Application of 2D IR Bioimaging: Hyperspectral Images of Formalin-Fixed Pancreatic Tissues and Observation of Slow Protein Degradation

Dicke, Sidney S.; Alperstein, Ariel M.; Schueler, Kathryn L.; Stapleton, Donald S.; Simonett, Shane P.; Fields, Caitlyn R.; Chalyavi, Farzaneh; Keller, Mark P.; Attie, Alan D.; Zanni, Martin T.

doi:10.1021/acs.jpcb.1c05554

Cited by 7 publications

(4 citation statements)

References 77 publications

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“…1D, a diagonal trace through the isotope labeled spectral region of the combination band is tracked over the course of the experiment. 43,64 Diagonal slicing can be interpreted similarly to FTIR spectra, although 2D IR spectra are more sensitive to secondary structures. 50,[64][65][66] When a secondary structure forms, like an a-helix or a b-sheet, the backbone carbonyl (amide I) vibrations from multiple amino acids couple to one another, creating delocalized vibrational modes.…”

Section: Resultsmentioning

confidence: 99%

“…43,64 Diagonal slicing can be interpreted similarly to FTIR spectra, although 2D IR spectra are more sensitive to secondary structures. 50,[64][65][66] When a secondary structure forms, like an a-helix or a b-sheet, the backbone carbonyl (amide I) vibrations from multiple amino acids couple to one another, creating delocalized vibrational modes. 43,50 That delocalization also increases the transition dipole strength, m, of the delocalized mode.…”

Section: Resultsmentioning

confidence: 99%

“…Higher sensitivity arises because 2D IR spectra scale as m 4 , whereas FTIR scale as m 2 . 50,64,65 Thus, unlike an FTIR spectrum, one does not expect the integrated area of a 2D IR spectrum to remain constant. 66,67 After 5 minutes (red trace), a peak is visible in the labeled region above 1600 cm À1 , indicative of a-helical secondary structure.…”

Section: Resultsmentioning

confidence: 99%

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Metastable intermediate during hIAPP aggregation catalyzed by membranes as detected with 2D IR spectroscopy

et al. 2022

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Metastable intermediate during hIAPP aggregation catalyzed by membranes as detected with 2D IR spectroscopy

et al. 2022

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“…Nonlinear spectroscopies can be turned into microscopy experiments by tightly focusing the laser beams and raster scanning the sample, thus creating an image that is a collection of many independently measured spectra. Generating hyperspectral images at 1 kHz is possible for some samples, such as animal tissues, but extremely time-consuming. − The limiting factors are signal-to-noise and the time it takes to collect each spectrum, which are interdependent. In the opposite extreme, ultrafast TA microscopy experiments have used lasers with MHz repetition rates, but modulated at 10s of kHz together with lock-in detection that effectively reduces the repetition rate and is incompatible with array detectors.…”

Section: Khz Lasers Open New Approaches To Time Resolved Spectroscopymentioning

confidence: 99%

Breaking Barriers in Ultrafast Spectroscopy and Imaging Using 100 kHz Amplified Yb-Laser Systems

Donaldson

Greetham

Middleton³

et al. 2023

Acc. Chem. Res.

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Conspectus Ultrafast spectroscopy and imaging have become tools utilized by a broad range of scientists involved in materials, energy, biological, and chemical sciences. Commercialization of ultrafast spectrometers including transient absorption spectrometers, vibrational sum frequency generation spectrometers, and even multidimensional spectrometers have put these advanced spectroscopy measurements into the hands of practitioners originally outside the field of ultrafast spectroscopy. There is now a technology shift occurring in ultrafast spectroscopy, made possible by new Yb-based lasers, that is opening exciting new experiments in the chemical and physical sciences. Amplified Yb-based lasers are not only more compact and efficient than their predecessors but also, most importantly, operate at many times the repetition rate with improved noise characteristics in comparison to the previous generation of Ti:sapphire amplifier technologies. Taken together, these attributes are enabling new experiments, generating improvements to long-standing techniques, and affording the transformation of spectroscopies to microscopies. This Account aims to show that the shift to 100 kHz lasers is a transformative step in nonlinear spectroscopy and imaging, much like the dramatic expansion that occurred with the commercialization of Ti:sapphire laser systems in the 1990s. The impact of this technology will be felt across a great swath of scientific communities. We first describe the technology landscape of amplified Yb-based laser systems used in conjunction with 100 kHz spectrometers operating with shot-to-shot pulse shaping and detection. We also identify the range of different parametric conversion and supercontinuum techniques which now provide a path to making pulses of light optimal for ultrafast spectroscopy. Second, we describe specific instances from our laboratories of how the amplified Yb-based light sources and spectrometers are transformative. For multiple probe time-resolved infrared and transient 2D IR spectroscopy, the gain in temporal span and signal-to-noise enables dynamical spectroscopy measurements from femtoseconds to seconds. These gains widen the applicability of time-resolved infrared techniques across a range of topics in photochemistry, photocatalysis, and photobiology as well as lower the technical barriers to implementation in a laboratory. For 2D visible spectroscopy and microscopy with white light, as well as 2D IR imaging, the high repetition rates of these new Yb-based light sources allow one to spatially map 2D spectra while maintaining high signal-to-noise in the data. To illustrate the gains, we provide examples of imaging applications in the study of photovoltaic materials and spectroelectrochemistry.

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Isotope Reverse-Labeled Infrared Spectroscopy as a Probe of In-Cell Protein Structure

Wat,

Pizzala,

Reppert

2024

J. Phys. Chem. B

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While recent years have seen great progress in determining the three-dimensional structure of isolated proteins, monitoring protein structure inside live cells remains extremely difficult. Here, we examine the utility of Fourier transform infrared (FTIR) spectroscopy as a probe of protein structure in live bacterial cells. Selective isotope enrichment is used both to distinguish recombinantly expressed NuG2b protein from the cellular background and to examine the conformation of specific residues in the protein. To maximize labeling flexibility and to improve spectral resolution between label and main-band peaks, we carry out isotope-labeling experiments in “reverse-labeling” mode: cells are initially grown in 13C-enriched media, with specific 12C-labeled amino acids added when protein expression is induced. 1 Because FTIR measurements require only around 20 μL of sample and each measurement takes only a few minutes to complete, isotope-labeling costs are minimal, allowing us to label multiple different residues in parallel in simultaneously grown cultures. For the stable NuG2b protein, isotope difference spectra from live bacterial cultures are nearly identical to spectra from isolated proteins, confirming that the structure of the protein is unperturbed by the cellular environment. By combining such measurements with site-directed mutagenesis, we further demonstrate that the local conformation of individual amino acids can be monitored, allowing us to determine, for example, whether a specific site in the protein contributes to α-helix or β-sheet structures.

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Application of 2D IR Bioimaging: Hyperspectral Images of Formalin-Fixed Pancreatic Tissues and Observation of Slow Protein Degradation

Cited by 7 publications

References 77 publications

Metastable intermediate during hIAPP aggregation catalyzed by membranes as detected with 2D IR spectroscopy

Metastable intermediate during hIAPP aggregation catalyzed by membranes as detected with 2D IR spectroscopy

Breaking Barriers in Ultrafast Spectroscopy and Imaging Using 100 kHz Amplified Yb-Laser Systems

Isotope Reverse-Labeled Infrared Spectroscopy as a Probe of In-Cell Protein Structure

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