Optical photothermal infrared spectroscopy and discrete wavenumber imaging for high content screening of single cells.

Major drawbacks of direct mid-infrared spectroscopic imaging of single cells in an aqueous buffer are strong water absorption, low resolution typically above 10 µm, and Mie scattering effects. This study demonstrates how an indirect detection principle can overcome these drawbacks using the optical photothermal infrared (O-PTIR) technique for high-resolution discrete wavenumber imaging and fingerprint spectroscopy of cultivated cells as a model system in a simple liquid sample chamber. The O-PTIR spectra of six leukemia- and cancer-derived cell lines showed main IR bands near 1648, 1547, 1447, 1400, 1220, and 1088 cm-1. Five spectra of approximately 260 single cells per cell type were averaged, the O-PTIR data set was divided into leukemia-derived cells (THP-1, HL 60, Jurkat, and Raji) and cancer cells (HeLa and HepaRG), and partial least squares linear discriminant analysis (PLS-LDA) was applied in the spectral range 800-1800 cm-1 to train three classification models. A leukemia versus cancer cell model showed an accuracy of 90.0%, the HeLa versus HepaRG cell model had an accuracy of 95.4%, and the model for the distinction of leukemia cells had an accuracy of 75.4%. IR bands in linear discriminants (LDs) of the models were correlated with second derivative spectra that resolved more than 25 subbands. The IR and second derivative spectra of proteins, DNA, RNA and lipids were collected as references to confirm band assignments. O-PTIR images of single cells at a 200 nm step size were acquired at 1086, 1548, and 1746 cm-1 to visualize the nucleic acid, protein, and lipid distribution, respectively. Variations in subcellular features and in the lipid-to-protein and nucleic acid-to-protein ratios were identified that were consistent with biomolecular information in LDs. In conclusion, O-PTIR can provide high-quality spectra and images with submicron resolution of single cells in aqueous buffers that offer prospects in high-content screening applications.

Raman Spectroscopy Profiling of Splenic T-Cells in Sepsis and Endotoxemia in Mice.

Sepsis is a life-threatening condition that results from an overwhelming and disproportionate host response to an infection. Currently, the quality and extent of the immune response are evaluated based on clinical symptoms and the concentration of inflammatory biomarkers released or expressed by the immune cells. However, the host response toward sepsis is heterogeneous, and the roles of the individual immune cell types have not been fully conceptualized. During sepsis, the spleen plays a vital role in pathogen clearance, such as bacteria by an antibody response, macrophage bactericidal capacity, and bacterial endotoxin detoxification. This study uses Raman spectroscopy to understand the splenic T-lymphocyte compartment profile changes during bona fide bacterial sepsis versus hyperinflammatory endotoxemia. The Raman spectral analysis showed marked changes in splenocytes of mice subjected to septic peritonitis principally in the DNA region, with minor changes in the amino acids and lipoprotein areas, indicating significant transcriptomic activity during sepsis. Furthermore, splenocytes from mice exposed to endotoxic shock by injection of a high dose of lipopolysaccharide showed significant changes in the protein and lipid profiles, albeit with interindividual variations in inflammation severity. In summary, this study provided experimental evidence for the applicability and informative value of Raman spectroscopy for profiling the immune response in a complex, systemic infection scenario. Importantly, changes within the acute phase of inflammation onset (24 h) were reliably detected, lending support to the concept of early treatment and severity control by extracorporeal Raman profiling of immunocyte signatures.

Revisiting the interaction of heme with hemopexin.

In hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity of <1 pM in a 1:1 ratio. However, several lines of evidence point to a higher stoichiometry and lower affinity than determined 50 years ago. Here, we re-analyzed these data. SPR and UV/Vis spectroscopy were used to monitor the interaction of heme with the human protein. The heme-binding sites of hemopexin were characterized using hemopexin-derived peptide models and competitive displacement assays. We obtained a KD value of 0.32 ± 0.04 nM and the ratio for the interaction was determined to be 1:1 at low heme concentrations and at least 2:1 (heme:hemopexin) at high concentrations. We were able to identify two yet unknown potential heme-binding sites on hemopexin. Furthermore, molecular modelling with a newly created homology model of human hemopexin suggested a possible recruiting mechanism by which heme could consecutively bind several histidine residues on its way into the binding pocket. Our findings have direct implications for the potential administration of hemopexin in hemolytic disorders.

Vibrational Spectroscopic Investigation of Blood Plasma and Serum by Drop Coating Deposition for Clinical Application.

In recent decades, vibrational spectroscopic methods such as Raman and FT-IR spectroscopy are widely applied to investigate plasma and serum samples. These methods are combined with drop coating deposition techniques to pre-concentrate the biomolecules in the dried droplet to improve the detected vibrational signal. However, most often encountered challenge is the inhomogeneous redistribution of biomolecules due to the coffee-ring effect. In this study, the variation in biomolecule distribution within the dried-sample droplet has been investigated using Raman and FT-IR spectroscopy and fluorescence lifetime imaging method. The plasma-sample from healthy donors were investigated to show the spectral differences between the inner and outer-ring region of the dried-sample droplet. Further, the preferred location of deposition of the most abundant protein albumin in the blood during the drying process of the plasma has been illustrated by using deuterated albumin. Subsequently, two patients with different cardiac-related diseases were investigated exemplarily to illustrate the variation in the pattern of plasma and serum biomolecule distribution during the drying process and its impact on patient-stratification. The study shows that a uniform sampling position of the droplet, both at the inner and the outer ring, is necessary for thorough clinical characterization of the patient's plasma and serum sample using vibrational spectroscopy.

Biochemical Analysis of Leukocytes after In Vitro and In Vivo Activation with Bacterial and Fungal Pathogens Using Raman Spectroscopy.

Biochemical information from activated leukocytes provide valuable diagnostic information. In this study, Raman spectroscopy was applied as a label-free analytical technique to characterize the activation pattern of leukocyte subpopulations in an in vitro infection model. Neutrophils, monocytes, and lymphocytes were isolated from healthy volunteers and stimulated with heat-inactivated clinical isolates of Candida albicans, Staphylococcus aureus, and Klebsiella pneumoniae. Binary classification models could identify the presence of infection for monocytes and lymphocytes, classify the type of infection as bacterial or fungal for neutrophils, monocytes, and lymphocytes and distinguish the cause of infection as Gram-negative or Gram-positive bacteria in the monocyte subpopulation. Changes in single-cell Raman spectra, upon leukocyte stimulation, can be explained with biochemical changes due to the leukocyte's specific reaction to each type of pathogen. Raman spectra of leukocytes from the in vitro infection model were compared with spectra from leukocytes of patients with infection (DRKS-ID: DRKS00006265) with the same pathogen groups, and a good agreement was revealed. Our study elucidates the potential of Raman spectroscopy-based single-cell analysis for the differentiation of circulating leukocyte subtypes and identification of the infection by probing the molecular phenotype of those cells.

Spatiotemporal Organization of Biofilm Matrix Revealed by Confocal Raman Mapping Integrated with Non-negative Matrix Factorization Analysis.

Biofilms are microbial aggregates of microorganisms surrounded by a hydrogel-like matrix formed by extracellular polymeric substances (EPS). The formation of biofilms is intrinsically complex, from the attachment of microbial cells to the dispersion of the biofilm. Meanwhile, the three-dimensional framework built up by EPS changes with time and protects the microorganisms against environmental stress. Simultaneously acquiring chemical and structural information within the biofilm matrix is vital for the cognition and regulation of biofilms, yet it remains a great challenge due to the sample complexity and the limited approaches. In this study, confocal Raman microscopy and non-negative matrix factorization (NMF) analysis were combined to investigate spatiotemporal organization of Escherichia coli biofilms during development at molecular-level detail. The alternating non-negative least-squares (ANLS) approach was incorporated with the sequential coordinate-wise descent (SCD) algorithm to realize the NMF analysis for the large-scale hyperspectral data set. As a result, three components, including bacteria, protein, and polyhydroxybutyrate (PHB), were successfully resolved from the spectra of E. coli biofilm. Furthermore, the structural changes of biofilms could be visualized and quantified by their abundances derived from the NMF analysis, which might be related to the nutrient and oxygen gradient and physiological functions. This methodology provides a comprehensive understanding of the chemical constituents and their spatiotemporal distribution within the biofilm matrix. Furthermore, it also shows great potential for the analysis of unknown and complex biological samples with 3D Raman mapping.

Detection and Differentiation of Bacterial and Fungal Infection of Neutrophils from Peripheral Blood Using Raman Spectroscopy.

Neutrophils are important cells of the innate immune system and the major leukocyte subpopulation in blood. They are responsible for recognizing and neutralizing invading pathogens, such as bacteria or fungi. For this, neutrophils are well equipped with pathogen recognizing receptors, cytokines, effector molecules, and granules filled with reactive oxygen species (ROS)-producing enzymes. Depending on the pathogen type, different reactions are triggered, which result in specific activation states of the neutrophils. Here, we aim to establish a label-free method to indirectly detect infections and to identify the cause of infection by spectroscopic characterization of the neutrophils. For this, isolated neutrophils from human peripheral blood were stimulated in an in vitro infection model with heat-inactivated Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacterial pathogens as well as with heat-inactivated and viable fungi (Candida albicans). Label-free and nondestructive Raman spectroscopy was used to characterize neutrophils on a single cell level. Phagocytized fungi could be visualized in a few high-resolution false color images of individual neutrophils using label-free Raman spectroscopic imaging. Using a high-throughput screening Raman spectroscope (HTS-RS), Raman spectra of more than 2000 individual neutrophils from three different donors were collected in each treatment group, yielding a data set of almost 20 000 neutrophil spectra. Random forest classification models were trained to differentiate infected and noninfected cells with high accuracy (90%). Among the neutrophils challenged with pathogens, even the cause of infection, bacterial or fungal, was predicted correctly with 92% accuracy. Therefore, Raman spectroscopy enables reliable neutrophil phenotyping and infection diagnosis in a label-free manner. In contrast to the microbiological diagnostic standard, where the pathogen is isolated in time-consuming cultivation, this Raman-based method could potentially be blood-culture independent, thus saving precious time in bloodstream infection diagnostics.

Vibrational spectroscopy as a powerful tool for follow-up immunoadsorption therapy treatment of dilated cardiomyopathy - a case report.

Dilated cardiomyopathy (DCM) is a leading cardiomyopathy condition and is the leading reason for heart transplantation. Due to high etiologic and genetic heterogeneity of the pathologies, different therapeutic treatment strategies are available and have been successful for different treatments. Immunoadsorption (IA) therapy removes the circulating anticardiac antibodies and improves the left ventricular function in substantial proportion of DCM patients. Powerful, non-invasive analytical tools are highly desired to investigate the efficiency and success of IA therapy. In this contribution, we followed the changes of a female DCM patient undergoing IA therapy at different treatment time points in a label-free, non-invasive manner from blood samples (plasma and serum) on the basis of vibrational spectroscopy (Raman scattering and IR absorption). Chemometric methods, including dimension reduction and statistical modeling, were used to interpret spectral data. The impact of different time points of the IA treatment can be identified in both the plasma and serum, using both techniques, with high accuracy. The removal of antibodies of immunoglobulin G (IgG) group during IA therapy and their restoration was reflected in both Raman and FTIR spectra. Relative changes in the spectral bands assigned to IgG agreed well with the immunoturbidimetry measurement of total IgG. Successful clinical treatment was accompanied by spectral differences between vibrational spectra obtained at initial disease state and 11 months after the IA treatment. The long-term follow-up of the patient reveals the stabilization of the health state after therapy. It is noteworthy that the treatment time points were distinguished with a better accuracy using spectra from plasma compared to those from serum samples, which might indicate the involvement of corresponding proteins in the coagulation. Vibrational spectroscopy is a powerful tool for personalized medicine to follow-up the treatment success of IA therapy for the DCM disorder.

High-Throughput Screening Raman Spectroscopy Platform for Label-Free Cellomics.

We present a high-throughput screening Raman spectroscopy (HTS-RS) platform for a rapid and label-free macromolecular fingerprinting of tens of thousands eukaryotic cells. The newly proposed label-free HTS-RS platform combines automated imaging microscopy with Raman spectroscopy to enable a rapid label-free screening of cells and can be applied to a large number of biomedical and clinical applications. The potential of the new approach is illustrated by two applications. (1) HTS-RS-based differential white blood cell count. A classification model was trained using Raman spectra of 52 218 lymphocytes, 48 220 neutrophils, and 7 294 monocytes from four volunteers. The model was applied to determine a WBC differential for two volunteers and three patients, producing comparable results between HTS-RS and machine counting. (2) HTS-RS-based identification of circulating tumor cells (CTCs) in 1:1, 1:9, and 1:99 mixtures of Panc1 cells and leukocytes yielded ratios of 55:45, 10:90, and 3:97, respectively. Because the newly developed HTS-RS platform can be transferred to many existing Raman devices in all laboratories, the proposed implementation will lead to a significant expansion of Raman spectroscopy as a standard tool in biomedical cell research and clinical diagnostics.

Photonic monitoring of treatment during infection and sepsis: development of new detection strategies and potential clinical applications.

Despite the strong decline in the infection-associated mortality since the development of the first antibiotics, infectious diseases are still a major cause of death in the world. With the rising number of antibiotic-resistant pathogens, the incidence of deaths caused by infections may increase strongly in the future. Survival rates in sepsis, which occurs when body response to infections becomes uncontrolled, are still very poor if an adequate therapy is not initiated immediately. Therefore, approaches to monitor the treatment efficacy are crucially needed to adapt therapeutic strategies according to the patient's response. An increasing number of photonic technologies are being considered for diagnostic purpose and monitoring of therapeutic response; however many of these strategies have not been introduced into clinical routine, yet. Here, we review photonic strategies to monitor response to treatment in patients with infectious disease, sepsis, and septic shock. We also include some selected approaches for the development of new drugs in animal models as well as new monitoring strategies which might be applicable to evaluate treatment response in humans in the future. Figure Label-free probing of blood properties using photonics.

Raman Spectroscopic Characterization of Packaged L. pneumophila Strains Expelled by T. thermophila.

The intracellular lifestyle of L. pneumophila within protozoa is considered to be a fundamental process that supports its survival in nature. However, after ingesting the cells of L. pneumophila, some protozoa expel them as compressed live cells in the form of small rounded pellets. The pellets of tightly packaged viable but not culturable forms (VBNCFs) as well as highly infectious mature intracellular forms (MIFs) of L. pneumophila are considered as infectious particles most likely capable to cause human infection. Since L. pneumophila cells are hardly culturable from these pellets, detection methods for packaged live L. pneumophila forms remaining in water should be cultivation free. Hence, we demonstrate the potential of Raman microspectroscopy to directly sort pellets containing L. pneumophila cells, expelled by T. thermophila, and to characterize them on the basis of their Raman spectra.

Raman spectroscopic differentiation of planktonic bacteria and biofilms.

Both biofilm formations as well as planktonic cells of water bacteria such as diverse species of the Legionella genus as well as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli were examined in detail by Raman microspectroscopy. Production of various molecules involved in biofilm formation of tested species in nutrient-deficient media such as tap water was observed and was particularly evident in the biofilms formed by six Legionella species. Biofilms of selected species of the Legionella genus differ significantly from the planktonic cells of the same organisms in their lipid amount. Also, all Legionella species have formed biofilms that differ significantly from the biofilms of the other tested genera in the amount of lipids they produced. We believe that the significant increase in the synthesis of this molecular species may be associated with the ability of Legionella species to form biofilms. In addition, a combination of Raman microspectroscopy with chemometric approaches can distinguish between both planktonic form and biofilms of diverse bacteria and could be used to identify samples which were unknown to the identification model. Our results provide valuable data for the development of fast and reliable analytic methods based on Raman microspectroscopy, which can be applied to the analysis of tap water-adapted microorganisms without any cultivation step.

In-depth structure-function profiling of the complex formation between clotting factor VIII and heme.

BACKGROUND AND AIMS: Blood disorders, such as sickle cell disease, and other clinical conditions are often accompanied by intravascular hemolytic events along with the development of severe coagulopathies. Hemolysis, in turn, leads to the accumulation of Fe(II/III)-protoporphyrin IX (heme) in the intravascular compartment, which can trigger a variety of proinflammatory and prothrombotic reactions. As such, heme binding to the blood coagulation proteins factor VIII (FVIII), fibrinogen, and activated protein C with functional consequences has been demonstrated earlier. METHODS: We herein present an in-depth characterization of the FVIII-heme interaction at the molecular level and its (patho-)physiological relevance through the application of biochemical, biophysical, structural biology, bioinformatic, and diagnostic tools. RESULTS: FVIII has a great heme-binding capacity with seven heme molecules associating with the protein. The respective binding sites were identified by investigating heme binding to FVIII-derived peptides in combination with molecular docking and dynamic simulation studies of the complex as well as cryo-electron microscopy, revealing three high-affinity and four moderate heme-binding motifs (HBMs). Furthermore, the relevance of the FVIII-heme complex formation was characterized in physiologically relevant assay systems, revealing a ~ 50 % inhibition of the FVIII cofactor activity even in the protein-rich environment of blood plasma. CONCLUSION: Our study provides not only novel molecular insights into the FVIII-heme interaction and its physiological relevance, but also strongly suggests the reduction of the intrinsic pathway and the accentuation of the final clotting step (by, for example, fibrinogen crosslinking) in hemolytic conditions as well as a future perspective in the context of FVIII substitution therapy of hemorrhagic events in hemophilia A patients.

Combined dielectrophoresis-Raman setup for the classification of pathogens recovered from the urinary tract.

Rapid and effective methods of pathogen identifications are of major interest in clinical microbiological analysis to administer timely tailored antibiotic therapy. Raman spectroscopy as a label-free, culture-independent optical method is suitable to identify even single bacteria. However, the low bacteria concentration in body fluids makes it difficult to detect their characteristic molecular fingerprint directly in suspension. Therefore, in this study, Raman spectroscopy is combined with dielectrophoresis, which enables the direct translational manipulation of bacteria in suspensions with spatial nonuniform electrical fields so as to perform specific Raman spectroscopic characterization. A quadrupole electrode design is used to capture bacteria directly from fluids in well-defined microsized regions. With live/dead fluorescence viability staining, it is verified, that the bacteria survive this procedure for the relevant range of field strengths. The dielectrophoretic enrichment of bacteria allows for obtaining high quality Raman spectra in dilute suspensions with an integration time of only one second. As proof-of-principle study, the setup was tested with Escherichia coli and Enterococcus faecalis, two bacterial strains that are commonly encountered in urinary tract infections. Furthermore, to verify the potential for dealing with real world samples, pathogens from patients' urine have been analyzed. With the additional help of multivariate statistical analysis, a robust classification model could be built and allowed the classification of those two strains within a few minutes. In contrast, the standard microbiological diagnostics are based on very time-consuming cultivation tests. This setup holds the potential to reduce the crucial parameter diagnosis time by orders of magnitude.

Intestinal epithelial barrier integrity investigated by label-free techniques in ulcerative colitis patients.

The intestinal epithelial barrier, among other compartments such as the mucosal immune system, contributes to the maintenance of intestinal homeostasis. Therefore, any disturbance within the epithelial layer could lead to intestinal permeability and promote mucosal inflammation. Considering that disintegration of the intestinal epithelial barrier is a key element in the etiology of ulcerative colitis, further assessment of barrier integrity could contribute to a better understanding of the role of epithelial barrier defects in ulcerative colitis (UC), one major form of chronic inflammatory bowel disease. Herein, we employ fast, non-destructive, and label-free non-linear methods, namely coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF), and two-photon fluorescence lifetime imaging (2P-FLIM), to assess the morpho-chemical contributions leading to the dysfunction of the epithelial barrier. For the first time, the formation of epithelial barrier gaps was directly visualized, without sophisticated data analysis procedures, by the 3D analysis of the colonic mucosa from severely inflamed UC patients. The results were compared with histopathological and immunofluorescence images and validated using transmission electron microscopy (TEM) to indicate structural alterations of the apical junction complex as the underlying cause for the formation of the epithelial barrier gaps. Our findings suggest the potential advantage of non-linear multimodal imaging is to give precise, detailed, and direct visualization of the epithelial barrier in the gastrointestinal tract, which can be combined with a fiber probe for future endomicroscopy measurements during real-time in vivo imaging.

Tuning the corona-core ratio of polyplex micelles for selective oligonucleotide delivery to hepatocytes or hepatic immune cells.

Targeted delivery of oligonucleotides or small molecular drugs to hepatocytes, the liver's parenchymal cells, is challenging without targeting moiety due to the highly efficient mononuclear phagocyte system (MPS) of the liver. The MPS comprises Kupffer cells and specialized sinusoidal endothelial cells, efficiently clearing nanocarriers regardless of their size and surface properties. Physiologically, this non-parenchymal shield protects hepatocytes; however, these local barriers must be overcome for drug delivery. Nanocarrier structural properties strongly influence tissue penetration, in vivo pharmacokinetics, and biodistribution profile. Here we demonstrate the in vivo biodistribution of polyplex micelles formed by polyion complexation of short interfering (si)RNA with modified poly(ethylene glycol)-block-poly(allyl glycidyl ether) (PEG-b-PAGE) diblock copolymer that carries amino moieties in the side chain. The ratio between PEG corona and siRNA complexed PAGE core of polyplex micelles was chemically varied by altering the degree of polymerization of PAGE. Applying Raman-spectroscopy and dynamic in silico modeling on the polyplex micelles, we determined the corona-core ratio (CCR) and visualized the possible micellar structure with varying CCR. The results for this model system reveal that polyplex micelles with higher CCR, i.e., better PEG coverage, exclusively accumulate and thus allow passive cell-type-specific targeting towards hepatocytes, overcoming the macrophage-rich reticuloendothelial barrier of the liver.

Toward a spectroscopic hemogram: Raman spectroscopic differentiation of the two most abundant leukocytes from peripheral blood.

The first response to infection in the blood is mediated by leukocytes. As a result crucial information can be gained from a hemogram. Conventional methods such as blood smears and automated sorting procedures are not capable of recording detailed biochemical information of the different leukocytes. In this study, Raman spectroscopy has been applied to investigate the differences between the leukocyte subtypes which have been obtained from healthy donors. Raman imaging was able to visualize the same morphological features as standard staining methods without the need of any label. Unsupervised statistical methods such as principal component analysis and hierarchical cluster analysis were able to separate Raman spectra of the two most abundant leukocytes, the neutrophils and lymphocytes (with a special focus on CD4(+) T-lymphocytes). For the same cells a classification model was built to allow an automated Raman-based differentiation of the cell type in the future. The classification model could achieve an accuracy of 94% in the validation step and could predict the identity of unknown cells from a completely different donor with an accuracy of 81% when using single spectra and with an accuracy of 97% when using the majority vote from all individual spectra of the cell. This marks a promising step toward automated Raman spectroscopic blood analysis which holds the potential not only to assign the numbers of the cells but also to yield important biochemical information.

Specific intracellular signature of SARS-CoV-2 infection using confocal Raman microscopy.

SARS-CoV-2 infection remains spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers.

Label-free multimodal imaging of infected Galleria mellonella larvae.

Non-linear imaging modalities have enabled us to obtain unique morpho-chemical insights into the tissue architecture of various biological model organisms in a label-free manner. However, these imaging techniques have so far not been applied to analyze the Galleria mellonella infection model. This study utilizes for the first time the strength of multimodal imaging techniques to explore infection-related changes in the Galleria mellonella larvae due to massive E. faecalis bacterial infection. Multimodal imaging techniques such as fluorescent lifetime imaging (FLIM), coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second harmonic generation (SHG) were implemented in conjunction with histological HE images to analyze infection-associated tissue damage. The changes in the larvae in response to the infection, such as melanization, vacuolization, nodule formation, and hemocyte infiltration as a defense mechanism of insects against microbial pathogens, were visualized after Enterococcus faecalis was administered. Furthermore, multimodal imaging served for the analysis of implant-associated biofilm infections by visualizing biofilm adherence on medical stainless steel and ePTFE implants within the larvae. Our results suggest that infection-related changes as well as the integrity of the tissue of G. mellonella larvae can be studied with high morphological and chemical contrast in a label-free manner.

COVID-19 Diagnostics: Past, Present, and Future.

In winter of 2020, SARS-CoV-2 emerged as a global threat, impacting not only health but also financial and political stability. To address the societal need for monitoring the spread of SARS-CoV-2, many existing diagnostic technologies were quickly adapted to detect SARS-CoV-2 RNA and antigens as well as the immune response, and new testing strategies were developed to accelerate time-to-decision. In parallel, the infusion of research support accelerated the development of new spectroscopic methods. While these methods have significantly reduced the impact of SARS-CoV-2 on society when coupled with behavioral changes, they also lay the groundwork for a new generation of platform technologies. With several epidemics on the horizon, such as the rise of antibiotic-resistant bacteria, the ability to quickly pivot the target pathogen of this diagnostic toolset will continue to have an impact.