Correlative imaging to resolve molecular structures in individual cells: Substrate validation study for super-resolution infrared microspectroscopy.

Light microscopy has been a favorite tool of biological studies for almost a century, recently producing detailed images with exquisite molecular specificity achieving spatial resolution at nanoscale. However, light microscopy is insufficient to provide chemical information as a standalone technique. An increasing amount of evidence demonstrates that optical photothermal infrared microspectroscopy (O-PTIR) is a valuable imaging tool that can extract chemical information to locate molecular structures at submicron resolution. To further investigate the applicability of sub-micron infrared microspectroscopy for biomedical applications, we analyzed the contribution of substrate chemistry to the infrared spectra acquired from individual neurons grown on various imaging substrates. To provide an example of correlative immunofluorescence/O-PTIR imaging, we used immunofluorescence to locate specific organelles for O-PTIR measurement, thus capturing molecular structures at the sub-cellular level directly in cells, which is not possible using traditional infrared microspectroscopy or immunofluorescence microscopy alone.

Optical Photothermal Infrared Microspectroscopy Discriminates for the First Time Different Types of Lung Cells on Histopathology Glass Slides.

The debate of whether a glass substrate can be used in Fourier transform infrared spectroscopy is strongly linked to its potential clinical application. Histopathology glass slides of 1 mm thickness absorb the mid-IR spectrum in the rich fingerprint spectral region. Thus, it is important to assess whether emerging IR techniques can be employed to study biological samples placed on glass substrates. For this purpose, we used optical photothermal infrared (O-PTIR) spectroscopy to study for the first time malignant and non-malignant lung cells with the purpose of identifying IR spectral differences between these cells placed on standard pathology glass slides. The data in this feasibility study showed that O-PTIR can be used to obtain good-quality IR spectra from cells from both the lipid region (3000-2700 cm-1) and the fingerprint region between 1770 and 950 cm-1 but with glass contributions from 1350 to 950 cm-1. A new single-unit dual-range (C-H/FP) quantum cascade laser (QCL) IR pump source was applied for the first time, delivering a clear synergistic benefit to the classification results. Furthermore, O-PTIR is able to distinguish between lung cancer cells and non-malignant lung cells both in the lipid and fingerprint regions. However, when these two spectral ranges are combined, classification accuracies are enhanced with Random Forest modeling classification accuracy results ranging from 96 to 99% across all three studied cell lines. The methodology described here for the first time with a single-unit dual-range QCL for O-PTIR on glass is another step toward its clinical application in pathology.

Correction: Empirical study on the effects of acquisition parameters for FTIR hyperspectral imaging of brain tissue.

Correction for 'Empirical study on the effects of acquisition parameters for FTIR hyperspectral imaging of brain tissue' by J. Sacharz et al., Anal. Methods, 2020, 12, 4334-4342, DOI: 10.1039/C9AY01200A.

Analysis of Fixed and Live Single Cells Using Optical Photothermal Infrared with Concomitant Raman Spectroscopy.

This paper reports the first use of a novel completely optically based photothermal method (O-PTIR) for obtaining infrared spectra of both fixed and living cells using a quantum cascade laser (QCL) and optical parametric oscillator (OPO) laser as excitation sources, thus enabling all biologically relevant vibrations to be analyzed at submicron spatial resolution. In addition, infrared data acquisition is combined with concomitant Raman spectra from exactly the same excitation location, meaning the full vibrational profile of the cell can be obtained. The pancreatic cancer cell line MIA PaCa-2 and the breast cancer cell line MDA-MB-231 are used as model cells to demonstrate the capabilities of the new instrumentation. These combined modalities can be used to analyze subcellular structures in both fixed and, more importantly, live cells under aqueous conditions. We show that the protein secondary structure and lipid-rich bodies can be identified on the submicron scale.

Imaging Isotopically Labeled Bacteria at the Single-Cell Level Using High-Resolution Optical Infrared Photothermal Spectroscopy.

We report that the cellular uptake of stable isotope-labeled compounds by bacteria can be probed at the single-cell level using infrared spectroscopy, and this monitors the chemical vibrations affected by the incorporation of "heavy" atoms by cells and thus can be used to understand microbial systems. This presents a significant advancement as most studies have focused on evaluating communities of cells due to the poor spatial resolution achieved by classical infrared microspectrometers, and to date, there is no study evaluating the incorporation of labeled compounds by bacteria at single-cell levels using infrared spectroscopy. The development of new technologies and instrumentations that provide information on the metabolic activity of a single bacterium is critical as this will allow for a better understanding of the interactions between microorganisms as well as the function of individual members and their interactions in different microbial communities. Thus, the present study demonstrates the ability of a novel far-field infrared imaging technique, optical photothermal infrared (O-PTIR) spectroscopy, as a tool to monitor the uptake of 13C-glucose and 15N-ammonium chloride by Escherichia coli bacteria at single-cell levels using spectral signatures recorded via single-point and imaging modes. An additional novelty is that imaging was achieved using six vibrational bands in the amide I and II regions, which were analyzed with chemometrics by employing partial least squares-discriminant analysis to predict 13C/12C and 15N/14N simultaneously.

Comparing infrared spectroscopic methods for the characterization of Plasmodium falciparum-infected human erythrocytes.

Malaria, caused by parasites of the species Plasmodium, is among the major life-threatening diseases to afflict humanity. The infectious cycle of Plasmodium is very complex involving distinct life stages and transitions characterized by cellular and molecular alterations. Therefore, novel single-cell technologies are warranted to extract details pertinent to Plasmodium-host cell interactions and underpinning biological transformations. Herein, we tested two emerging spectroscopic approaches: (a) Optical Photothermal Infrared spectroscopy and (b) Atomic Force Microscopy combined with infrared spectroscopy in contrast to (c) Fourier Transform InfraRed microspectroscopy, to investigate Plasmodium-infected erythrocytes. Chemical spatial distributions of selected bands and spectra captured using the three modalities for major macromolecules together with advantages and limitations of each method is presented here. These results indicate that O-PTIR and AFM-IR techniques can be explored for extracting sub-micron resolution molecular signatures within heterogeneous and dynamic samples such as Plasmodium-infected human RBCs.

Orientation Matters: Polarization Dependent IR Spectroscopy of Collagen from Intact Tendon Down to the Single Fibril Level.

Infrared (IR) spectroscopy has been used for decades to study collagen in mammalian tissues. While many changes in the spectral profiles appear under polarized IR light, the absorption bands are naturally broad because of tissue heterogeneity. A better understanding of the spectra of ordered collagen will aid in the evaluation of disorder in damaged collagen and in scar tissue. To that end, collagen spectra have been acquired with polarized far-field (FF) Fourier Transform Infrared (FTIR) imaging with a Focal Plane Array detector, with the relatively new method of FF optical photothermal IR (O-PTIR), and with nano-FTIR spectroscopy based on scattering-type scanning near-field optical microscopy (s-SNOM). The FF methods were applied to sections of intact tendon with fibers aligned parallel and perpendicular to the polarized light. The O-PTIR and nano-FTIR methods were applied to individual fibrils of 100-500 nm diameter, yielding the first confirmatory and complementary results on a biopolymer. We observed that the Amide I and II bands from the fibrils were narrower than those from the intact tendon, and that both relative intensities and band shapes were altered. These spectra represent reliable profiles for normal collagen type I fibrils of this dimension, under polarized IR light, and can serve as a benchmark for the study of collagenous tissues.

Empirical study on the effects of acquisition parameters for FTIR hyperspectral imaging of brain tissue.

Fourier transform infrared (FTIR) spectroscopic imaging is a powerful technique for molecular imaging of pathologies associated with the nervous systems including multiple sclerosis research. However, there is no standard methodology or standardized protocol for FTIR imaging of tissue sections that maximize the ability to discriminate between the molecular, white and granular layers, which is essential in the investigation of the mechanism of demyelination process. Tissue sections are heterogeneous, complex and delicate, hence the parameters to generate high quality images in minimal time becomes essential in the modern clinical laboratory. This article presents an FTIR spectroscopic imaging study of post-mortem human brain tissue testing the effects of various measurement parameters and data analysis methods on image quality and acquisition time. Hyperspectral images acquired from the same region of a tissue using a range of the most common optical and collection parameters in different combinations were compared. These included magnification (4× and 15×), number of co-added scans (1, 4, 8, 16, 32, 64 and 128 scans) and spectral resolution (4, 8 and 16 cm-1). Images were compared in terms of acquisition time, signal-to-noise (S/N) ratio, and accuracy of the discrimination between three major tissue types in a section from the cerebellum (white matter, granular and molecular layers). In the latter case, unsupervised k-means cluster (KMC) analysis was employed to generate images from the hyperspectral images, which were compared to a reference image. The classification accuracy for tissue class discrimination was highest for the 4× magnifying objective, with 4 cm-1 spectral resolution and 128 co-added scans. The 15× magnifying objective gave the best accuracy for a spectral resolution of 4 cm-1 and 64 scans (96.3%), which was just above what was achieved using the 4× magnifying objective, with 4 cm-1 spectral resolution and 32 and 64 co-added scans (95.4 and 95.6%, respectively). These findings were correlated with a decrease in S/N ratio with increasing number of scans and was generally lower for the 15× objective. However, longer scan times were required using the 15× magnifying objective, which did not justify the very small improvement in the classification of tissue types.

Detection of High-Explosive Materials within Fingerprints by Means of Optical-Photothermal Infrared Spectromicroscopy.

As we live under a constant threat of global terrorism, the effective detection of highly energetic materials is one of the critical procedures needed at a variety of locations, including airports, border checkpoints, and entrances to high-security buildings. In this work, the application of optical-photothermal infrared (O-PTIR) spectromicroscopy for the detection of highly explosive materials within fingerprints is described. High-explosive (HE) materials (e.g., PETN, RDX, C-4, or TNT) were used to prepare contaminated fingerprints. These were subsequently deposited on various objects, including microscopic glass slides, a table, a mug, etc. Samples deposited on glass slides were directly sent for analyses; for other samples, adhesive tapes were used to lift off fingermarks. In cases of difficulty in locating fingerprints, additional powders were used to enhance their visibility. Experiments were performed with a mIRage IR microscope working in a noncontact, far-field reflection mode, offering submicron IR spectroscopy and imaging. Fast imaging (several characteristic absorbances were selected for every substance of interest) was used to locate "suspicious" particles among various residues present in fingerprints. Subsequently, spectra were collected for those particles. Reflection mode O-PTIR spectra taken from powdered and nonenhanced fingerprints were of comparable quality to transmission mode FTIR spectra collected for pure HEs. On the basis of the performed experiments, we consider O-PTIR spectromicroscopy to open a new avenue for the nondestructive, efficient, and reliable analysis of exogenous substances deposited within fingerprints. The real significance of O-PTIR is in its ability to deliver high-quality, spatially resolved FTIR transmission-like spectra below the diffraction limit of infrared wavelengths, doing so in an easy-to-use reflection (far-field) mode. Collected spectra are also searchable and interpretable in both commercial and institutional IR databases without mathematical modeling.

Super-Resolution Infrared Imaging of Polymorphic Amyloid Aggregates Directly in Neurons.

Loss of memory during Alzheimer's disease (AD), a fatal neurodegenerative disorder, is associated with neuronal loss and the aggregation of amyloid proteins into neurotoxic ß-sheet enriched structures. However, the mechanism of amyloid protein aggregation is still not well understood due to many challenges when studying the endogenous amyloid structures in neurons or in brain tissue. Available methods either require chemical processing of the sample or may affect the amyloid protein structure itself. Therefore, new approaches, which allow studying molecular structures directly in neurons, are urgently needed. A novel approach is tested, based on label-free optical photothermal infrared super-resolution microspectroscopy, to study AD-related amyloid protein aggregation directly in the neuron at sub-micrometer resolution. Using this approach, amyloid protein aggregates are detected at the subcellular level, along the neurites and strikingly, in dendritic spines, which has not been possible until now. Here, a polymorphic nature of amyloid structures that exist in AD transgenic neurons is reported. Based on the findings of this work, it is suggested that structural polymorphism of amyloid proteins that occur already in neurons may trigger different mechanisms of AD progression.

Optical Photothermal Infrared Microspectroscopy with Simultaneous Raman - A New Non-Contact Failure Analysis Technique for Identification of <10 µm Organic Contamination in the Hard Drive and other Electronics Industries.

Optical Photothermal Infrared (O-PTIR) spectroscopy is a new technique for measuring submicron spatial resolution IR spectra with little or no sample preparation. This speeds up analysis times benefiting high-volume manufacturers through gaining insight into process contamination that occurs during development and on production lines. The ability to rapidly obtain far-field non-contact IR spectra at high spatial resolution facilitates the chemical identification of small organic contaminants that are not possible to measure with conventional Fourier transform infrared (FT-IR) microspectroscopy. The unique pump-probe system architecture also facilitates submicron simultaneous IR + Raman microscopy from the same spot with the same spatial resolution. With these unique capabilities, O-PTIR is finding utilization in the high-volume and high-value industries of high-tech componentry (memory storage, electronics, displays, etc.).

Two-dimensional correlation analysis of highly spatially resolved simultaneous IR and Raman spectral imaging of bioplastics composite using optical photothermal Infrared and Raman spectroscopy.

Optical photothermal infrared (O-PTIR) and Raman spectroscopy and imaging was used to explore the spatial distributions of molecular constituents of a laminate sample consisting of the bioplastics, polyhydroxyalkanoate (PHA) and polylactic acid (PLA), near the interfacial boundary. Highly spatially resolved simultaneous IR and Raman spectra were sequentially collected at 100 nm increments along a line traversing the interface. The set of spectra were subjected to 2D-COS analysis to extract the detailed nature of the spatial distribution of the laminate constituents. It was revealed that the laminate is not a simple binary system of two non-interacting polymers, but consists of different constituents with more complex spatial distributions. Some portion of PLA seems to penetrate into the PHA layer. The crystallinity of PHA near the interface is reduced compared to the rest of the PHA layer. The result suggests the existence of some partial molecular mixing even for these seemingly immiscible polymer pairs. The mixing probably occurs at the segmental level confined to only several hundred nanometers of space at the interface. Such partial mixing may explain the high compatibility between the two bioplastics.

Automated Osteosclerosis Grading of Clinical Biopsies Using Infrared Spectroscopic Imaging.

Osteosclerosis and myefibrosis are complications of myeloproliferative neoplasms. These disorders result in excess growth of trabecular bone and collagen fibers that replace hematopoietic cells, resulting in abnormal bone marrow function. Treatments using imatinib and JAK2 pathway inhibitors can be effective on osteosclerosis and fibrosis; therefore, accurate grading is critical for tracking treatment effectiveness. Current grading standards use a four-class system based on analysis of biopsies stained with three histological stains: hematoxylin and eosin (H&E), Masson's trichrome, and reticulin. However, conventional grading can be subjective and imprecise, impacting the effectiveness of treatment. In this Article, we demonstrate that mid-infrared spectroscopic imaging may serve as a quantitative diagnostic tool for quantitatively tracking disease progression and response to treatment. The proposed approach is label-free and provides automated quantitative analysis of osteosclerosis and collagen fibrosis.

Determination of the secondary structure of proteins in different environments by FTIR-ATR spectroscopy and PLS regression.

The secondary structures of proteins (alpha-helical, beta-sheet, beta-turn, and random coil) in the solid state and when bound to polymer beads, containing immobilized phenyl and butyl ligands such as those as commonly employed in hydrophobic interaction chromatography, have been investigated using FTIR-ATR spectroscopy and partial least squares (PLS) methods. Proteins with known structural features were used as models, including 12 proteins in the solid state and 7 proteins adsorbed onto the hydrophobic surfaces. A strong PLS correlation was achieved between predictions derived from the experimental data for 4 proteins adsorbed onto the phenyl-modified beads and reference data obtained from the X-ray crystallographic structures with r(2) values of 0.9974, 0.9864, 0.9924, and 0.9743 for alpha-helical, beta-sheet, beta-turn, and random coiled structures, respectively. On the other hand, proteins adsorbed onto the butyl sorbent underwent greater secondary structural changes compared to the phenyl sorbent as evidenced from the poorer PLS r(2) values (r(2) are 0.9658, 0.9106, 0.9571, and 0.9340). The results thus indicate that the secondary structures for these proteins were more affected by the butyl sorbent, whereas the secondary structure remains relatively unchanged for the proteins adsorbed onto the phenyl sorbent. This study has important ramifications for understanding the nature of protein secondary structural changes following adsorption onto hydrophobic sorbent surfaces. This knowledge could also enable the development of useful protocols for enhancing the chromatographic purification of proteins in their native bioactive states.

Fourier-transform infrared (FTIR) spectroscopy for monitoring and determining the degree of crystallisation of polyhydroxyalkanoates (PHAs).

FTIR spectroscopy has been used to monitor and determine the degree of crystallisation in a sample of polyhydroxybutyrate-co-14%valerate (PHB-co-14%HV). Time series spectra of solution-cast films of the polymer revealed spectral changes attributed to the onset of crystallisation. Curve fitting was used to obtain an absolute measure of crystallinity. Mean centred principal-component analysis (PCA) revealed that 99.9% of the spectral variance could be attributed to factor 1. The loadings plot for factor 1 contained features attributable to crystalline and amorphous phases. These features were opposite in sign, indicating that changes in the spectra with the onset of crystallisation are simultaneous and opposite in direction, i.e. as the crystalline band increases the amorphous band decreases. Cross-peaks in asynchronous 2D correlation maps indicate there are likely to be very minor components that are changing out of phase. The presence of these minor components is supported by examination of the loadings of higher factors in the PCA model. PCA has been shown to be suitable for determining the number of dynamic spectral features and has enabled relative and objective monitoring of crystallisation kinetics.