The effects of lithium on human red blood cells studied using optical spectroscopy and laser trap.

Lithium has been the treatment of choice for patients with bipolar disorder. However, lithium overdose happens more frequently since it has a very narrow therapeutic range in blood, necessitating investigation of its adverse effects on blood cells. The possible changes that lithium exposure may have on functional and morphological characteristics of human red blood cells (RBCs) have been studied ex vivo using single-cell Raman spectroscopy, optical trapping, and membrane fluorescent probe. The Raman spectroscopy was performed with excitation at 532 nm light, which also results in simultaneous photoreduction of intracellular hemoglobin (Hb). The level of photoreduction of lithium-exposed RBCs was observed to decline with lithium concentration, indicating irreversible oxygenation of intracellular Hb from lithium exposure. The lithium exposure may also have an effect on RBC membrane, which was investigated via optical stretching in a laser trap and the results suggest lower membrane fluidity for the lithium-exposed RBCs. The membrane fluidity of RBCs was further studied using the Prodan generalized polarization method and the results verify the reduction of membrane fluidity upon lithium exposure.

The effects of short term hyperglycemia on human red blood cells studied using Raman spectroscopy and optical trap.

Management of postprandial hyperglycemia is important for preventing severe complications like cardiovascular disease in diabetes patients. The associated glycemic instability in postprandial hyperglycemia may also cause disorders in circulating red blood cells (RBCs). Therefore, effects of short-term hyperglycemic stress on RBCs such as occur in the postprandial condition, have been studied here ex vivo using single-cell Raman spectroscopy and optical trapping. RBCs incubated in high glucose containing media relevant to postprandial hyperglycemia were studied for changes with respect to controls by analyzing the single-cell Raman spectra acquired with Raman optical tweezers with 532 nm excitation light. Use of 532 nm light for exciting Raman spectra also results in simultaneous photoreduction of intracellular hemoglobin (Hb). The level of photoreduction was noticed to be limited in hyperglycemia-exposed cells in comparison to the control. Since this suggests formation of permanently oxidized Hb in hyperglycemia-exposed RBCs, a fluorescence study was performed which showed elevated levels of oxidative stress in these cells. The changes in the RBC membrane, which may result due to higher levels of oxidative stress, were investigated using optical stretching experiments under the laser trap. The results indicated a loss of elasticity for the RBC membrane due to hyperglycemic exposure.

The glycolipid GM1 reshapes asymmetric biomembranes and giant vesicles by curvature generation.

The ganglioside GM1 is present in neuronal membranes at elevated concentrations with an asymmetric spatial distribution. It is known to generate curvature and can be expected to strongly influence the neuron morphology. To elucidate these effects, we prepared giant vesicles with GM1 predominantly present in one leaflet of the membrane, mimicking the asymmetric GM1 distribution in neuronal membranes. Based on pulling inward and outward tubes, we developed a technique that allowed the direct measurement of the membrane spontaneous curvature. Using vesicle electroporation and fluorescence intensity analysis, we were able to quantify the GM1 asymmetry across the membrane and to subsequently estimate the local curvature generated by the molecule in the bilayer. Molecular-dynamics simulations confirm the experimentally determined dependence of the membrane spontaneous curvature as a function of GM1 asymmetry. GM1 plays a crucial role in connection with receptor proteins. Our results on curvature generation of GM1 point to an additional important role of this ganglioside, namely in shaping neuronal membranes.

Asymmetric Ionic Conditions Generate Large Membrane Curvatures.

Biological membranes possess intrinsic asymmetry. This asymmetry is associated not only with leaflet composition in terms of membrane species but also with differences in the cytosolic and periplasmic solutions containing macromolecules and ions. There has been a long quest for understanding the effect of ions on the physical and morphological properties of membranes. Here, we elucidate the changes in the mechanical properties of membranes exposed to asymmetric buffer conditions and the associated curvature generation. As a model system, we used giant unilamellar vesicles (GUVs) with asymmetric salt and sugar solutions on the two sides of the membrane. We aspirated the GUVs into micropipettes and attached small beads to their membranes. An optical tweezer was used to exert a local force on a bead, thereby pulling out a membrane tube from the vesicle. The assay allowed us to measure the spontaneous curvature and the bending rigidity of the bilayer in the presence of different ions and sugar. At low sugar/salt (inside/out) concentrations, the membrane spontaneous curvature generated by NaCl and KCl is close to zero, but negative in the presence of LiCl. In the latter case, the membrane bulges away from the salt solution. At high sugar/salt conditions, the membranes were observed to become more flexible and the spontaneous curvature was enhanced to even more negative values, comparable to those generated by some proteins. Our findings reveal the reshaping role of alkali chlorides on biomembranes.

Effects of acute hypoxic exposure on oxygen affinity of human red blood cells.

Adaptation of red blood cells subjected to acute hypoxia, crucial for managing high altitude syndrome and pulmonary diseases, has been investigated. For this, red blood cells were exposed to the acute hypoxic condition by purging nitrogen over increasing time periods from 15 to 60 min and thereafter equilibrated with atmospheric oxygen for 10 min. Raman spectra of these red blood cells were then recorded and analyzed to look for changes in the level of oxygenation compared to unexposed cells. A decreasing oxygen affinity for the cells was observed with increasing time of exposure to the hypoxic condition. This change in oxygen affinity for the red blood cells may result from metabolic adjustment of the cells under the hypoxic condition to promote increased concentration of intracellular 2, 3-diphosphoglycerate.

Microfluidic sorting with a moving array of optical traps.

Optical sorting was demonstrated by selective trapping of a set of microspheres (having specific size or composition) from a flowing mixture and guiding these in the desired direction by a moving array of optical traps. The approach exploits the fact that whereas the fluid drag force varies linearly with particle size, the optical gradient force has a more complex dependence on the particle size and also on its optical properties. Therefore, the ratio of these two forces is unique for different types of flowing particles. Selective trapping of a particular type of particles can thus be achieved by ensuring that the ratio between fluid drag and optical gradient force on these is below unity whereas for others it exceeds unity. Thereafter, the trapped particles can be sorted using a motion of the trapping sites towards the output. Because in this method the trapping force seen by the selected fraction of particles can be suitably higher than the fluid drag force, the particles can be captured and sorted from a fast fluid flow (about 150 µm/s). Therefore, even when using a dilute particle suspension, where the colloidal trafficking issues are naturally minimized, due to high flow rate a good throughput (about 30 particles/s) can be obtained. Experiments were performed to demonstrate sorting between silica spheres of different sizes (2, 3, and 5 µm) and between 3 µm size silica and polystyrene spheres.

Optical orientation and rotation of trapped red blood cells with Laguerre-Gaussian mode.

We report the use of Laguerre-Gaussian (LG) modes for controlled orientation and rotation of optically trapped red blood cells (RBCs). For LG modes with increasing topological charge the resulting increase in size of the intensity annulas led to trapping of the cells at larger tilt angle with respect to the beam axis and thus provided additional control on the stable orientation of the cells under trap. Further, the RBCs could also be driven as micro-rotors by a transfer of orbital angular momentum from the LG trapping beam having large topological charge or by rotating the profile of LG mode having fractional topological charge.

Long-distance optical guiding of colloidal particles using holographic axilens.

We report the use of an aspheric holographic optical element (axilens) that essentially combines the properties of the long focal depth of an axicon and the high energy concentration of a conventional spherical lens for long-distance guiding of microscopic objects. With the use of the axilens, polystyrene spheres (~6 µm diameter) could be transported over a distance of ~16 mm that was ~3 times longer compared with that obtained using a spherical lens of focal length identical to the mean focal length of the axilens. Further, due to the availability of good on-axis power density, even objects having very marginally higher refractive index than the medium (differing only at third decimal place) could be guided with a guiding speed of ~5 µm/s.

Long-distance axial trapping with Laguerre-Gaussian beams.

We show that the axial spread of the focal volume of a tightly focused beam propagating through a glass-water interface is much reduced for Laguerre-Gaussian (LG) modes as compared to the TEM(00) mode. Therefore, use of the LG beam helps in achieving a significant improvement of the axial trapping range in optical tweezers. We demonstrate the use of LG modes to manipulate biological cells from the bottom layer of the medium to the top surface layer. Exposure of the cells to a higher oxygen concentration at the surface layer is used for estimation of the intramembrane oxygen diffusion rate.

Effects of mobile phone emissions on human red blood cells.

Raman spectroscopy was performed on GSM 900 and 1800 MHz mobile phone signal exposed red blood cells (RBCs). The observed changes in the Raman spectra of mobile signal exposed RBCs compared to unexposed control suggest reduced hemoglobin-oxygen affinity for the exposed cells. The possible mechanism may involve activation of the voltage gated membrane Ca2+ channels by the mobile phone emissions resulting in an increase in the levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) in cells via altered metabolic activities. Further studies carried out with fluorescent Ca2+ indicator confirmed increased intracellular Ca2+ level in the exposed cells. Since intracellular ATP level influences the shape and mechanics of RBCs, exposed cells were studied using diffraction phase microscopy and optical tweezers. Detectable changes in shape and mechanical properties were observed due to mobile signal exposure.

Simultaneous photoreduction and Raman spectroscopy of red blood cells to investigate the effects of organophosphate exposure.

Simultaneous photoreduction and Raman spectroscopy with 532 nm laser has been used to study the effects of organophosphate (chlorpyrifos [CPF]) exposure on human red blood cells (RBCs). Since in RBCs, auto-oxidation causes oxidative stress, which, in turn, is balanced by the cellular detoxicants, any possible negative effect of CPF on this balance should results in an increased level of damaged (permanently oxygenated) hemoglobin. Therefore, when 532 nm laser, at a suitable power, was applied to photoreduce the cells, only common oxygenated form of hemoglobin got photoreduced leaving the permanently oxygenated hemoglobin detectable in the Raman spectra simultaneously excited by the same laser. Using the technique effects of CPF to build up oxidative stress on RBCs could be detected at concentrations as low as 10 ppb from a comparison of relative strengths of different Raman bands. Experiments performed using simultaneously exposing the cells, along with CPF, to H2 O2 (oxidative agent) and/or 3-Aminotriazole (inhibitor of anti-oxidant catalase), suggested role of CPF to suppress the cellular anti-oxidant mechanism. Since the high level of damaged hemoglobin produced by the action of CPF (at concentrations >100 ppm) is expected to cause membrane damage, atomic force microscopy (AFM) was used to identify such damages.Upper panel: Raman spectra of normal, photoreduced CPF exposed and unexposed RBCs. Lower panel: The weak Fe-O2 Raman band for CPF exposed cells shown on the left. The AFM images of unexposed and exposed cells are shown on the right. Scale bar, 2.5 µm.

Red blood cell membrane damage by light-induced thermal gradient under optical trap.

Rapid membrane damage of optically trapped red blood cells (RBCs) was observed at trapping powers ≥280 mW. An excellent agreement between the estimated laser-induced thermal gradient across trapped cell's membrane and that typically required for membrane electropermeabilization suggests a mechanism involving temperature gradient-induced electropermeabilization of membrane. Also the rapid collapse of the trapped cell due to membrane rupture was seen to cause shock waves in the surroundings permeabilizing nearby untrapped cells. When the experiments were carried out with RBCs collected from type II diabetic patients, a noticeable change in the damage rate compared to normal RBCs was seen suggesting a novel optical diagnosis method for the disease.

Changes in hemoglobin-oxygen affinity with shape variations of red blood cells.

Shape variations of red blood cells (RBCs) are known to occur upon exposure to various drugs or under diseased conditions. The commonly observed discocytic RBCs can be transformed to echinocytic or stomatocytic shape under such conditions. Raman spectra of the three major shape variations, namely discocyte, echinocyte, and stomatocyte, of RBCs were studied while subjecting the cells to oxygenated and deoxygenated conditions. Analysis of the recorded spectra suggests an increased level of hemoglobin (Hb)-oxygen affinity for the echinocytes. Also, some level of Hb degradation could be noticed for the deoxygenated echinocytes. The effects may arise from a reduced level of intracellular adenosine triphosphate in echinocytic cells and an increased fraction of submembrane Hb.

The use of optical trap and microbeam to investigate the mechanical and transport characteristics of tunneling nanotubes in tumor spheroids.

The use of optical trap and microbeam for investigating mechanical and transport properties of inter cellular tunneling nanotubes (TnTs) in tumor spheroids has been demonstrated. TnTs in tumor spheroids have been visualized by manipulating TnT connected cells using optical tweezers. Functionality of the TnTs for transferring cytoplasmic vesicles and injected dye molecules by optoporation method has been studied. Further, the TnTs could be longitudinally stretched by manipulating the connected cells and their elastic response was studied. Manipulation of cells at the surface of tumor spheroid using optical tweezers and injection of fluorescent dye into a trapped cell using optoporation technique.

Studies on erythrocytes in malaria infected blood sample with Raman optical tweezers.

Raman spectroscopy was performed on optically trapped red blood cells (RBCs) from blood samples of healthy volunteers (h-RBCs) and from patients suffering from P. vivax infection (m-RBCs). A significant fraction of m-RBCs produced Raman spectra with altered characteristics relative to h-RBCs. The observed spectral changes suggest a reduced oxygen-affinity or right shifting of the oxygen-dissociation curve for the intracellular hemoglobin in a significant fraction of m-RBCs with respect to its normal functional state.

Optical trapping of spermatozoa using Laguerre-Gaussian laser modes.

We report results of a study on the use of Laguerre-Gaussian (LG) modes for optical trapping of spermatozoa. The results show that for a given trap beam power the first-order LG mode (LG(01)) leads to lower photodamage to the cells without compromising the trapping efficiency.

Hemoglobin degradation in human erythrocytes with long-duration near-infrared laser exposure in Raman optical tweezers.

Near-infrared laser (785-nm)-excited Raman spectra from a red blood cell, optically trapped using the same laser beam, show significant changes as a function of trapping duration even at trapping power level of a few milliwatts. These changes in the Raman spectra and the bright-field images of the trapped cell, which show a gradual accumulation of the cell mass at the trap focus, suggest photoinduced aggregation of intracellular heme. The possible role of photoinduced protein denaturation and hemichrome formation in the observed aggregation of heme is discussed.

Rotation of transparent, nonbirefringent objects by transfer of the spin angular momentum of light.

The transfer of the spin angular momentum of light to small objects has been discussed only for absorbing or birefringent objects. However, we show that it can also be transferred to objects that are nonabsorbing and nonbirefringent. Our estimate of the magnitude of the spin angular momentum transferred to a transparent, nonbirefringent sodium bromate crystal predicts rotational speeds in reasonable agreement with experimental results.

Controlled rotation of biological microscopic objects using optical line tweezers.

Controlled, continuous rotation of cells or intracellular objects was achieved using optical tweezers with an elliptic beam profile (line tweezers), which was generated by placing a cylindrical lens in the path of the trapping beam. By rotating the cylindrical lens, rotation of the elliptic trapping beam and hence of the object trapped therein was achieved. Compared to previously reported techniques for rotation of microscopic objects, this approach is much simpler, gives better utilization of available laser power and also allows much easier control of the trap beam profile. We have used this approach for rotation of biological objects varying in size from 2 to 40 microm. At 25 mW trapping beam power at the object plane E. coli bacteria could be rotated at speeds approaching 10 Hz and an intracellular object (presumably a calcium oxalate crystal) trapped inside Elodea densa plant cell could be rotated with speeds of up to 4 Hz. To our knowledge, this is the first report for rotation of an intracellular object.