Single-molecule Detection Experiments Research Articles

Rapid, single-molecule assays in nano/micro-fluidic chips with arrays of closely spaced parallel channels fabricated by femtosecond laser machining.

Cost-effective pharmaceutical drug discovery depends on increasing assay throughput while reducing reagent needs. To this end, we are developing an ultrasensitive, fluorescence-based platform that incorporates a nano/micro-fluidic chip with an array of closely spaced channels for parallelized optical readout of single-molecule assays. Here we describe the use of direct femtosecond laser machining to fabricate several hundred closely spaced channels on the surfaces of fused silica substrates. The channels are sealed by bonding to a microscope cover slip spin-coated with a thin film of poly(dimethylsiloxane). Single-molecule detection experiments are conducted using a custom-built, wide-field microscope. The array of channels is epi-illuminated by a line-generating red diode laser, resulting in a line focus just a few microns thick across a 500 micron field of view. A dilute aqueous solution of fluorescently labeled biomolecules is loaded into the device and fluorescence is detected with an electron-multiplying CCD camera, allowing acquisition rates up to 7 kHz for each microchannel. Matched digital filtering based on experimental parameters is used to perform an initial, rapid assessment of detected fluorescence. More detailed analysis is obtained through fluorescence correlation spectroscopy. Simulated fluorescence data is shown to agree well with experimental values.

Near-field driving of a optical monopole antenna

Nanosized optical antennas have the potential to confine and enhance optical electromagnetic fields, making nano-antennas essential tools for applications in integrated nano-optical devices and high-resolution microscopy. The size, shape and material of the nano-antenna, together with the optical frequency, determine the antenna response and its resonances. Here, we discuss a λ/4 long optical nano-antenna, analogous to the radio frequency monopole antenna. The antenna is fabricated at the end of a near-field aperture-type fibre probe by focused-ion-beam milling in two sequential steps. Illumination through the fibre creates a localized evanescent excitation source, with the advantage of a lower background compared to 'apertureless' techniques, which require far-field excitation. Previously, we have studied the field localization, antenna excitation conditions and antenna resonances, both in experiment, by near-field single-molecule detection experiments, and in theory, by finite integration technique simulations. In this study we investigate the importance of both polarization conditions and antenna position in creating an efficient local driving field for the monopole antenna. It is shown that the antenna is driven by the field component along the antenna axis. Next we show the advantage of the antenna over the aperture: upon reduction of the diameter the antenna gains local field intensity, while the aperture field decreases rapidly. Finally, the highly localized field near the antenna apex is probed by single molecules and detected molecular emission features below 30 nm FWHM are presented.

Spontaneous Emission Control with Planar Dielectric Structures: An Asset for Ultrasensitive Fluorescence Analysis

We report on the interest of spontaneous emission control with planar dielectric multi-layer structures to increase the fluorescence collection efficiency in single-molecule detection experiments and ultrasensitive analysis. The effect of a very simple Fabry Perot microcavity on the radiation pattern of Eu3+ chelate molecules is introduced and discussed. Then the fluorescence correlation spectroscopy (FCS) technique is used to detect Cyanine 5 molecules on a simple mirror and Rhodamine 6G (R6G) molecules in a microcavity, both in liquid solutions. The respective advantages of those dielectric structures for increasing the count rate per molecule are described. The detection enhancement is used to demonstrate photon antibunching on the nanosecond time scale with a dilute solution of R6G molecules (10-9 M) in the microcavity.

Imaging Single Lipid Molecules in Living Cells Using Two Photon Excitation

A Two Photon Laser Scanning Microscope was used for detecting single Cy3-labeled lipids on the plasma membrane of living human coronary artery smooth muscle cells. For Two-Photon-Excitation of Cy3 the fs-laser system was tuned to 700nm [1]. Figure A shows a fluorescence image of blank HASM cells taken with a mean power of 6mW at the sample and a dwell time of 50ms per pixel. The spots around the nucleus originate from autofluorescence of mitochondria. After taking this blank image the cells were incubated with a vesicle solution of POPC/DMPE-Cy3 at a molar ratio of 103.Lipid exchange between the vesicles and the cells resultedin a concentration of Cy3-labeled lipids of ∼1/50 μm2 on the cell membrane [2]. The vesicles were removed out of the measurement chamber by flushing with PBS buffer. For single molecule detection experiments a scanning region with almost no autofluorescence was chosen as indicated in Figure A. A sequence of images as shown Figure B was taken with a dwell time of 50ms per pixel. The fluorescence peaks represent single Cy3-labeled lipids diffusing on the cell membrane. Imaging the diffusion of single fluorescence labeled lipids by TPE has been accomplished for an artificial lipid membrane [3]. Here we show that single molecule detection with TPE is also possible on cells in vivo. This opens the perspective to utilize the advantages of Two Photon Excitation, e.g. the reduced background, increased lateral and axial resolution and the possibility of synchronous multicolor experiments, for single molecule imaging in living biological cells.

Manipulating the biochemical nanoenvironment around single molecules contained within vesicles

A method to study single-molecule reactions confined in a biomimetic container is described. The technique combines rapid vesicle preparation, optical trapping and fluorescence confocal microscopy for performing simultaneous single-vesicle trapping and single-molecule detection experiments. The collisional environment between a single enzyme and substrate inside a vesicle is characterized by a Brownian dynamics Monte Carlo simulation.

Optical collection efficiency function in single-molecule detection experiments

The optical collection efficiency function for an optical system such as that used in single-molecule detection experiments is studied. Closed analytical expressions based on a geometrical optics approximation are presented. Comparison is made with exact wave optics calculations.

Statistics of Single-Molecule Detection

A new mathematical approach for the calculation of the photon detection statistics in single-molecule detection experiments is presented. The approach is based on a path integral representation of the involved probability distributions. A numerical Monte Carlo sampling method for computing the involved path integrals is given. The general ideas are applied to the modeling of measured burst size distributions in single-molecule detection experiments with B-phycoerythrin.

Comparison of One-Focus and Two-Foci Setup in Single-Molecule Detection Experiments

Single molecule detection in fluid flow has become a widely used technique in ultrasensitive fluorescence detection. The detection of a single molecule in a fluid flow at room temperature is limited by effects such as background signals from the surrounding liquid and possible contamination in it, photobleaching effects which restrict the overall number of detectable photons from one molecule, and diffusion can cause the molecule to miss the exciting laser beam when flowing along the capillary. A method for enhancing the single molecule detection efficiency is the use of multiple excitation-detection regions along the capillary and the application of an analysis for correlating the photon detection data from the different regions. In this note we demonstrate that, at a constant laser power, no improvement results using time-correlated detection from multiple-foci regions. (AIP) {copyright} {ital 1997} {ital Society for Applied Spectroscopy}