Search

We report on the study of single biological cells with a confocal micro-Raman spectroscopy system that uses optical trapping and shifted excitation Raman difference technique. A tunable diode laser was used to capture a living cell in solution, confine it in the confocal excitation volume, and then excite the Raman scattering. The optical trapping allows us to lift the cell well off the cover plate so that the fluorescence interference from the plate can be effectively reduced. In order to further remove the interference of the fluorescence and stray light from the trapped cell, we employed a shifted excitation Raman difference technique with slightly tuned laser frequencies. With this system, high-quality Raman spectra were obtained from single optically trapped biological cells including E. coli bacteria, yeast cells, and red blood cells. A significant difference between control and heat-treated E. coli B cells was observed due to the denaturation of biomolecules.

We measured the Raman spectra of single optically trapped highly refractive and nontransparent microscopic particles suspended in a liquid using an inverted confocal laser-tweezers-Raman-spectroscopy system. A low-power diode-laser beam of TEM00 mode was used both for optical trapping and Raman excitation of refractive, absorptive, and reflective metal particles. To form a stable trap for a nontransparent particle, the beam focus was located near the top of the particle and the particle was pushed against a glass plate by the axial repulsive force. Raman spectra from single micron-sized crystals with high index of refraction including silicon, germanium, and KNbO3, and from absorptive particles of black and color paints were recorded. Surface-enhanced Raman scattering of R6G and phenylalanine molecules absorbed on the surface of a trapped cluster of silver particles was also demonstrated.

In this study we report on the dynamic motion of a nano-sized colloidal particle captured in a polarized optical trap. A polystyrene sphere (300nm-diameter) that is electrically charged in solution was trapped with an optical tweezers formed by a linearly polarized TEM00 Gaussian beam, while the Brownian displacements of the trapped particle in x and y directions were measured so that the position of the particle's mass center can be mapped on the transverse plane and the corss-correlation between x and y displacements can be calculated. We found that the position's fluctuation of the trapped nano-sized particle in the parallel direction to the laser polarization is significantly larger than that in the normal direction, which suggests that there exists an additional random electric force parallel to the laser polarization direction exerting on the charged particle beside the known radiation forces on the dielectric particle. This asymmetry in dynamic motion is significant when the particle size is well less than the wavelength of the trapping laser. However, in an optical trap formed by a circularly polarized beam, this asymmetry in dynamic motion was observed to disappear. We present both the experimental results and a theoretical analysis.

We report on a rapid method for reagentless identification and discrimination of single bacterial cells in aqueous solutions using a combination of laser tweezers and confocal Raman spectroscopy (LTRS). The optical trapping enables capturing of individual bacteria in aqueous solution in the focus of the laser beam and levitating the captured cell well off the cover plate, thus maximizing the excitation and collection of Raman scattering from the cell and minimizing the unwanted background from the cover plate and environment. Raman spectral patterns excited by a near-infrared laser beam provide intrinsic molecular information for reagentless analysis of the optically isolated bacterium. In our experiments, six species of bacteria were used to demonstrate the capability of the confocal LTRS in the identification and discrimination between the diverse bacterial species at various growth conditions. We show that synchronized bacterial cells can be well-discriminated among the six species using principal component analyses (PCA). Unsynchronized bacterial cells that are cultured at stationary phases can also be well-discriminated by the PCA, as well as by a hierarchical cluster analysis (HCA) of their Raman spectra. We also show that unsynchronized bacteria selected from random growth phases can be classified with the help of a generalized discriminant analysis (GDA). These findings demonstrate that the LTRS may find valuable applications in rapid sensing of microbial cells in diverse aqueous media.

We demonstrated that food-borne bacteria and microorganisms existing in aqueous environment can be rapidly discriminated from the other food particles by Raman spectroscopy and sorted to a new chamber by optical manipulation.

Direct estimation of the number of channels on a membrane path is important for channel biophysics. It is a classical question and has been addressed elegantly by previous studies. Especially, pioneering researchers took the advantage of fluctuations in membrane conductance caused by ion channels opening and closing. The number of channels, the single-channel current, and the probability of the channel opening can be obtained using the method of non-stationary or stationary fluctuation analysis. Here, we developed a method to count the number of channels by simultaneous electrical recording of channel opening and optical recording of the fluorescence from the green fluorescent protein(GFP) tagged to the channel. Based on the number of channels and the macroscopic current, we first tuned this method using the cyclic-nucleotide gated (CNG) channel, of which the single channel conductance and open probability are well characterized. Then we applied the I-F relationship to the hyperpolarization-activated, cAMP-regulated HCN channel, of which the estimation of single channel conductance has been controversial. We estimated that the number of channels on a piece of membrane patch could read 10,000 to 20,000 and the single channel conductance for mHCN2 channel is about 1.82 pico Siemens.

The authors of the work: 'Chromosomal analysis and identification based on optical tweezers and raman spectroscopy' [Opt. Express 14, 5385 (2006], claim that they have been able to identify and differentiate between three human chromosomes with an optical-tweezer - Raman Spectroscopic experimental (LTRS) set-up. The results and conclusions as they are presented in the paper are questionable, however, when the spectral data and data analysis are studied in greater detail.

High-density samples of fluorescent quantum dots (QDs) were imaged using an apertureless near-field optical microscopy technique. QD fluorescence was modulated by oscillating a silicon atomic force microscope tip above an illuminated sample and a lock-in amplifier was used to suppress background from the excitation laser. Spatial resolution near 10nm and a peak signal-to-noise ratio (SNR) of ∼60 were achieved. Individual QDs within high-density ensembles were still easily resolved (SNR>5) at a density of 14QDs∕μm2. These results have favorable implications for the eventual nanoscale imaging of viable biological systems, such as cellular membranes.

We report on a novel technique for sorting and identification of single biological cells and food-borne bacteria based on laser tweezers and Raman spectroscopy (LTRS). With this technique, biological cells of different physiological states in a sample chamber were identified by their Raman spectral signatures and then they were selectively manipulated into a clean collection chamber with optical tweezers through a microchannel. As an example, we sorted the live and dead yeast cells into the collection chamber and validated this with a standard staining technique. We also demonstrated that bacteria existing in spoiled foods could be discriminated from a variety of food particles based on their characteristic Raman spectra and then isolated with laser manipulation. This label-free LTRS sorting technique may find broad applications in microbiology and rapid examination of food-borne diseases.

We report on real-time Raman spectroscopic studies of optically trapped living cells and organelles using an inverted confocal laser-tweezers-Raman-spectroscopy (LTRS) system. The LTRS system was used to hold a single living cell in a physiological solution or to hold a functional organelle within a living cell and consequently measured its Raman spectra. We have measured the changes in Raman spectra of a trapped yeast cell as the function of the temperature of the bathing solution and studied the irreversible cell degeneration during the heat denaturation. In addition, we measured the in-vitro Raman spectra of the nuclei within living pine cells and B. sporeformer, Strep. salivarius, and E. coli bacteria suspended in solution and showed the possibility of using LTRS system as a sensor for rapid identification of microbes in a fluid.

We report on the development and testing of a compact laser tweezers Raman spectroscopy (LTRS) system. The system combines optical trapping and near-infrared Raman spectroscopy for manipulation and identification of single biological cells in solution. A low-power diode laser at 785 nm was used for both trapping and excitation for Raman spectroscopy of the suspended microscopic particles. The design of the LTRS system provides high sensitivity and permits real-time spectroscopic measurements of the biological sample. The system was calibrated by use of polystyrene microbeads and tested on living blood cells and on both living and dead yeast cells. As expected, different images and Raman spectra were observed for the different cells. The LTRS system may provide a valuable tool for the study of fundamental cellular processes and the diagnosis of cellular disorders.