Resolution Limit in Infrared Chemical Imaging

Phal, Yamuna; Pfister, Luke; Carney, P. Scott; Bhargava, Rohit

doi:10.1021/acs.jpcc.2c00740

Cited by 18 publications

(16 citation statements)

References 54 publications

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“…Spectroscopic imaging has been used for improved the spatial resolution of single-molecule localization microscopy by using dyes having different excitation and emission spectra ( 28 , 29 ). Here, we show that spectroscopic MIP imaging could resolve subdiffraction objects based on their distinct absorption spectra and potentially be used for improving the imaging resolution, as both SNR and spectral distance are high ( 30 ). To further explore the chemical composition, we extract the spectrum of indicated pixels, as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Video-rate mid-infrared photothermal imaging by single-pulse photothermal detection per pixel

et al. 2023

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By optically sensing absorption-induced photothermal effect, mid-infrared (IR) photothermal (MIP) microscope enables super-resolution IR imaging of biological systems in water. However, the speed of current sample-scanning MIP system is limited to milliseconds per pixel, which is insufficient for capturing living dynamics. By detecting the transient photothermal signal induced by a single IR pulse through fast digitization, we report a laser-scanning MIP microscope that increases the imaging speed by three orders of magnitude. To realize single-pulse photothermal detection, we use synchronized galvo scanning of both mid-IR and probe beams to achieve an imaging line rate of more than 2 kilohertz. With video-rate speed, we observed the dynamics of various biomolecules in living organisms at multiple scales. Furthermore, by using hyperspectral imaging, we chemically dissected the layered ultrastructure of fungal cell wall. Last, with a uniform field of view more than 200 by 200 square micrometer, we mapped fat storage in free-moving Caenorhabditis elegans and live embryos.

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Section: Resultsmentioning

confidence: 99%

Video-rate mid-infrared photothermal imaging by single-pulse photothermal detection per pixel

et al. 2023

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“…This suggests that htt aggregates are organized into a β-sheet enriched core, surrounded by a ɑ-helix dominant outer layer. By tapping into the additional dimension of spectral information, we could detect the aggregation core enriched in β-sheet components, despite being of a lower size than the visible probe beam (73, 74) ( Fig. S7 ).…”

Section: Resultsmentioning

confidence: 99%

Nanoscale Structural Mapping of Protein Aggregates in Live Cells Modeling Huntington’s Disease

Guo,

Chiesa,

Yin

et al. 2023

Preprint

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Protein aggregation, in the form of amyloid fibrils, is intimately correlated with many neurodegenerative diseases. Despite recent advances in structural biology, it remains challenging to acquire structural information of proteins in live cells. Tagging with fluorescent proteins, like green fluorescent protein (GFP), is routinely used for protein visualization. Yet, this method alone cannot provide detailed structural information on the protein system of interest, and tagging proteins has the potential to perturb native structure and function. Here, by fluorescence-detected as well as label-free scattering-based mid-infrared photothermal (MIP) microscopy, we demonstrate nanoscale mapping of secondary structure of protein aggregates in a yeast model of Huntington’s disease. We first used GFP as a highly sensitive photothermal reporter to validate β-sheet enrichment in huntingtin (htt) protein aggregates. We then obtained label-free structural maps of protein aggregates. Our data showed that the fluorescent protein tag indeed perturbed the secondary structure of the aggregate, evident by a spectral shift. Live cell MIP spectroscopy further revealed the fine spatial distribution of structurally distinct components in protein aggregates, featuring a 246-nm diameter core highly enriched in β-sheet surrounded by a ɑ-helix-rich shell. Interestingly, this structural partition exists only in presence of the [RNQ+] prion, a prion that acts to facilitate the formation of other amyloid prions. Indeed, when htt is induced to aggregate in the absence of this prion ([rnq-] state), it forms non-toxic amyloid aggregates exclusively. These results showcase the potential of MIP for unveiling detailed and subtle structural information on protein systems in live cells.SignificanceProtein aggregation is a hallmark of neurodegenerative diseases, such as Huntington’s Disease. Understanding the nature of neurotoxic aggregates could lead to better therapeutic approaches. The limited progress in this direction is partly due to the lack of tools for extracting structural information in the physiological context of the aggregates. Here, we report a photothermally detected mid-infrared micro-spectroscopy technique able to dissect the secondary structure of aggregates of the huntingtin protein in live cells. We describe for the first time a nanoscale partition of secondary structures between β-rich core and ɑ-rich shell of the aggregates. This work demonstrates the potential of mid-infrared photothermal microscopy for structural and functional mapping of proteins in live cells.

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“…15–17 However, for optical techniques such as infrared microscopy and confocal Raman microscopy, the diffraction limit of light imposes a maximum resolution of about a μm, thereby preventing the nanoscale imaging necessary to fully characterise biological features. 18,19 Furthermore, infrared spectroscopy typically has penetration depths in biological samples of up to 1 μm, and therefore can only provide information averaged over the “bulk” of a sample rather than surface specific information. 20…”

Section: Introductionmentioning

confidence: 99%

“…[15][16][17] However, for optical techniques such as infrared microscopy and confocal Raman microscopy, the diffraction limit of light imposes a maximum resolution of about a μm, thereby preventing the nanoscale imaging necessary to fully characterise biological features. 18,19 Furthermore, infrared spectroscopy typically has penetration depths in biological samples of up to 1 μm, and therefore can only provide information averaged over the "bulk" of a sample rather than surface specific information. 20 One method to circumvent Abbe's diffraction limit is the use of "near-field" methods based on atomic force microscopy (AFM) which has proven useful in the imaging of the mechanical properties of cells and interactions between cells and their environment.…”

Section: Introductionmentioning

confidence: 99%

Photoinduced force microscopy as a novel method for the study of microbial nanostructures

Davies-Jones,

Davies,

Graf

et al. 2024

Nanoscale

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Resolution Limit in Infrared Chemical Imaging

Cited by 18 publications

References 54 publications

Video-rate mid-infrared photothermal imaging by single-pulse photothermal detection per pixel

Video-rate mid-infrared photothermal imaging by single-pulse photothermal detection per pixel

Nanoscale Structural Mapping of Protein Aggregates in Live Cells Modeling Huntington’s Disease

Photoinduced force microscopy as a novel method for the study of microbial nanostructures

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