Label-free visualization of unfolding and crosslinking mediated protein aggregation in nonenzymatically glycated proteins.

Nonenzymatic glycation (NEG) unfolds and crosslinks proteins, resulting in aggregation. Label-free evaluation of such structural changes, without disturbing molecular integrity, would be beneficial for understanding the fundamental mechanisms of protein aggregation. The current study demonstrates the assessment of NEG-induced protein aggregation by combining autofluorescence (AF) spectroscopy and imaging. The methylglyoxal (MG) induced protein unfolding and the formation of cross-linking advanced glycation end-products (AGEs) leading to aggregation were evaluated using deep-UV-induced-autofluorescence (dUV-AF) spectroscopy in proteins with distinct structural characteristics. Since the AGEs formed on proteins are fluorescent, the study demonstrated the possibility of autofluorescence imaging of NEG-induced protein aggregates. Autofluorescence spectroscopy can potentially reveal molecular alterations such as protein unfolding and cross-linking. In contrast, AGE-based autofluorescence imaging offers a means to visually explore the structural arrangement of aggregates, regardless of whether they are amyloid or non-amyloid in nature.

Characterization and classification of ductal carcinoma tissue using four channel based stokes-mueller polarimetry and machine learning.

Interaction of polarized light with healthy and abnormal regions of tissue reveals structural information associated with its pathological condition. Even a slight variation in structural alignment can induce a change in polarization property, which can play a crucial role in the early detection of abnormal tissue morphology. We propose a transmission-based Stokes-Mueller microscope for quantitative analysis of the microstructural properties of the tissue specimen. The Stokes-Mueller based polarization microscopy provides significant structural information of tissue through various polarization parameters such as degree of polarization (DOP), degree of linear polarization (DOLP), and degree of circular polarization (DOCP), anisotropy (r) and Mueller decomposition parameters such as diattenuation, retardance and depolarization. Further, by applying a suitable image processing technique such as Machine learning (ML) output images were analysed effectively. The support vector machine image classification model achieved 95.78% validation accuracy and 94.81% testing accuracy with polarization parameter dataset. The study's findings demonstrate the potential of Stokes-Mueller polarimetry in tissue characterization and diagnosis, providing a valuable tool for biomedical applications.

Spectroscopic evaluation of sesame and mustard oils treated with Murchana method.

In recent years, there has been a growing interest in traditional medicinal practices such as Ayurveda, which emphasizes the use of natural ingredients for various therapeutic purposes. Vegetable oils are an integral part of our diet and have several applications in the cosmetics and healthcare industries. These oils have also been prescribed in ancient Ayurveda texts to treat various health problems. Ayurveda prescribes a processing technique called 'Murchana' to improve the therapeutic nature of the oils. Spectroscopic techniques have been used for quality assessment in many fields. High sensitivity and a low detection rate make spectroscopy a formidable analytical technique. This study focusses on the spectroscopic analysis of sesame and mustard oils prepared using the ayurvedic processing method 'Murchana'. Spectroscopic analysis techniques including UV-Vis absorbance spectroscopy, fluorescence spectroscopy, and FTIR spectroscopy were employed to study the oils. Origin software was used to plot graphs of the spectra. The results indicated that the murchana process may reduce the components of the oil responsible for its oxidation, thereby increasing the shelf life of the oils. However, further investigations, including other spectroscopy and chromatography techniques, will prove beneficial in ascertaining the effects of the murchana process on vegetable oils. The study's findings also suggest that spectroscopic techniques can be used to supplement chemical techniques to investigate the characteristics of vegetable oils.

The revolution of PDMS microfluidics in cellular biology.

Microfluidics is revolutionizing the way research on cellular biology has been traditionally conducted. The ability to control the cell physicochemical environment by adjusting flow conditions, while performing cellular analysis at single-cell resolution and high-throughput, has made microfluidics the ideal choice to replace traditional in vitro models. However, such a revolution only truly started with the advent of polydimethylsiloxane (PDMS) as a microfluidic structural material and soft-lithography as a rapid manufacturing technology. Indeed, before the "PDMS age," microfluidic technologies were: costly, time-consuming and, more importantly, accessible only to specialized laboratories and users. The simplicity of molding PDMS in various shapes along with its inherent properties (transparency, biocompatibility, and gas permeability) has spread the applications of innovative microfluidic devices to diverse and important biological fields and clinical studies. This review highlights how PDMS-based microfluidic systems are innovating pre-clinical biological research on cells and organs. These devices were able to cultivate different cell lines, enhance the sensitivity and diagnostic effectiveness of numerous cell-based assays by maintaining consistent chemical gradients, utilizing and detecting the smallest number of analytes while being high-throughput. This review will also assist in identifying the pitfalls in current PDMS-based microfluidic systems to facilitate breakthroughs and advancements in healthcare research.

Revealing the Structural Organization of Gamma-irradiated Starch Granules Using Polarization-resolved Second Harmonic Generation Microscopy.

Starch is a semi-crystalline macromolecule with the presence of amorphous and crystalline components. The amorphous amylose and crystalline amylopectin regions in starch granules are susceptible to certain physical modifications, such as gamma irradiation. Polarization-resolved second harmonic generation (P-SHG) microscopy in conjunction with SHG-circular dichroism (CD) was used to assess the three-dimensional molecular order and inherent chirality of starch granules and their reaction to different dosages of gamma irradiation. For the first time, the relationship between starch achirality (χ21/χ16 and χ22/χ16) and chirality (χ14/χ16) determining susceptibility tensor ratios has been elucidated. The results showed that changes in the structure and orientation of long-chain amylopectin were supported by the decrease in the SHG anisotropy factor and the χ22/χ16 ratio. Furthermore, SHG-CD illustrated the molecular tilt angle by revealing the arrangement of amylopectin molecules pointing either upward or downward owing to molecular polarity.

Possibilities of simulation of coronavirus SARS-CoV-2 by using light scattering approach.

Issues related to human coronavirus (SARS CoV-2) are a burning topic of research in present times. Due to its easily contagious nature, real experimentation under laboratory conditions requires a high level of biosafety. A powerful algorithm serves as a potential tool for the analysis of these particles. We attempted to simulate the light scattering from coronavirus (SARS CoV-2) model. Different images were modelled using a modified version of a Monte Carlo code. The results indicate that spikes on the viruses exhibit a significant scattering profile and that the presence of spikes during modelling contributes to the distinctiveness of the scattering profiles.

Stokes-Mueller polarization-based analysis of model SARS-CoV-2 virions.

Understanding the virology of the coronavirus at the structural level has gained utmost importance to overcome the constant and long-term health complications induced by them. In this work, the light scattering properties of SARS-CoV-2 of size 140 nm were simulated by using discrete dipole approximation (DDA) for two incident wavelengths 200 nm and 350 nm, respectively. Three different 3-dimensional (3D) models of SARS-CoV-2 corresponding to 15, 20, and 40 numbers of spike proteins on the viral capsid surface were constructed as target geometries for the DDA calculations. These models were assessed by employing Stokes-Mueller polarimetry to obtain individual polarization properties such as degree of polarization (DOP), degree of linear polarization (DOLP), and degree of circular polarization (DOCP). Irrespective of its spike numbers, all the coronavirus models were found to display higher DOP and DOCP values and negligibly small DOLP values for circularly polarized incident light, indicating the presence of chiral structures. On the other hand, the lack of understanding about the dependence of the Mueller matrix on its microstructural properties was overcome by transforming 16 Mueller elements into sub-matrices with specific structural and physical properties using Lu-Chipman-based Mueller matrix polar decomposition method. The obtained properties such as retardance, diattenuation, and depolarization were used for investigating the composition and microstructural information. The approach presented in this work has the potential to understand the virology of the coronavirus at the structural level and, therefore, will be beneficial in developing effective detection strategies by exploiting their characteristic electromagnetic scattering signatures.

Types of spectroscopy and microscopy techniques for cancer diagnosis: a review.

Cancer is a life-threatening disease that has claimed the lives of many people worldwide. With the current diagnostic methods, it is hard to determine cancer at an early stage, due to its versatile nature and lack of genomic biomarkers. The rapid development of biophotonics has emerged as a potential tool in cancer detection and diagnosis. Using the fluorescence, scattering, and absorption characteristics of cells and tissues, it is possible to detect cancer at an early stage. The diagnostic techniques addressed in this review are highly sensitive to the chemical and morphological changes in the cell and tissue during disease progression. These changes alter the fluorescence signal of the cell/tissue and are detected using spectroscopy and microscopy techniques including confocal and two-photon fluorescence (TPF). Further, second harmonic generation (SHG) microscopy reveals the morphological changes that occurred in non-centrosymmetric structures in the tissue, such as collagen. Again, Raman spectroscopy is a non-destructive method that provides a fingerprinting technique to differentiate benign and malignant tissue based on Raman signal. Photoacoustic microscopy and spectroscopy of tissue allow molecule-specific detection with high spatial resolution and penetration depth. In addition, terahertz spectroscopic studies reveal the variation of tissue water content during disease progression. In this review, we address the applications of spectroscopic and microscopic techniques for cancer detection based on the optical properties of the tissue. The discussed state-of-the-art techniques successfully determines malignancy to its rapid diagnosis.

Spectroscopic methods for assessment of hand sanitizers.

Sanitization of inanimate objects or body surfaces using disinfectant is essential for eliminating disease-causing pathogens and maintaining personal hygiene. With the advent of health emergencies, the importance and high demand for hand sanitizers (HS) are observed in everyday life. It is also important to know the constituent added to formulate HS, as the presence of harsh chemicals can cause skin irritation. In this study, different spectroscopic techniques were used to assess several commercially available HS along with the in-house prepared HS as per the WHO protocol. Fourier transform infrared spectroscopy and Raman spectroscopy identified the different HS chemical bonds and quantified the amount of alcohol and water in the HS. Varying amount of alcohols in HS, calibration profile was generated to identify its amount in commercial samples. Further, the commercial samples were also checked for contaminants whose presence in the HS might bring down its sanitization efficacy. Supplementary Information: The online version contains supplementary material available at 10.1007/s11696-022-02208-x.

Recent trends in smartphone-based detection for biomedical applications: a review.

Smartphone-based imaging devices (SIDs) have shown to be versatile and have a wide range of biomedical applications. With the increasing demand for high-quality medical services, technological interventions such as portable devices that can be used in remote and resource-less conditions and have an impact on quantity and quality of care. Additionally, smartphone-based devices have shown their application in the field of teleimaging, food technology, education, etc. Depending on the application and imaging capability required, the optical arrangement of the SID varies which enables them to be used in multiple setups like bright-field, fluorescence, dark-field, and multiple arrays with certain changes in their optics and illumination. This comprehensive review discusses the numerous applications and development of SIDs towards histopathological examination, detection of bacteria and viruses, food technology, and routine diagnosis. Smartphone-based devices are complemented with deep learning methods to further increase the efficiency of the devices. Graphical Abstract.

Label-Free Characterization of Collagen Crosslinking in Bone-Engineered Materials Using Nonlinear Optical Microscopy.

Engineered biomaterials provide unique functions to overcome the bottlenecks seen in biomedicine. Hence, a technique for rapid and routine tests of collagen is required, in which the test items commonly include molecular weight, crosslinking degree, purity, and sterilization induced structural change. Among them, the crosslinking degree mainly influences collagen properties. In this study, second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy are used in combination to explore the collagen structure at molecular and macromolecular scales. These measured parameters are applied for the classification and quantification among the different collagen scaffolds, which were verified by other conventional methods. It is demonstrated that the crosslinking status can be analyzed from SHG images and presented as the coherency of collagen organization that is correlated with the mechanical properties. Also, the comparative analyses of SHG signal and relative CARS signal of amide III band at 1,240 cm−1 to δCH2 band at 1,450 cm−1 of these samples provide information regarding the variation of the molecular structure during a crosslinking process, thus serving as nonlinear optical signatures to indicate a successful crosslinking.

Stokes polarimetry-based second harmonic generation microscopy for collagen and skeletal muscle fiber characterization.

The complete polarization state of second harmonic (SH) light was measured and characterized by collagen type I and skeletal muscle fiber using a Stokes vector-based SHG microscope. The polarization states of the SH signal are analyzed in a pixel-by-pixel manner and displayed through two dimensional (2D) Stokes vector images. Various polarization parameters are reconstructed using Stokes values to quantify the polarization properties of SH light. Also, the measurements are extended for different input polarization states to investigate the molecular structure of second harmonic generation (SHG) active molecules such as collagen type I and myosin.

Advances in adaptive optics-based two-photon fluorescence microscopy for brain imaging.

Deep tissue imaging using two-photon fluorescence (TPF) techniques have revolutionized the optical imaging community by providing in depth molecular information at the single-cell level. These techniques provide structural and functional aspects of mammalian brain at unprecedented depth and resolution. However, wavefront distortions introduced by the optical system as well as the biological sample (tissue) limit the achievable fluorescence signal-to-noise ratio and resolution with penetration depth. In this review, we discuss on the advances in TPF microscopy techniques for in vivo functional imaging and offer guidelines as to which technologies are best suited for different imaging applications with special reference to adaptive optics.

Light emitting diode (LED) based fluorescence microscopy for tuberculosis detection: a review.

Since time immemorial, tuberculosis (TB) has intimidated the human race owing to its severity. Its socio-economic burden has led to it being a major cause of concern. It is one of the world's major causes of death from a single agent. Since most of the middle- and low-income countries are burdened with TB, sputum smear examination using conventional light microscopy is often the only resort for diagnosing TB. However, fluorescence microscopy is used as standard in most high-income countries, owing to its increased sensitivity. Light-emitting diodes (LEDs), being inexpensive, are increasingly gaining popularity as an alternative light source for fluorescence microscopy. It has been found to be highly efficient and has a lot of advantages over the conventional Ziehl-Neelsen-based bright field microscopy. In this review, we discuss about the usefulness of LED-based fluorescence microscopy in diagnosing TB and how it is superior to the other sources of light used.

Deciphering biophysical signatures for microbiological applications.

Identification and classification of microbes are vital for maintenance of normal and altered state of human health and have applications in pharmaceutical industries, food processing, clinical analysis, and treatment. Development of methods aimed towards achieving these goals must be rapid and reliable. Conventional physiochemical and morphology-based methods of identification are often ambiguous, while newer molecular methods such as flow cytometry and polymerase chain reaction, though reliable, are time and resource intensive. Spectroscopic methods provide advantages over conventional methods as these can be fast, non-destructive, and highly specific. Surface charge of bacteria is an important parameter which can reveal composition of cell wall and is attributed to the presence of carboxyl and phosphoryl groups. Interaction of the cell with the solvent and response to various stresses can hence be measured by the changes in surface charge. In this study, we have obtained auto-fluorescence spectra (tryptophan) and dynamic light scattering (DLS) measurements from common pathogenic strains of Pseudomonas aeruginosa and Staphylococcus aureus. Fluorescence emission spectra were obtained in the range of 300-550 nm at excitation wavelength of 280 nm and DLS measurements comprised zeta potential and size parameters. Both types of measurements were performed in physiological and stress-induced conditions such as heat, sonication, and antibiotic treatment with vancomycin and cetylpyridinium chloride. Effects of these antibiotics on membrane integrity and cell viability, as obtained by DLS measurements, were statistically significant and comparable with conventional methods. Multivariate analysis enabled clustering of 83% of the samples at the genera level, based on variances from auto-fluorescence and DLS measurements.

Polarization-resolved Stokes-Mueller imaging: a review of technology and applications.

Polarization microscopy, a powerful optical tool to study anisotropic properties of biomolecules, provides better microstructural information of a sample as compared with conventional optical microscopic techniques. The measurement and analysis of polarization states of light can be performed using both Jones matrix as well as Stokes algebra. Further, the details of optical properties of specimen are characterized by Mueller matrix. However, the application of Jones calculus is limited to perfectly polarized light, but Stokes-Mueller polarimetry is emerging as a promising tool for tissue imaging due to its application irrespective of polarization state of the light. In this review article, we explain the development of Stokes-Mueller formalism in context of linear optics. Furthermore, application of Mueller matrix decomposition (MMD) method to derive sample properties is demonstrated in several bio-medical studies.

Enantiomeric Recognition and Separation by Chiral Nanoparticles.

Chiral molecules are stereoselective with regard to specific biological functions. Enantiomers differ considerably in their physiological reactions with the human body. Safeguarding the quality and safety of drugs requires an efficient analytical platform by which to selectively probe chiral compounds to ensure the extraction of single enantiomers. Asymmetric synthesis is a mature approach to the production of single enantiomers; however, it is poorly suited to mass production and allows for only specific enantioselective reactions. Furthermore, it is too expensive and time-consuming for the evaluation of therapeutic drugs in the early stages of development. These limitations have prompted the development of surface-modified nanoparticles using amino acids, chiral organic ligands, or functional groups as chiral selectors applicable to a racemic mixture of chiral molecules. The fact that these combinations can be optimized in terms of sensitivity, specificity, and enantioselectivity makes them ideal for enantiomeric recognition and separation. In chiral resolution, molecules bond selectively to particle surfaces according to homochiral interactions, whereupon an enantiopure compound is extracted from the solution through a simple filtration process. In this review article, we discuss the fabrication of chiral nanoparticles and look at the ways their distinctive surface properties have been adopted in enantiomeric recognition and separation.

Polarization resolved second harmonic microscopy.

Second harmonic (SH) microscopy has proven to be a powerful imaging modality over the past years due to its intrinsic advantages as a multiphoton process with endogenous contrast specificity, which allows pinhole-less optical sectioning, non-invasive observation, deep tissue penetration, and the possibility of easier signal detection at visible wavelengths. Depending on the relative orientation between the polarization of the incoming light and the second-order susceptibility of non-centrosymmetric structures, SH microscopy provides the unique capacity to probe the absolute molecular structure of a broad variety of biological tissues without the necessity for additional labeling. In addition, SH microscopy, when working with polarimetry, provides clear and in-depth insights on the details of molecular orientation and structural symmetry. In this review, the working principles of the polarization resolving techniques and the corresponding implements of SH microscopy are elucidated, with focus on Stokes vector based polarimetry. An overview of the advancements on SH anisotropy measurements are also presented. Specifically, the recent progresses on the following three topics in polarization resolved SH microscopy will be elucidated, which include Stokes vector resolving for imaging molecular structure and orientation, 3-D structural chirality by SH circular dichroism, and correlation with fluorescence lifetime imaging (FLIM) for in vivo wound healing diagnosis. The potentials and challenges for future researches in exploring complex biological tissues are also discussed.

Types of advanced optical microscopy techniques for breast cancer research: a review.

A cancerous cell is characterized by morphological and metabolic changes which are the key features of carcinogenesis. Adenosine triphosphate (ATP) in cancer cells is primarily produced by aerobic glycolysis rather than oxidative phosphorylation. In normal cellular metabolism, nicotinamide adenine dinucleotide (NADH) is considered as a principle electron donor and flavin adenine dinucleotide (FAD) as an electron acceptor. During metabolism in a cancerous cell, a net increase in NADH is found as the pathway switched from oxidative phosphorylation to aerobic glycolysis. Often during initiation and progression of cancer, the developmental regulation of extracellular matrix (ECM) is restricted and becomes disorganized. Tumor cell behavior is regulated by the ECM in the tumor micro environment. Collagen, which forms the scaffold of tumor micro-environment also influences its behavior. Advanced optical microscopy techniques are useful for determining the metabolic characteristics of cancerous, normal cells and tissues. They can be used to identify the collagen microstructure and the function of NADH, FAD, and lipids in living system. In this review article, various optical microscopy techniques applied for breast cancer research are discussed including fluorescence, confocal, second harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS), and fluorescence lifetime imaging (FLIM).

Revealing molecular structure and orientation with Stokes vector resolved second harmonic generation microscopy.

We report on measurements and characterization of polarization properties of Second Harmonic (SH) signals using a four-channel photon counting based Stokes polarimeter. In this way, the critical polarization parameters can be obtained concurrently without the need of repeated image acquisition. The critical polarization parameters, including the degree of polarization (DOP), the degree of linear polarization (DOLP), and the degree of circular polarization (DOCP), are extracted from the reconstructed Stokes vector based SH images in a pixel-by-pixel manner. The measurements are further extended by varying the polarization states of the incident light and recording the resulting Stokes parameters of the SH signal. In turn this allows the molecular structure and orientation of the samples to be determined. Use of Stokes polarimetry is critical in determination of the full polarization state of light, and enables discrimination of material properties not possible with conventional crossed-polarized detection schemes. The combination of SHG microscopy and Stokes polarimeter hence makes a powerful tool to investigate the structural order of targeted specimens.