Viscoelastic and spectroscopic properties of single RBC are probed using dual beam optical tweezers and Raman
techniques, respectively. Complex response function of cell was measured by means of one and two particles
passive microrheology at different stretching states yielding local and overall mechanical properties of exactly
the same human erythrocyte. The frequency dependent response function (measured up to 10 kHz) was corrected
for the presence of the traps and spectral distribution of complex stiffness over controlled range of cell deformation
is calculated and discussed. The presence of non-thermal sources of membrane motions is also explored based
on comparison of passive and active microrheology experiments. In order to get insight into structural changes
of RBC due to deformation, Raman spectra of single cell were recorded. Evolution of Raman bands with
cell deformation was analyzed using sensitive 2D correlation method. The combination of force and Raman
spectroscopy is promising and potentially very powerful method to establish essential linkages between structure,
mechanical properties and functions of living cells.
Living cells and single molecules as DNA experiences numerous mechanical events, necessitating single molecule
force spectroscopy techniques to provide insight into cellular mechanics as a whole system. This paper shows
results on Raman spectroscopy of a single red blood cell which is gradually stretched using optically trapped
beads attached to the cell. The applied force is intended to simulate step-by-step deformation experienced by cells
in normal conditions (induced by blood flow) as they squeeze through microvasculature. To further improve the
sensitivity of the experiments and facilitate their interpretation, 2D correlation and principal component analysis
techniques were applied. The purpose of this work is to help unravel direct relationship between mechanical
deformation of RBC and chemical changes occurring in the cell structure on molecular level. We also obtained
Raman spectra from single DNA molecules in their natural aqueous environment as a first step to establish a
direct relationship between DNA's extension and structure in the low force, entropic regime.
It has been described that benign MCF10A breast cancer cell line suffers phenotypic changes toward malignancy when
are cultured in sparse conditions. Using Raman spectroscopy with an InVia Raman microscope (Renishaw) with a
backscattered configuration, we have studied the metabolic changes of confluent and sparse MCF10A cell cultures. We
used Principal Component Analysis and Partial Least Squares Discriminant Analyses to assess the different profiling of
the metabolic composition of breast cancer cells. The results indicated that Raman spectroscopy together with
multivariate analysis is a useful technique to distinguish metabolic changes in malignant transformation. The
identification of new metabolites, implementing the catalogue on the characterization of the different phenotypes
associated to cell malignancy using Raman spectroscopy is under study.
Retinal nervous tissue sustains a substantial damage during the autoimmune inflammatory processes characteristic
for Multiple Sclerosis (MS). The damage can be characterized non-surgically by Raman Spectroscopy,
a non-invasive optical imaging technology. We used non-resonant near-infrared Raman spectrosocopy to create
a spectral library of eight pivotal biomolecules known to be involved in neuroinflammation: Nicotinamide
Adenine Dinucliotide (NADH), Flavin Adenine Nucleotide (FAD), Lactate, Cytochrome C, Glutamate, N-Acetyl-
Aspartate (NAA), Phosphotidylcholine, with Advanced Glycolization End Products (AGEs) analyzed as a reference.
Principal Component Analysis (PCA) of 50 spectra taken of murine retinal tissue culture undergoing
an inflammatory response and healthy controls was used in order to characterize the molecular makeup of the
inflammation. The loading plots revealed a heavy influence of peaks related to Glutamate, NADH, and Phosphotidylcholine
to inflammation-related spectral changes. Partial Least Squares - Discriminant analysis (PLS-DA)
was performed to create a multivariate classifier for the spectral diagnosis of neuroinflammed tissue and yielded
a diagnostic sensitivity of 100% and specificity of 100%. We demonstrate then the effectiveness of combining Raman
spectroscopy with PCA and PLS-DA statistical techniques to detect and monitor neuroinflamation in retina.
With this technique Glutamate, NAA and NADH are detected in retina tissue as signs for neuroinflammation.
An understanding of the mechanisms of drug diffusion and uptake through cellular membranes is critical for elucidating drug action and in the development of effective drug delivery systems. We study these processes for emodin, a potential anticancer drug, in live cancer cells using surface-enhanced Raman scattering. Micrometer-sized silica beads covered by nanosized silver colloids are passively embedded into the cell and used as sensors of the drug. We demonstrate that the technique offers distinct advantages: the possibility to study the kinetics of drug diffusion through the cellular membrane toward specific cell organelles, the detection of lower drug concentrations compared to fluorescence techniques, and less damage imparted on the cell.
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