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Spectral unmixing for D2O labeled tissues from various organs a, Spectral characterization on CD vibrations in lipid and protein with solution standards. The isolated D-labeled lipids spectrum agrees with the CD peak in 12-d1-PA (left) and D-labeled protein spectrum matches with the C(α)D peak from d4-alanine (right). Experiments were repeated two times independently with similar results. b, Retrieval of CDP and CDL signals for different organs of D2O labeled mouse. The linear combinational algorithms for different organs are presented below each spectrum. Raw spectra were truncated to spectral region of 2080-2220 cm⁻¹ followed by polynomial baseline correction and normalization. Black solid lines are spectra for untreated tissues, red dashed lines are spectra for lipid components (protein were digested with Proteinase K) and blue dashed lines are spectra for protein components (lipids were removed by methanol wash). Experiments were repeated three times independently with similar results.
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Understanding metabolism is indispensable in unraveling the mechanistic basis of many physiological and pathological processes. However, in situ metabolic imaging tools are still lacking. Here we introduce a framework for mid-infrared (MIR) metabolic imaging by coupling the emerging high-information-throughput MIR microscopy with specifically desig...
Citations
... Vibrational imaging methods for spectral mapping provide a complementary and more sensitive tool when compared with Raman spectroscopic counterparts 4,5 because infrared absorption offers a much larger cross-section than that of Raman scattering 6 . Nowadays, MIR spectral imaging technologies have been widely used in chemical, medical, and bio-related fields, such as non-destructive material detection, stand-off gas analysis, high-throughout environmental monitoring, and label-free biomedical diagnosis [7][8][9][10] . Despite tremendous successes in these applications, MIR hyperspectral imaging has long been plagued by the time-consuming acquisition of threedimensional spectral data cubes 11 , which becomes more pronounced for demanding requirements with high spatial definition and broad spectral bands. ...
... Recently, an alternative approach based on quantum cascade lasers (QCLs) has emerged to implement MIR spectral imaging at an improved speed due to the agile spectral tuning capability for the light source 8,10,17 . For instance, the frame rate of QCL-based imaging at a single wavenumber reaches 50 Hz in 640 × 480 pixels 8 , which permits real-time visualization of datacubes only for several spectral bands. ...
Mid-infrared hyperspectral imaging has become an indispensable tool to spatially resolve chemical information in a wide variety of samples. However, acquiring three-dimensional data cubes is typically time-consuming due to the limited speed of raster scanning or wavelength tuning, which impedes real-time visualization with high spatial definition across broad spectral bands. Here, we devise and implement a high-speed, wide-field mid-infrared hyperspectral imaging system relying on broadband parametric upconversion of high-brightness supercontinuum illumination at the Fourier plane. The upconverted replica is spectrally decomposed by a rapid acousto-optic tunable filter, which records high-definition monochromatic images at a frame rate of 10 kHz based on a megapixel silicon camera. Consequently, the hyperspectral imager allows us to acquire 100 spectral bands over 2600-4085 cm⁻¹ in 10 ms, corresponding to a refreshing rate of 100 Hz. Moreover, the angular dependence of phase matching in the image upconversion is leveraged to realize snapshot operation with spatial multiplexing for multiple spectral channels, which may further boost the spectral imaging rate. The high acquisition rate, wide-field operation, and broadband spectral coverage could open new possibilities for high-throughput characterization of transient processes in material and life sciences.
... enable high-speed widefield MIP imaging of enzymatic activities and other cellular processes. Although we focus on caspase and phosphatase in this study, activity mapping of a broad category of enzyme species can be done by developing more spectrally resolvable enzymatic reaction reporters 24,28,60 (Supplementary Scheme 3). Additionally, enzyme activity profiling in other tissues, such as a tumor, is worth exploration. ...
Simultaneous spatial mapping of the activity of multiple enzymes in a living system can elucidate their functions in health and disease. However, methods based on monitoring fluorescent substrates are limited. Here, we report the development of nitrile (C≡N)-tagged enzyme activity reporters, named nitrile chameleons, for the peak shift between substrate and product. To image these reporters in real time, we developed a laser-scanning mid-infrared photothermal imaging system capable of imaging the enzymatic substrates and products at a resolution of 300 nm. We show that when combined, these tools can map the activity distribution of different enzymes and measure their relative catalytic efficiency in living systems such as cancer cells, Caenorhabditis elegans, and brain tissues, and can be used to directly visualize caspase–phosphatase interactions during apoptosis. Our method is generally applicable to a broad category of enzymes and will enable new analyses of enzymes in their native context.
... Vibrational spectroscopy imaging integrated with vibrational probes provides a new direction for single-cell metabolic analysis. Vibrational probes are biorthogonal, enabling selective detection of metabolic products without interference from cellular endogenous molecules 8,[29][30][31][32] . Due to their small size and bio-compatibility, vibrational probe-labeled small molecules are widely used to trace metabolic activities such as fatty acids, amino acids, and nucleic acids. ...
... Infrared (IR) absorption provides over eight orders of magnitude stronger absorption cross sections when compared with Raman scattering 35 , which promises higher sensitivity and speed. While there are a handful of reports integrating different IR probes to study metabolism using Fourier transform IR or discrete frequency IR 29,36 , the coarse resolution associated with these IR imaging modalities presents challenges to achieving single-cell or even sub-cellular metabolic analysis and co-registration of other imaging modalities such as fluorescence imaging. ...
... We further quantified the spatial resolution of OPTIR metabolic imaging by pinging to a small lipid droplet and acquiring a line profile along two, perpendicular directions (Fig. 1E). The fitting results indicated a resolution of around 500 nm, which is a 6 times improvement compared to previously reported IR-based metabolic imaging setups 29 . Another key advantage of OPTIR compared to direct IR imaging is the consistent sub-micrometer spatial resolution irrespective of broadly tuning IR wavelengths (4.3 to 10.6 μm), whereas direct IR imaging will experience 2.5 times reduced resolution when IR tuning in this range. ...
Understanding metabolic heterogeneity is the key to uncovering the underlying mechanisms of metabolic-related diseases. Current metabolic imaging studies suffer from limitations including low resolution and specificity, and the model systems utilized often lack human relevance. Here, we present a single-cell metabolic imaging platform to enable direct imaging of lipid metabolism with high specificity in various human-derived 2D and 3D culture systems. Through the incorporation of an azide-tagged infrared probe, selective detection of newly synthesized lipids in cells and tissue became possible, while simultaneous fluorescence imaging enabled cell-type identification in complex tissues. In proof-of-concept experiments, newly synthesized lipids were directly visualized in human-relevant model systems among different cell types, mutation status, differentiation stages, and over time. We identified upregulated lipid metabolism in progranulin-knockdown human induced pluripotent stem cells and in their differentiated microglia cells. Furthermore, we observed that neurons in brain organoids exhibited a significantly lower lipid metabolism compared to astrocytes.
... Most apparent from a method perspective, high-fidelity performance is maintained for~12-fold and~51-fold larger FOV (0.8 and 0.4 NA objectives respectively) compared to FT-IR imaging, as shown in Fig. 1e. Although the use of widefield measurements can provide larger fields of view, this design is neither limited by the size of the detector array nor by speckle or shot noise when an incident beam illuminates a large sample area 10,23 . Using the full intensity of the beam on each pixel allows highfidelity measurements (Fig. 1f) that correlate well with FT-IR reference spectra and provide high SNR rapidly, making imaging fast. ...
... The design proposed here can also take advantage of emerging developments. QCLs are gradually becoming available beyond the mid-IR fingerprint region, for instance, the cell-silent window (2000-2500 cm −1 ) enabling metabolic imaging with vibrational probes 10 , to the C-H and O-H stretch regions (2800-3500 cm −1 ). The stability of lasers is improving, and aided by balanced detection schemes 53 , will eventually enable better sensitivity to molecular configurations 54 , particularly by means of controlling polarization to measure circular dichroism 55 . ...
Chemical imaging, especially mid-infrared spectroscopic microscopy, enables label-free biomedical analyses while achieving expansive molecular sensitivity. However, its slow speed and poor image quality impede widespread adoption. We present a microscope that provides high-throughput recording, low noise, and high spatial resolution where the bottom-up design of its optical train facilitates dual-axis galvo laser scanning of a diffraction-limited focal point over large areas using custom, compound, infinity-corrected refractive objectives. We demonstrate whole-slide, speckle-free imaging in ~3 min per discrete wavelength at 10× magnification (2 μm/pixel) and high-resolution capability with its 20× counterpart (1 μm/pixel), both offering spatial quality at theoretical limits while maintaining high signal-to-noise ratios (>100:1). The data quality enables applications of modern machine learning and capabilities not previously feasible – 3D reconstructions using serial sections, comprehensive assessments of whole model organisms, and histological assessments of disease in time comparable to clinical workflows. Distinct from conventional approaches that focus on morphological investigations or immunostaining techniques, this development makes label-free imaging of minimally processed tissue practical.
... Although Raman microscopy, including stimulated Raman scattering (SRS) microscopy, is pushing the boundaries of metabolic imaging and optical super-multiplexing 4,5 , infrared microscopy has yet to unleash its full potential for modern bioimaging due to inherent limitations such as its coarse spatial resolution, strong water background and the low detectability of dilute samples. However, infrared microscopy has several prominent spectroscopic features that differentiate it from its Raman counterparts [6][7][8][9] . Infrared absorption cross-sections (σ IR ) are 10 8 to 10 10 times those of Raman scattering, promising much-increased sensitivity. ...
Bioimaging harnessing optical contrasts and chemical specificity is of vital importance in probing complex biology. Vibrational spectroscopy based on mid-infrared excitation can reveal rich chemical information about molecular distributions. However, its full potential for bioimaging is hindered by the achievable sensitivity. Here we report bond-selective fluorescence-detected infrared-excited (BonFIRE) spectro-microscopy. BonFIRE employs two-photon excitation in the mid- and near-infrared to upconvert vibrational excitations to electronic states for fluorescence detection, thus encoding vibrational information into fluorescence. The system utilizes tunable narrowband picosecond pulses to ensure high sensitivity, biocompatibility and robustness for bond-selective biological interrogations over a wide spectrum of reporter molecules. We demonstrate BonFIRE spectral imaging in both fingerprint and cell-silent spectroscopic windows with single-molecule sensitivity for common fluorescent dyes. We then demonstrate BonFIRE imaging on various intracellular targets in fixed and live cells, neurons and tissues, with promise for further vibrational multiplexing. For dynamic bioanalysis in living systems, we implement a high-frequency modulation scheme and demonstrate time-lapse BonFIRE microscopy of live HeLa cells. We expect BonFIRE to expand the bioimaging toolbox by providing a new level of bond-specific vibrational information and facilitate functional imaging and sensing for biological investigations.
... In comparison, IR-based spectroscopic imaging solutions are more suitable for detecting amyloid protein aggregates, such as tau fibrils and oligomers. First, IR absorption offers a cross-section of~10 −18 cm 2 that is ten orders of magnitude larger than Raman scattering 43 . An important advantage of IR lies in its exceptional sensitivity to changes in the chemical bond strength as well as variations in β-sheet protein secondary structure in amide bands since a change of 0.02% can be easily detected 9,44 . ...
Amyloid proteins are associated with a broad spectrum of neurodegenerative diseases. However, it remains a grand challenge to extract molecular structure information from intracellular amyloid proteins in their native cellular environment. To address this challenge, we developed a computational chemical microscope integrating 3D mid-infrared photothermal imaging with fluorescence imaging, termed Fluorescence-guided Bond-Selective Intensity Diffraction Tomography (FBS-IDT). Based on a low-cost and simple optical design, FBS-IDT enables chemical-specific volumetric imaging and 3D site-specific mid-IR fingerprint spectroscopic analysis of tau fibrils, an important type of amyloid protein aggregates, in their intracellular environment. Label-free volumetric chemical imaging of human cells with/without seeded tau fibrils is demonstrated to show the potential correlation between lipid accumulation and tau aggregate formation. Depth-resolved mid-infrared fingerprint spectroscopy is performed to reveal the protein secondary structure of the intracellular tau fibrils. 3D visualization of the β-sheet for tau fibril structure is achieved.
... By taking advantage of the superiority of the optical components and devices in the telecommunication region, we significantly improved the measurable number of spectral elements and the spectral resolution by maintaining a high SNR and measurement speed. The UC-TSIR spectrometer could enable various applications, particularly measurement of complex irreversible phenomena at a high temporal resolution 5,7,22 , statistical analysis of a large number of high contents spectral data 9,42,43 , and broadband hyperspectral image acquisition at a high frame rate [44][45][46] . It can also be applicable to other sensing techniques, such as MIR optical coherence tomography 47 for 3D deep-inside profiling of highly scattering media. ...
High-speed measurement confronts the extreme speed limit when the signal becomes comparable to the noise level. In the context of broadband mid-infrared spectroscopy, state-of-the-art ultrafast Fourier-transform infrared spectrometers, in particular dual-comb spectrometers, have improved the measurement rate up to a few MSpectra s−1, which is limited by the signal-to-noise ratio. Time-stretch infrared spectroscopy, an emerging ultrafast frequency-swept mid-infrared spectroscopy technique, has shown a record-high rate of 80 MSpectra s−1 with an intrinsically higher signal-to-noise ratio than Fourier-transform spectroscopy by more than the square-root of the number of spectral elements. However, it can measure no more than ~30 spectral elements with a low resolution of several cm−1. Here, we significantly increase the measurable number of spectral elements to more than 1000 by incorporating a nonlinear upconversion process. The one-to-one mapping of a broadband spectrum from the mid-infrared to the near-infrared telecommunication region enables low-loss time-stretching with a single-mode optical fiber and low-noise signal detection with a high-bandwidth photoreceiver. We demonstrate high-resolution mid-infrared spectroscopy of gas-phase methane molecules with a high resolution of 0.017 cm−1. This unprecedentedly high-speed vibrational spectroscopy technique would satisfy various unmet needs in experimental molecular science, e.g., measuring ultrafast dynamics of irreversible phenomena, statistically analyzing a large amount of heterogeneous spectral data, or taking broadband hyperspectral images at a high frame rate. We develop upconversion time-stretch infrared spectroscopy, which enables high-speed and high-resolution broadband mid-infrared spectroscopy with over 1000 spectral elements at a rate of more than 10 MSpectra s−1.
... To acquire photon-sparse images of selective Raman bands in various samples (SI Appendix, Table S1), we tuned the pump laser frequency with respect to the narrow transmission band of filters that otherwise rejected the Rayleigh scattered light (Methods). As previously shown by others (34) and the authors (32), we imaged select Raman bands by tuning the laser and accordingly shifting the Raman scattered spectrum with respect to a spectrally bound transmission window. In this way, we selectively imaged the breathing mode of the aromatic carbon ring (∼1,000 cm −1 ) for polystyrene (PS, SI Appendix, Fig. S3) and the bending asymmetric CH 3 (∼1,400 cm −1 ) for polydimethylsiloxane (PDMS, SI Appendix, Fig. S4). ...
... Raman scattering converts photons incident on to a cell to distinct vibrational fingerprints depending on the cell's molecular content (9,10), with emerging probes greatly facilitating related investigations (34,44). However, Raman scattering represents only a modest fraction of all research and clinical imaging to date. ...
Despite its massive potential, Raman imaging represents just a modest fraction of all research and clinical microscopy to date. This is due to the ultralow Raman scattering cross-sections of most biomolecules that impose low-light or photon-sparse conditions. Bioimaging under such conditions is suboptimal, as it either results in ultralow frame rates or requires increased levels of irradiance. Here, we overcome this tradeoff by introducing Raman imaging that operates at both video rates and 1,000-fold lower irradiance than state-of-the-art methods. To accomplish this, we deployed a judicially designed Airy light-sheet microscope to efficiently image large specimen regions. Further, we implemented subphoton per pixel image acquisition and reconstruction to confront issues arising from photon sparsity at just millisecond integrations. We demonstrate the versatility of our approach by imaging a variety of samples, including the three-dimensional (3D) metabolic activity of single microbial cells and the underlying cell-to-cell variability. To image such small-scale targets, we again harnessed photon sparsity to increase magnification without a field-of-view penalty, thus, overcoming another key limitation in modern light-sheet microscopy.
... While we focus on caspase and phosphatase in this study, activity mapping of a broad category of enzyme species, such as esterase, kinase, and other proteases, can be done by developing more spectrally resolvable enzymatic reaction reporters. For example, introducing other functional groups with bio-orthogonal IR absorbance, such as azide (-N 3 ) 51 and alkyne (C≡C) 30 , or utilizing isotope-labeled triple bonds 28 (e. g. ...
Enzymes are vital components in a variety of physiological and biochemical processes. Participation of various enzyme species are required for many biological events and signaling networks. Thus, spatially mapping the activity of multiple enzymes in a living system is significant for elucidating enzymatic functions in health and connections to diseases. Here, we report the development of nitrile (C≡N)-tagged enzyme activity reporters, named nitrile chameleons for the shifted peak between substrate and product. By real-time mid-infrared photothermal imaging of the enzymatic substrates and products at 300 nm resolution, our approach can map the activity distribution of different enzymes and quantitate the relative catalytic efficiency in living cancer cells, C. elegans, and brain tissues. An important finding is the direct visualization of caspase-phosphatase cooperation during apoptosis. Our method is generally applicable to a broad category of enzymes and will advance the discovery of potential targets for diagnosis and drug development.
... In most cases, coherent Raman scattering imaging requires tightly-focused laser beams with large excitation power, resulting in a high potential for photodamage 24 . In comparison, IR absorption offers a cross-section (~10 −18 cm 2 ) that is ten orders of magnitude larger than Raman scattering 25 . Furthermore, IR imaging can be implemented without a tight beam focus, featuring higher chemical sensitivity and reduced photodamage risk. ...
... Such metabolic imaging can be implemented with the assistance of the IR-active vibrational probes and by extending the spectral coverage to the cell-silent window (1800 cm −1 -2700 cm −1 ). For example, IR tags, such as the azide group, 13 C-edited carbonyl bond, and deuterium-labeled probes 25 , can be exploited to investigate various metabolic activities simply by adding another QCL laser module to cover both the fingerprint region and the cell-silent region. BS-IDT imaging of IR tags with a sub-micrometer resolution, high speed, and rich spectral information would contribute to unraveling the mechanisms of many biological processes 25,62 . ...
... For example, IR tags, such as the azide group, 13 C-edited carbonyl bond, and deuterium-labeled probes 25 , can be exploited to investigate various metabolic activities simply by adding another QCL laser module to cover both the fingerprint region and the cell-silent region. BS-IDT imaging of IR tags with a sub-micrometer resolution, high speed, and rich spectral information would contribute to unraveling the mechanisms of many biological processes 25,62 . Fifth, BS-IDT can also be deployed in material characterizations. ...
Recovering molecular information remains a grand challenge in the widely
used holographic and computational imaging technologies. To address this
challenge, we developed a computational mid-infrared photothermal microscope,
termed Bond-selective Intensity Diffraction Tomography (BS-IDT).
Based on a low-cost brightfield microscope with an add-on pulsed light source,
BS-IDT recovers both infrared spectra and bond-selective 3D refractive index
maps from intensity-only measurements. High-fidelity infrared fingerprint
spectra extraction is validated. Volumetric chemical imaging of biological cells
is demonstrated at a speed of ~20 s per volume, with a lateral and axial resolution
of ~350nm and ~1.1 μm, respectively. BS-IDT’s application potential is
investigated by chemically quantifying lipids stored in cancer cells and volumetric
chemical imaging on Caenorhabditis elegans with a large field of view
(~100 μm x 100μm).
