein- and kinesin-driven cargoes under known force loads. We can distinguish single and multiple motor-driven cargoes and show that the stall forces for inward and outward transported cargoes are similar. By combining the stall force observable with the ability to detect individual steps, we can characterize kinesin- and dynein-driven active transport in different force regimes.
optical tweezers; motor protein; particle tracking; organelle transport; mechanical manipulation
Stimulated Raman scattering (SRS) microscopy allows label-free chemical imaging based on vibrational spectroscopy. Narrowband excitation with picosecond lasers creates the highest signal levels and enables imaging speeds up to video-rate, but sacrifices chemical specify in samples with overlapping bands compared to broadband (multiplex) excitation. We develope a rapidly tunable picosecond optical parametric oscillator with an electro-optical tunable Lyot filter, and demonstrate multi-color SRS microscopy with synchronized line-by-line wavelength tuning to avoid spectral artifacts due to sample movement. We show sensitive imaging of three different kinds of polymer beads and live HeLa cells with moving intracellular lipid droplets.
Conventional histopathology with hematoxylin & eosin (H&E) has been the gold standard for histopathological diagnosis of a wide range of diseases. However, it is not performed in vivo and requires thin tissue sections obtained after tissue biopsy, which carries risk, particularly in the central nervous system. Here we describe the development of an alternative, multicolored way to visualize tissue in real time through the use of coherent Raman imaging (CRI), without the use of dyes. CRI relies on intrinsic chemical contrast based on vibrational properties of molecules and intrinsic optical sectioning by nonlinear excitation. We demonstrate that multi-color images originating from CH2 and CH3 vibrations of lipids and protein, as well as two-photon absorption of hemoglobin, can be obtained with subcellular resolution from fresh tissue. These stain-free histopathological images show resolutions similar to those obtained by conventional techniques, but do not require tissue fixation, sectioning or staining of the tissue analyzed.
Coherent anti-Stokes Raman scattering; CARS; Histology; In vivo microscopy; Stimulated Raman scattering; SRS
We demonstrate the application of CARS microscopy for the rapid, label-free chemical imaging of water-borne pathogens. Chemically selective images of cryptosporidium were acquired in just a few seconds using CARS microscopy, demonstrating its capability for the rapid detection of cryptosporidium at the single oocyst level. We discuss the applicability of such a technique in a near-real time automated water testing system.
Coherent anti-Stokes Raman scattering; microscopy; cryptosporidium oocyst
Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of gene expression and regulation, and the demonstration that a single-molecule event can determine the phenotype of a cell.
Imaging of nucleic acids is important for studying cellular processes such as cell division and apoptosis. A noninvasive label-free technique is attractive. Raman spectroscopy provides rich chemical information based on specific vibrational peaks. However, the signal from spontaneous Raman scattering is weak and long integration times are required, which drastically limits the imaging speed when used for microscopy. Coherent Raman scattering techniques, comprising coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, overcome this problem by enhancing the signal level by up to five orders of magnitude. CARS microscopy suffers from a nonresonant background signal, which distorts Raman spectra and limits sensitivity. This makes CARS imaging of weak transitions in spectrally congested regions challenging. This is especially the case in the fingerprint region, where nucleic acids show characteristic peaks. The recently developed SRS microscopy is free from these limitations; excitation spectra are identical to those of spontaneous Raman and sensitivity is close to shot-noise limited. Here we demonstrate the use of SRS imaging in the fingerprint region to map the distribution of nucleic acids in addition to proteins and lipids in single salivary gland cells of Drosophila larvae, and in single mammalian cells. This allows the imaging of DNA condensation associated with cell division and opens up possibilities of imaging such processes in vivo.
CARS; SRS; Microscopy; live cells; DNA
Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and readily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis.
Optical imaging in vivo with molecular specificity is important in biomedicine because of its high spatial resolution and sensitivity compared to MRI. Stimulated Raman scattering (SRS) microscopy allows highly sensitive optical imaging based on vibrational spectroscopy without adding toxic or perturbative labels. However, SRS tissue imaging in living animals and humans has not been feasible because of weak signals from thick tissues and motion blur due to limited acquisition speed. Here we make in vivo SRS imaging possible by significantly enhancing the collection of the backscattered signal and by increasing the imaging speed by three orders of magnitude, to video rate. This allows label-free in vivo imaging of water, lipid and protein in skin and mapping of penetration pathways of topically-applied drugs in mice and humans.
Label-free microscopy with chemical contrast and high acquisition speed up to video-rate has recently been made possible by stimulated Raman scattering (SRS) microscopy. While SRS imaging offers superb sensitivity, the spectral specificity of the original narrowband implementation is limited, making distinguishing chemical species with overlapping Raman bands difficult. Here we present a highly specific imaging method that allows mapping of a particular chemical species in the presence of interfering species based on tailored multiplex excitation of its vibrational spectrum. This is done by spectral modulation of a broadband pump beam at a high-frequency (>1MHz), allowing detection of the stimulated Raman gain signal of the narrowband Stokes beam with high sensitivity. Using the scheme, we demonstrate quantification of cholesterol in the presence of lipids, and real-time three-dimensional spectral imaging of protein, stearic acid and oleic acid in live C.elegans.
We use the time-lens concept to demonstrate a new scheme for synchronization of two pulsed light sources for biological imaging. An all fiber, 1064 nm time-lens source is synchronized to a picosecond solid-state Ti: Sapphire mode-locked laser by using the mode-locked laser pulses as the clock. We demonstrate the application of this synchronized source for CARS and SRS imaging by imaging mouse tissues. Synchronized two wavelength pulsed source is an important technical difficulty for CARS and SRS imaging. The time-lens source demonstrated here may provide an all fiber, user friendly alternative for future SRS imaging.
The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy, including stimulated Raman scattering and two photon absorption, and pump-probe microscopy, including stimulated emission, excited state absorption and ground state depletion, provide distinct and powerful image contrasts for non-fluorescent species. Thanks to high-frequency modulation transfer scheme, they exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles, excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques.
We have developed a multiplex sequencing-by-synthesis method combining terminal-phosphate labeled fluorogenic nucleotides (TPLFNs) and resealable microreactors. In the presence of phosphatase, the incorporation of a non-fluorescent TPLFN into a DNA primer by DNA polymerase results in a fluorophore. We immobilize DNA templates within polydimethylsiloxane (PDMS) microreactors, sequentially introduce one of the four identically labeled TPLFNs, seal the microreactors, allow template-directed TPLFN incorporation, and measure the signal from the fluorophores trapped in the microreactors. This workflow allows sequencing in a manner akin to pyrosequencing but without constant monitoring of each microreactor. With cycle times of <10 minutes, we demonstrate 30 base reads with ∼99% raw accuracy. “Fluorogenic pyrosequencing” combines benefits of pyrosequencing, such as rapid turn-around, native DNA generation, and single-color detection, with benefits of fluorescence-based approaches, such as highly sensitive detection and simple parallelization.
We present a fiber-format picosecond light source for coherent anti-Stokes Raman scattering microscopy. Pulses from a Yb-doped fiber amplifier are frequency converted by four-wave mixing (FWM) in normal-dispersion photonic crystal fiber to produce a synchronized two-color picosecond pulse train. We show that seeding the FWM process overcomes the deleterious effects of group-velocity mismatch and allows efficient conversion into narrow frequency bands. The source generates more than 160 mW of nearly transform-limited pulses tunable from 775 to 815 nm. High-quality coherent Raman images of animal tissues and cells acquired with this source are presented.
Efficient drug delivery to the skin is essential for the treatment of major dermatologic diseases, such as eczema, psoriasis and acne. However, many compounds penetrate the skin barrier poorly and require optimized formulations to ensure their bioavailability. Here, stimulated Raman scattering (SRS) microscopy, a recently-developed, label-free chemical imaging tool, is used to acquire high resolution images of multiple chemical components of a topical formulation as it penetrates into mammalian skin. This technique uniquely provides label-free, non-destructive, three-dimensional images with high spatiotemporal resolution. It reveals novel features of (trans)dermal drug delivery in the tissue environment: different rates of drug penetration via hair follicles as compared to the intercellular pathway across the stratum corneum are directly observed, and the precipitation of drug crystals on the skin surface is visualized after the percutaneous penetration of the co-solvent excipient in the formulation. The high speed three-dimensional imaging capability of SRS thus reveals features that cannot be seen with other techniques, providing both kinetic information and mechanistic insight into the (trans)dermal drug delivery process.
Skin; topical drug delivery; stimulated Raman scattering microscopy; skin penetration pathways; dermatopharmacokinetics