A major challenge in performing quantitative biological studies using Raman spectroscopy lies in overcoming the influence of the dominant sample fluorescence background. Moreover, the prediction accuracy of a calibration model can be severely compromised by the quenching of the endogenous fluorophores due to the introduction of spurious correlations between analyte concentrations and fluorescence levels. Apparently, functional models can be obtained from such correlated samples, which cannot be used successfully for prospective prediction. This work investigates the deleterious effects of photobleaching on prediction accuracy of implicit calibration algorithms, particularly for transcutaneous glucose detection using Raman spectroscopy. Using numerical simulations and experiments on physical tissue models, we show that the prospective prediction error can be substantially larger when the calibration model is developed on a photobleaching correlated dataset compared to an uncorrelated one. Furthermore, we demonstrate that the application of shifted subtracted Raman spectroscopy (SSRS) reduces the prediction errors obtained with photobleaching correlated calibration datasets compared to those obtained with uncorrelated ones.
One of the most promising ways to study the biochemistry of single floating cells is to combine the techniques of
optical tweezers and Raman spectroscopy (OTRS). This can reveal the information that is lost when ensemble
averages are made over cell populations, like in biochemical assays. However, the interpretation of the acquired
data is often ambiguous. Indeed, the trapped living cell continues to move and rotate in the optical trap not only
because of the Brownian motion, but also because of its inherent biological motility and the variation of its shape
and size. This affects both Rayleigh and Raman light scattering. We propose the use of Rayleigh scattering to
monitor the growth of a single optically trapped yeast cell, while OTRS measurements are being performed. For
this purpose, we added a quadrant photodiode to our OTRS setup. The cell orientation in the optical trap is
shown to vary as the cell growth proceeds, especially when it becomes asymmetrical (budding) or it changes its
size or shape considerably (living and growing cell). Control experiments, performed using heat-treated cells and
polystyrene beads, confirm that this behavior is a consequence of the cell growth. These measurements have to
be taken into account in the interpretation of Raman spectra so as not to incorrectly attribute variations in the
spectra to change in the biochemical constituents of the cell if they are in fact due to a change of the orientation
of the cell in the trap.
Living cells show a variety of morphological traits upon which numerous identification techniques have already
been developed. However most of them involve lengthy biochemical procedures and can compromise the viability
of the cell. We demonstrate a method to differentiate cells only on the basis of its trapping dynamics while it is
being drawn into an optical trap (Optical Trapping Dynamics). Since it relies only on the inherent properties of
the optical trap, without requiring external markers or biochemically sensitive spectroscopic techniques, it can be
readily combined with existing optical tweezers setups. We applied it to the study of the yeast cell-cycle stages,
showing, in particular, how it can be amenable for the measurement of the budding index of a cell population.
Living cells initiate a stress response in order to survive environmentally stressful conditions. We monitored changes in the Raman spectra of an optically trapped Saccharomyces cerevisiae yeast cell under normal and hyperosmotic stress conditions. When the yeast cells were challenged with a high concentration of glucose so as to exert hyperosmotic stress, it was shown that two chemical substances - glycerol and ethanol - could be monitored in real time in a single cell. The volume of the detection area of our confocal microspectrometer is approximately 1 fL. The average quantities of detected glycerol and ethanol are about 300 attomol and 700 attomol respectively. This amounts to the detection of approximately 108 glycerol molecules and 4 X 108 ethanol molecules after 36 min of hyper osmotic stress. Besides this, we also optically trapped a single yeast cell for up to three hours under normal conditions and monitored the changes in the Raman spectra during the lag phase of its growth and the G1 phase of its cell cycle. During the lag phase the cell synthesises new proteins and the observed behavior of the peaks corresponding to these proteins as well as those of RNA served as a sensitive indicator of the adaptation of the cell to its changed environment. The changes observed in the Raman spectra of a trapped yeast cell in the late G1 phase or the beginning of S phase corresponded to the growth of a bud.
Gradient radiation forces exerted by strongly focused cylindrical
vector beams of radial and azimuthal polarizations on dielectric
spheres of different radii and refractive indices were calculated.
The effect of longitudinal and transversal components of the
focused electrical field on trapping properties was studied.
Experiments on optical trapping were performed using low-mode
optical fiber excited with Laguerre-Gaussian beam as a source of
the trapping beams.