Aseefhali Bankapur

Raman Tweezers Spectroscopy of Live, Single Red and White Blood Cells

An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC) and white blood cells (WBC) under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm) is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW). Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip.

Probing oxidative stress in single erythrocytes with Raman Tweezers

Raman Tweezers have been successfully applied to characterize chemically-induced oxidative stress on optically-trapped live, single erythrocytes. There is significant enhancement in Raman peak intensities corresponding to SS and C-S stretching modes that are induced by oxidative stress. This is consistent with the formation of mixed disulphides between protein SH groups and low-molecular-mass thiols such as glutathione during oxidative damage to cells. Enhancement in glutathione level as a protective response against oxidative stress has been observed. Principal component analysis of the data yields good discrimination between spectra of normal and stress-induced red blood cells.

Micro-Raman Spectroscopy of Silver Nanoparticle Induced Stress on Optically-Trapped Stem Cells

We report here results of a single-cell Raman spectroscopy study of stress effects induced by silver nanoparticles in human mesenchymal stem cells (hMSCs). A high-sensitivity, high-resolution Raman Tweezers set-up has been used to monitor nanoparticle-induced biochemical changes in optically-trapped single cells. Our micro-Raman spectroscopic study reveals that hMSCs treated with silver nanoparticles undergo oxidative stress at doping levels in excess of 2 µg/ml, with results of a statistical analysis of Raman spectra suggesting that the induced stress becomes more dominant at nanoparticle concentration levels above 3 µg/ml.

A Micro-Raman Study of Live, Single Red Blood Cells (RBCs) Treated with AgNO3 Nanoparticles

Silver nanoparticles (Ag NPs) are known to exhibit broad antimicrobial activity. However, such activity continues to raise concerns in the context of the interaction of such NPs with biomolecules. In a physiological environment NPs interact with individual biological cells either by penetrating through the cell membrane or by adhering to the membrane. We have explored the interaction of Ag NPs with single optically-trapped, live erythrocytes (red blood cells, RBCs) using Raman Tweezers spectroscopy. Our experiments reveal that Ag NPs induce modifications within an RBC that appear to be irreversible. In particular we are able to identify that the heme conformation in an RBC transforms from the usual R-state (oxy-state) to the T-state (deoxy-state). We rationalize our observations by proposing a model for the nanoparticle cytotoxicity pathway when the NP size is larger than the membrane pore size. We propose that the interaction of Ag NPs with the cell surface induces damage brought about by alteration of intracellular pH caused by the blockage of the cell membrane transport.

Probing differentiation in cancer cell lines by single-cell micro-Raman spectroscopy

Abstract. Single-cell micro-Raman spectroscopy has been applied to explore cell differentiation in single, live, and malignant cells from two tumor cell lines. The spectra of differentiated cells exhibit substantial enhancement primarily in the intensities of protein peaks with concomitant decrease in intensities of O─P─O asymmetric stretching peaks in DNA/RNA. Principal component analyses show that the spectral score of differentiated cells tends to asymptotically approach that of spectra obtained from normal neural stem cells/progenitors. This lends credence to the notion that the observed spectral changes are specific to differentiation, since upon differentiation, malignant cells become less malignant and tend toward benignity.

A hybrid LIBS–Raman system combined with chemometrics: an efficient tool for plastic identification and sorting

AbstractClassification of plastics is of great importance in the recycling industry as the littering of plastic wastes increases day by day as a result of its extensive use. In this paper, we demonstrate the efficacy of a combined laser-induced breakdown spectroscopy (LIBS)–Raman system for the rapid identification and classification of post-consumer plastics. The atomic information and molecular information of polyethylene terephthalate, polyethylene, polypropylene, and polystyrene were studied using plasma emission spectra and scattered signal obtained in the LIBS and Raman technique, respectively. The collected spectral features of the samples were analyzed using statistical tools (principal component analysis, Mahalanobis distance) to categorize the plastics. The analyses of the data clearly show that elemental information and molecular information obtained from these techniques are efficient for classification of plastics. In addition, the molecular information collected via Raman spectroscopy exhibits clearly distinct features for the transparent plastics (100% discrimination), whereas the LIBS technique shows better spectral feature differences for the colored samples. The study shows that the information obtained from these complementary techniques allows the complete classification of the plastic samples, irrespective of the color or additives. This work further throws some light on the fact that the potential limitations of any of these techniques for sample identification can be overcome by the complementarity of these two techniques. Graphical Abstractᅟ

Effect of infrared light on live blood cells: Role of β-carotene

We have utilized Raman tweezers to measure and assign micro-Raman spectra of optically trapped, live red blood cells (RBCs), white blood cells (WBCs) and platelets. Various types of WBCs- both granulocytes, lymphocytes, and their different types have been studied. The Raman bands are assigned to different biomolecules of blood cells. The Raman spectra thus obtained has been enabled detection of β-carotene in these blood cells, the spectral features of which act as a signature that facilitates experimental probing of the effect of 785nm laser light on different blood cells as a function of incident laser power in the mW range. The spectral changes that we obtain upon laser irradiation indicate that, both haemoglobin as well as the cell membrane sustains damage. In case of lymphocytes and platelets the peaks corresponding to β-carotene showed drastic changes. Thorough analysis of the spectral changes indicates possibility of free radical induced damage of β-carotene in lymphocytes and platelets. Among different blood cells, RBCs have a power threshold of only 10mW. The power threshold for other types of blood cells is somewhat higher, but always below about 30mW. These values are likely to serve as useful guides for Raman tweezers based experiments on live cells.

Effect of biocompatible nucleants in rapid crystallization of natural amino acids using a CW Nd:YAG laser

Laser-induced crystallization is emerging as an alternative technique to crystallize biomolecules. However, its applications are limited to specific small molecules and some simple proteins, possibly because of the need to use high-intensity, pulsed lasers and relatively long laser irradiation time. Both these factors tend to denature biological molecules. If the laser-intensity and time required to crystallize biomolecules were to be reduced, laser-induced crystallization may well become of widespread utility. We report here the crystallization of nineteen natural amino acids by a laser-induced method in combination with one of three nucleants: aluminum, coconut coir, and peacock feather barbule. We have utilized a low-power, continuous wave (CW) Nd:YAG laser (λ = 1064 nm). The advantages of our method are (i) the use of very small laser powers (60 mW), and (ii) the ability to obtain diffraction quality crystals within a mere few seconds. For most amino acids our method yields several orders of magnitude reduction in crystallization time. The use of biocompatible nucleants like coir fibres and peacock feather barbules are novel; their non-toxic nature may find broad applicability in rapid crystallization of diverse biological molecules.

Micro-Raman spectroscopy for identification and classification of UTI bacteria

Urinary tract infection (UTI) is one of the major clinical problems known to mankind, especially among adult women. Conventional methods for identification of UTI causing bacteria are time consuming and expensive. Therefore, a rapid and cost-effective method is desired. In the present study, five bacteria (one Gram-positive and four Gram-negative), most commonly known to cause UTI, have been identified and classified using micro-Raman spectroscopy combined with principal component analysis (PCA).

Laser-assisted crystallization: an alternative tool to crystallize biomolecules

Knowledge of 3D structures is a prerequisite for developing structure-based drug design and discovery of novel drugs and pharmaceutical products. The human genome contains protein-coding genes in between 20,000 to 25,000 and many proteins playing a key role in the living mechanisms have not been crystallized so far. Developing high throughput, automatic and rapid protein crystallization method is critical, and laser-induced crystallization is one of the promising alternatives in this regard. We have used an ultrafast laser (800 nm wavelength, 60 fs pulse duration, 5.2 MHz repetition rate, 300 mW average power) to crystallize NaCl, five chalcone compounds, and a lysozyme protein [1]. Recently, we conducted a systematic study in which we successfully crystallized biomolecules using a continuous wave Nd:YAG (wavelength = 1064 nm) laser using low cost and readily available absorbing nucleants like copper wire, aluminum wire, copper particles, aluminum particles, graphite particles, as well as multi-walled carbon nanotubes (MCWNT). We illustrate the potential of our approach by first crystallizing standard small molecules NaCl and KCl – and glycine in solution form under varying conditions of laser power and irradiation time. Optimized values of these parameters are then utilized to crystallize 20 amino acids and lysozyme protein. The prepared crystals were further characterized using single crystal X-ray diffraction and Raman spectroscopy. The current study elucidates that the femtosecond laser based crystallization offers advantages such as use of very low laser powers (~4 mW/cm^2), extremely rapid crystallization (~3 seconds), use of biomolecules in low concentration (0.5 M), etc. Though high-throughput technologies have evolved for crystallization of macromolecules, such crystallization still requires time periods ranging from several hours to weeks to confirm whether or not nucleation has occurred. Our technique might offer new avenues to understand the nucleation mechanism and to have better control over the crystallization process. This kind of approach may facilitate the development of protocols that enable preparation of diffraction quality crystals of small molecules, and of seeding crystals for peptides and proteins on significantly shorter timescales than hitherto possible. In our laboratory, we are exploring this technique to obtain the crystals of protein molecules. [1] T. Shilpa et al. [2015], Proc. Indian Natl. Sci. Acad., 81, 517-523.