Optical Photothermal Infrared (O-PTIR) is a rapidly emerging technique for super-resolution infrared spectroscopy that uses a short wavelength visible probe beam to detect infrared absorption by a sample with 10-30X better spatial resolution than conventional infrared spectroscopy. O-PTIR has been used in biomedical applications including cancer, neurodegenerative disease, cellular metabolism research, and other areas. O-PTIR has recently been combined fluorescence imaging to provide targeted IR spectroscopic analysis of fluorescently labeled regions of cells and tissue and also to rapidly detect IR absorption over a wide sample area. This presentation will overview O-PTIR technology and survey a variety of applications.
We present a novel widefield super-resolution imaging technique based on Fluorescence Enhanced Photothermal IR (FE-PTIR) spectroscopy. The key enabling innovation is the realization that fluorescence emission, is on average, up to 100x more sensitive to temperature changes to than current O-PTIR contrast mechanisms of expansion and refractive index changes up on sample heating my wavelength specific absorption of the pump IR beam. Improved sensitivity allows the defocusing of the IR beam to cover an area of up to 70x70um, with measurement via 2D high sensitivity fluorescence camera, providing for true, rapid widefield chemical imaging with <500nm spatial resolution . FE-PTIR thus allows the IR spectroscopic analysis of specifically labeled regions of biological cells and tissue, for example to study conformational stages of a specifically labeled class of target proteins. FE-PTIR can enable the study protein misfolding associated with neurodegenerative diseases. Various examples from these applications will be provided.
Direct imaging of metabolism at sub-cellular resolution provides insights to understand single-cell physiology at spatially resolved manner. Here, we presented a metabolic profiling platform based on Optical Photothermal Infrared (OPTIR) spectroscopic imaging that enables in situ lipid metabolism characterization at sub-cellular resolution. We applied the developed metabolic imaging platform to study lipid synthesis from azide-labeled fatty acids across various systems including neuroglioma cells, human-induced pluripotent stem cells (iPSCs), iPSC-derived microglia cells, and iPSC-derived brain organoids. The specificity of the developed platform was further improved by coupling a fluorescence module that enables cell type specific-metabolic profiling. Collectivity, the proposed metabolic imaging platform opens a vast potential for in situ metabolism characterization at the single-cell level.
We developed an optical infrared photothermal (OPTIR) metabolic imaging platform that enables in situ lipid metabolism characterization at sub-cellular resolution. The cell-type specific lipid metabolic imaging was achieved through the fluorescence module integrated into the OPTIR system. We successfully imaged newly-synthesized lipids after azide-palmitic acid treatment in cells across different model systems including neuroglioma, human-induced pluripotent stem cells (iPSCs), iPSC-derived microglia cells, and iPSC-derived brain organoids. The metabolic heterogeneity in different cell types in brain organoids was observed. Collectivity, the proposed metabolic imaging platform opens a vast potential for in situ metabolism characterization at the single-cell level.
We demonstrated the potential of applying infrared spectroscopy for neuroscience by increasing both the spatial resolution and the specificity of chemical structure analysis in tissues and cells, thus overcoming the current limitations of conventional fluorescence microscopy and infrared microspectroscopy. Specifically, we used a novel instrumental setup that combines optical photothermal infrared spectroscopy (OPTIR) with an epifluorescent imaging module (EF) to allow IR spectroscopic analysis of fluorescently microglia cells surrounding amyloid plaques in comparison to microglial cells in amyloid-free tissue. We have demonstrated the capability of EF-OPTIR microscopy to locate specific cells and amyloid proteins and assess amyloid structures with sub-micron spatial resolution.
Optical Photothermal Infrared (O-PTIR) spectroscopy has established itself as a cutting edge vibrational microspectroscopy tool, offering significant advantages over traditional FTIR/QCL and Raman spectroscopic tools, providing submicron simultaneous IR+Raman microscopy, in non-contact mode with high sensitivity. A new modality, “counter-propagating” has been engineered to provide for enhanced IR (and Raman) spatial resolution and sensitivity, with the use of high NA refractive objectives. This improves spatial resolution to ~300nm for both IR and Raman, whilst also improving sensitivity, image quality and facilitating immersion objective studies in fluid. Further, we coupled a widefield epifluorescence to facilitate a novel concept – fluorescence guided (or fluorescence co-located) O-PTIR microspectroscopy. Several life sciences examples from bacteria, cells and tissues will be provided to demonstrate these capabilities and how they can enable new experiments and research findings.
We have developed a new multimodal microscope enabling “Fluorescence-Guided” Optical Photothermal Infrared (O-PTIR) spectroscopy. O-PTIR has proven itself a breakthrough in IR spectroscopy, providing IR spectroscopic analysis with spatial resolution ~20x better than traditional IR in a non-contact optical configuration. We have now coupled O-PTIR with fluorescence microscopy, a long established, cornerstone technique in life science research. The new fluorescence guided O-PTIR instrument allows: (1) a sample to be analyzed via fluorescence microscopy to map sample regions that are labeled one or more fluorophores; and (2) use fluorescence images to direct infrared spectroscopic analysis in the vicinity of the fluorescently labeled regions. We have used fluorescence guided O-PTIR to perform spectroscopic analysis of the protein secondary structure of fluorescently labeled amyloid plaques in mouse brain tissue, with the goal of providing insights in neurodegenerative disease research.
The recent advent of Optical Photothermal IR (O-PTIR), has enabled for the first time, submicron infrared microscopy in far-field reflection mode with the combination of Raman for simultaneous and true correlative IR+Raman microscopy. These unique and exciting synergistic capabilities are now spawning interest in life science application [1]. In this presentation, examples of life science applications, ranging from live cell imaging of epithelial cheek cells in water, with submicron resolution of organelles, single bacteria IR imaging to ultra-high resolution images of breast tissue calcifications showing previously unknown levels of heterogeneity of calcifications that with traditional FTIR/QCL microscopes are not possible.
Atomic Force Microscopy (AFM) and infrared (IR) spectroscopy have been combined in a single instrument capable of producing sub-micron spatial resolution IR spectra and images. This new capability enables the sprectroscopic characterization of microdomain-forming polymers at levels not previously possible. Films of poly(3-hydroxybutyrate-co-3-hydroxyheanoate) were solution cast on ZnSe prisms. Dramitic differences in the IR spectra are observed in the 1200-1300 cm-1 range as a funstion of position on a spatial scale of less than one micron. This spectral region is particularly sensitive to the polymer crystallinity, enabling the identification of crystalline and amorphous domains within a single spherulite of this polymer.
We demonstrate the flexibility of radical-beam ion-beam etching for anisotropic low-damage dry etching and in situ processing of GaAs/AlGaAs structures. High anisotropy and low damage are verified by the light-current characteristics of etched laser facets and from Schottky diode characteristics of etched surfaces. These data show that damage is reduced by increasing the ratio of chemical to physical etching components. We also demonstrate direct- write patterning by selective chlorine etching of focused ion beam-damaged GaAs. The depths and linewidths are measured by atomic force microscopy. The enhanced etching is correlated to the implant-damage distribution. A selectivity of 10 to 1 is attained for doses equal or above 5 X 1014 cm-2, which corresponds to the amorphization threshold in GaAs.
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