The electronic properties of single human red blood cells under mechanical deformations were investigated using a combination of dual beam optical tweezers and UV-vis absorption spectroscopy. The mechanical deformations were induced by two near-infrared optical traps with different trapping powers and trap configurations. The deformations were applied in two ways: locally, due to the mechanical forces around the traps, and by stretching the cell by moving the traps in opposite directions. In the presence of local deformations, the single cell undergoes a transition from an oxygenated state to a partially deoxygenated state. This process was found to be reversible and strongly power-dependent. Stretching the cell caused an opposite effect, indicating that the electronic response of the whole cell is dominated by the local interaction with the trapping beams. Results are discussed considering light-induced local heating, the Stark effect, and biochemical alterations due to mechanical forces, and are compared with reports of previous Raman spectroscopy studies. The information gained by the analysis of a single red blood cell's electronic response facilitates the understanding of fundamental physiological processes and sheds further light on the cell's mechanochemistry. This information may offer new opportunities for the diagnosis and treatment of blood diseases.
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.
We studied fluctuations of an optically trapped bead connected to a single DNA molecule anchored between the
bead and a cover glass or between two optically trapped beads. Power spectral densities of the bead position for
different extensions of the molecule were compared with the power spectral density of the position fluctuations of
the same bead without the molecule attached. Experiments showed that the fluctuations of the DNA molecule
extended up to 80% by a force of 3 pN include the colored noise contribution with spectral dependence 1/fα
with α~ 0.75.
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.
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.
We show that Surface Enhanced Raman spectroscopy (SERS) combined with 2D correlation and multivariate analysis
provides considerable progress in using Raman microspectroscopy for cutting edge biomedical research applications
such as treatment delivering in cancer living cells, the diagnosis of retina neuroinflamed tissue and the study of elastic
properties of single DNA molecules.
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.
We subject micrometer-sized, optically trapped colloidal particles in a non-polar liquid to a sinusoidally varying electric
field, and measure their resulting movement. From this movement, we calculate the electrophoretic mobility and charge
of the particle in the liquid. The use of high frequencies of the electric field (well above the corner frequency of the
optical tweezers) allows us to estimate the electrical charge of colloidal particles with an accuracy of the order of the
electron charge in a time interval of only 10 ms. This technique can be used to provide valuable information about the
dynamics of the poorly understood processes that lead to the charge on colloidal particles in non-polar liquids.
Optical tweezers technique combined with local confocal luminescence spectroscopy is suggested as a tool for
investigation of local optical fields. Utilizing this method plasmon-enhanced optical fields inside a pair of dielectric
2 μm spheres partially covered by 70 nm silver nanoparticles are visualized via field enhanced luminescence of
rhodamine dye solution. Positions of the particles are controlled with submicrometer accuracy by two optical
traps formed by strongly focused laser beams with λ=980nm. A supplementary beam from CW laser with
λ=532nm provided for luminescence excitation is also focused into the sample cavity just to the trapping area.
In order to obtain spatial filtering of the signal and separate luminescence signal from an area near the spheres
pin-hole based confocal system is designed. The focal volume available for luminescence signal collection turns
out to be approximately 3μm x 3 μm x 5 μm. Since optical field is enhanced in the region near plasmon-active 2
μm spheres the enhancement of luminescence intensity is observed. Collective plasmonic effects in two-particle
measurements are also considered.
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.
The combination of Raman spectroscopy and Optical Tweezers has been used to trap living cells and collect information
about their biochemical state. Cells can continue living in such traps for periods of hours, allowing acquisition of time
resolved Raman spectra. However no spatial information can be acquired as the cells continue to rotate and move in the
single beam trap.
Here we describe the development of Holographic Optical Tweezers (HOT) for the controlled movement of floating cells
in order to construct their Raman images. Instead of a single trap, rapidly programmable multiple trapping points can be
produced around the periphery of the cells to impede the rotational motion of the cell. By trapping and scanning the cell
using HOT relative to a fixed Raman exciting laser, a point by point image of the cell can be constructed. We use an
interactive program that permits us to position the trapping points relative to the live image feed we see from the
microscope, using point and click. To demonstrate the possibilities of this technique images are shown of floating Jurkat
cells.
Following the recent advances in nano-optics, optical manipulation by evanescent fields instead of conventional propagating fields has recently awaken an increasing interest. The main advantages of using low dimensionality fields are (i) the possibility of integrating on a chip applications involving optical forces but also (ii) the absence of limitation by the diffraction limit for the trapping volume. Previous works have investigated theoretically and experimentally the guiding of dielectric and metallic beads at an interface sustaining an extended surface wave. In this work, we study theoretically the radiation forces exerted on Rayleigh dielectric beads under local evanescent illumination. Especially, we consider the configuration where a three-dimensional Gaussian beam is totally reflected at the interface of a glass prism. The results point out the illumination parameters where the gradient forces exceed the scattering force and allow for a stable trapping. The effect of the Goos-Haenchen shift on the location of the trapping site is also discussed.
We report quantitative measurements of the radiation forces exerted on a micrometer dielectric sphere by a Surface Plasmon Polariton (SPP) excited at a gold/water interface. We separate the contributions of the two constituents of the plasmon wave - the electromagnetic field and the charge-density oscillations - to the total radiation force. Measurements performed with a Photonic Force Microscope (PFM) show an enhanced attraction to the surface compared to a conventional evanescent wave at the dielectric interface (102 enhancement factor).
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.
The trapping of micro-objects by optical radiation forces, so-called optical tweezers, has become widely used in physical, chemical and biological experiments where accurate and non-invasive manipulation is required. Recent advances in beam shaping render it possible for instance to rotate or to dynamically manipulate independently several elements. Today, one of the remaining challenges of conventional optical tweezers is the direct manipulation of systems with sizes belonging to the sub-wavelength or Rayleigh regime. Indeed, the diffraction limit prevents in that case from achieving a commensurable trapping volume and thus does not allow for minimizing the fluctuations in position of the trapped object due to its strong Brownian motion. In order to overcome this limitation, it has been proposed to use evanescent fields instead of the usual propagating fields. Recent advances in optics of noble metal nano-structures have recently provided new configurations to achieve nano-optical tweezers. Especially, tightly localized modes resulting from the coupling between resonant noble metal nanostructures may offer the gradient forces able to trap and manipulate Rayleigh objects. In this work, we calculate the radiation forces exerted on a nanometric dielectric sphere when exposed to a patterned optical near-field landscape at an interface decorated with resonant gold nanostructures. By comparing their magnitude with other forces that affect the movement of the particle, we discuss the practical ability of our configuration for multiple parallel optical manipulation.
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.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.