Precision proteoform design for 4R tau isoform selective templated aggregation.

Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.

Optical Photothermal Infrared - Fluorescence In Situ Hybridization (OPTIR-FISH).

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we present a comprehensive protocol for simultaneous cell identification and metabolic analysis with the OPTIR-FISH platform by combining rRNA-tagged FISH probes and isotope-labeled substrates. Fluorescence imaging provides cell identification by the specific binding of rRNA-tagged FISH probes, while OPTIR imaging provides metabolic activities within single cells by isotope-induced red shift on OPTIR spectra. Using bacteria cultured with 13C-glucose as a test bed, the protocol outlines microbial culture with isotopic labeling, fluorescence in situ hybridization (FISH), sample preparation, optimization of the OPTIR-FISH imaging setup, and data acquisition. We also demonstrate how to perform image analysis and interpret spectral data at the single-cell level with high throughput. This protocol's standardized and detailed nature will greatly facilitate its adoption by researchers from diverse backgrounds and disciplines within the broad single-cell metabolism research community.

Single-cell mapping of lipid metabolites using an infrared probe in human-derived model systems.

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.

Precision Proteoform Design for 4R Tau Isoform Selective Templated Aggregation.

Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau, folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naïve 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.

Fluorescently Guided Optical Photothermal Infrared Microspectroscopy for Protein-Specific Bioimaging at Subcellular Level.

Infrared spectroscopic imaging is widely used for the visualization of biomolecule structures, and techniques such as optical photothermal infrared (OPTIR) microspectroscopy can achieve <500 nm spatial resolution. However, these approaches lack specificity for particular cell types and cell components and thus cannot be used as a stand-alone technique to assess their properties. Here, we have developed a novel tool, fluorescently guided optical photothermal infrared microspectroscopy, that simultaneously exploits epifluorescence imaging and OPTIR to perform fluorescently guided IR spectroscopic analysis. This novel approach exceeds the diffraction limit of infrared microscopy and allows structural analysis of specific proteins directly in tissue and single cells. Experiments described herein used epifluorescence to rapidly locate amyloid proteins in tissues or neuronal cultures, thus guiding OPTIR measurements to assess amyloid structures at the subcellular level. We believe that this new approach will be a valuable addition to infrared spectroscopy providing cellular specificity of measurements in complex systems for studies of structurally altered protein aggregates.

High-Throughput Antimicrobial Susceptibility Testing of Escherichia coli by Wide-Field Mid-Infrared Photothermal Imaging of Protein Synthesis.

Antimicrobial resistance poses great threats to global health and economics. Current gold-standard antimicrobial susceptibility testing (AST) requires extensive culture time (36-72 h) to determine susceptibility. There is an urgent need for rapid AST methods to slow down antimicrobial resistance. Here, we present a rapid AST method based on wide-field mid-infrared photothermal imaging of protein synthesis from 13C-glucose in Escherichia coli. Our wide-field approach achieved metabolic imaging for hundreds of bacteria at the single-cell resolution within seconds. The perturbed microbial protein synthesis can be probed within 1 h after antibiotic treatment in E. coli cells. The susceptibility of antibiotics with various mechanisms of action has been probed through monitoring protein synthesis, which promises great potential of the proposed platform toward clinical translation.

Mid-Infrared Photothermal-Fluorescence In Situ Hybridization for Functional Analysis and Genetic Identification of Single Cells.

Simultaneous identification and metabolic analysis of microbes with single-cell resolution and high throughput are necessary to answer the question of "who eats what, when, and where" in complex microbial communities. Here, we present a mid-infrared photothermal-fluorescence in situ hybridization (MIP-FISH) platform that enables direct bridging of genotype and phenotype. Through multiple improvements of MIP imaging, the sensitive detection of isotopically labeled compounds incorporated into proteins of individual bacterial cells became possible, while simultaneous detection of FISH labeling with rRNA-targeted probes enabled the identification of the analyzed cells. In proof-of-concept experiments, we showed that the clear spectral red shift in the protein amide I region due to incorporation of 13C atoms originating from 13C-labeled glucose can be exploited by MIP-FISH to discriminate and identify 13C-labeled bacterial cells within a complex human gut microbiome sample. The presented methods open new opportunities for single-cell structure-function analyses for microbiology.

Nanosecond-resolution photothermal dynamic imaging via MHZ digitization and match filtering.

Photothermal microscopy has enabled highly sensitive label-free imaging of absorbers, from metallic nanoparticles to chemical bonds. Photothermal signals are conventionally detected via modulation of excitation beam and demodulation of probe beam using lock-in amplifier. While convenient, the wealth of thermal dynamics is not revealed. Here, we present a lock-in free, mid-infrared photothermal dynamic imaging (PDI) system by MHz digitization and match filtering at harmonics of modulation frequency. Thermal-dynamic information is acquired at nanosecond resolution within single pulse excitation. Our method not only increases the imaging speed by two orders of magnitude but also obtains four-fold enhancement of signal-to-noise ratio over lock-in counterpart, enabling high-throughput metabolism analysis at single-cell level. Moreover, by harnessing the thermal decay difference between water and biomolecules, water background is effectively separated in mid-infrared PDI of living cells. This ability to nondestructively probe chemically specific photothermal dynamics offers a valuable tool to characterize biological and material specimens.

Bond-selective imaging by optically sensing the mid-infrared photothermal effect.

Mid-infrared (IR) spectroscopic imaging using inherent vibrational contrast has been broadly used as a powerful analytical tool for sample identification and characterization. However, the low spatial resolution and large water absorption associated with the long IR wavelengths hinder its applications to study subcellular features in living systems. Recently developed mid-infrared photothermal (MIP) microscopy overcomes these limitations by probing the IR absorption-induced photothermal effect using a visible light. MIP microscopy yields submicrometer spatial resolution with high spectral fidelity and reduced water background. In this review, we categorize different photothermal contrast mechanisms and discuss instrumentations for scanning and widefield MIP microscope configurations. We highlight a broad range of applications from life science to materials. We further provide future perspective and potential venues in MIP microscopy field.

Background-Suppressed High-Throughput Mid-Infrared Photothermal Microscopy via Pupil Engineering.

Mid-infrared photothermal (MIP) microscopy has been a promising label-free chemical imaging technique for functional characterization of specimens owing to its enhanced spatial resolution and high specificity. Recently developed wide-field MIP imaging modalities have drastically improved speed and enabled high-throughput imaging of micron-scale subjects. However, the weakly scattered signal from subwavelength particles becomes indistinguishable from the shot-noise as a consequence of the strong background light, leading to limited sensitivity. Here, we demonstrate background-suppressed chemical fingerprinting at a single nanoparticle level by selectively attenuating the reflected light through pupil engineering in the collection path. Our technique provides over 3 orders of magnitude background suppression by quasi-darkfield illumination in the epi-configuration without sacrificing lateral resolution. We demonstrate 6-fold signal-to-background noise ratio improvement, allowing for simultaneous detection and discrimination of hundreds of nanoparticles across a field of view of 70 µm × 70 µm. A comprehensive theoretical framework for photothermal image formation is provided and experimentally validated with 300 and 500 nm PMMA beads. The versatility and utility of our technique are demonstrated via hyperspectral dark-field MIP imaging of S. aureus and E. coli bacteria and MIP imaging of subcellular lipid droplets inside C. albicans and cancer cells.

Real-time imaging of surface chemical reactions by electrochemical photothermal reflectance microscopy.

Traditional electrochemical measurements based on either current or potential responses only present the average contribution of an entire electrode's surface. Here, we present an electrochemical photothermal reflectance microscope (EPRM) in which a potential-dependent nonlinear photothermal signal is exploited to map an electrochemical process with sub-micron spatial resolution. By using EPRM, we are able to monitor the photothermal signal of a Pt electrode during the electrochemical reaction at an imaging speed of 0.3 s per frame. The potential-dependent photothermal signal, which is sensitive to the free electron density, clearly revealed the evolution of surface species on the Pt surface. Our results agreed well with the reported spectroelectrochemical techniques under similar conditions but with a much faster imaging speed. We further mapped the potential oscillation during the oxidation of formic acid on the Pt surface. The photothermal images from the Pt electrode well matched the potential change. This technique opens new prospects for real-time imaging of surface chemical reaction to reveal the heterogeneity of electrochemical reactivity, which enables broad applications to the study of catalysis, energy storage, and light harvest systems.

Bond-selective transient phase imaging via sensing of the infrared photothermal effect.

Phase-contrast microscopy converts the phase shift of light passing through a transparent specimen, e.g., a biological cell, into brightness variations in an image. This ability to observe structures without destructive fixation or staining has been widely utilized for applications in materials and life sciences. Despite these advantages, phase-contrast microscopy lacks the ability to reveal molecular information. To address this gap, we developed a bond-selective transient phase (BSTP) imaging technique that excites molecular vibrations by infrared light, resulting in a transient change in phase shift that can be detected by a diffraction phase microscope. By developing a time-gated pump-probe camera system, we demonstrate BSTP imaging of live cells at a 50 Hz frame rate with high spectral fidelity, sub-microsecond temporal resolution, and sub-micron spatial resolution. Our approach paves a new way for spectroscopic imaging investigation in biology and materials science.

Ultrafast chemical imaging by widefield photothermal sensing of infrared absorption.

Infrared (IR) imaging has become a viable tool for visualizing various chemical bonds in a specimen. The performance, however, is limited in terms of spatial resolution and imaging speed. Here, instead of measuring the loss of the IR beam, we use a pulsed visible light for high-throughput, widefield sensing of the transient photothermal effect induced by absorption of single mid-IR pulses. To extract these transient signals, we built a virtual lock-in camera synchronized to the visible probe and IR light pulses with precisely controlled delays, allowing submicrosecond temporal resolution determined by the probe pulse width. Our widefield photothermal sensing microscope enabled chemical imaging at a speed up to 1250 frames/s, with high spectral fidelity, while offering submicrometer spatial resolution. With the capability of imaging living cells and nanometer-scale polymer films, widefield photothermal microscopy opens a new way for high-throughput characterization of biological and material specimens.

Fingerprinting a Living Cell by Raman Integrated Mid-Infrared Photothermal Microscopy.

Vibrational spectroscopic imaging techniques, based on infrared absorption or Raman scattering, allow for noninvasive chemically specific visualization of biological systems. The infrared and Raman modalities with different selection rules provide complementary information. Specifically, infrared microscopy provides strong signals in the fingerprint region, but suffers from low spatial resolution. Raman microscopy provides submicrometer resolution, but requires a long acquisition time. We developed a system that combines the strengths of both techniques by integrating confocal Raman microspectroscopy to the recently developed mid-infrared photothermal microscopy. This hybrid system is capable of fast infrared photothermal imaging of living cells with submicrometer resolution to identify points of interest, followed by a full-spectrum Raman analysis of the identified objects. In addition, a fingerprint photothermal spectrum can be acquired by scanning the wavelengths of the infrared laser. Comprehensive vibrational fingerprint mapping of live cells, demonstrated in adipocytes and single bacteria, promises broad applications of this technology in biology and material science.

Bond-Selective Imaging of Cells by Mid-Infrared Photothermal Microscopy in High Wavenumber Region.

Using a visible beam to probe the thermal effect induced by infrared absorption, mid-infrared photothermal (MIP) microscopy allows bond-selective chemical imaging at submicron spatial resolution. Current MIP microscopes cannot reach the high wavenumber region due to the limited tunability of the existing quantum cascade laser source. We extend the spectral range of MIP microscopy by difference frequency generation (DFG) from two chirped femtosecond pulses. Flexible wavelength tuning in both C-D and C-H regions was achieved with mid-infrared power up to 22.1 mW and spectral width of 29.3 cm-1. Distribution of fatty acid in live human lung cancer cells was revealed by MIP imaging of the C-D bond at 2192 cm-1.