Single-Molecule Detection by Laser-Induced Fluorescence Technique with a Position-Sensitive Photon-Counting Apparatus

Abstract

A new method of detecting single-molecule fluorescence has been implemented using a conventional optical microscope and a position-sensitive photon-counting apparatus. The samples were solvent-free fluorescent dyes adsorbed on a solid substrate in air at room temperature by spraying microdroplets of a dye solution followed by solvent evaporation. At the concentration used, each droplet contained a very small number of dye molecules. Quantized fluorescence intensity was determined and the number of occurrences of the quantized intensity agreed well with that calculated from Poisson's formula, thus confirming success of the position-sensitive single-molecule fluorescence detection.

Fluorescence microscopy in superfluid helium: Single molecule imaging

“…In order to reduce the background and to emphasize the signal from the single molecule only very small volumes must be probed. 16 Here, we describe two new optical systems which have improved resolution and a much higher collection efficiency. All these methods have in common that only a single small volume element is studied at a given time.…”

Alterations of Single Molecule Fluorescence Lifetimes in Near-Field Optical Microscopy

“…Note added in proof: Single molecule detection by conventional optical microscopy was recently reported (29). This method could be used as an independent technique to explore unperturbed inhomogeneous lifetime distributions.…”

Stepping rotation of F 1 -ATPase visualized through angle-resolved single-fluorophore imaging

“…Individual behaviors can be assessed by single-fluorophore imaging (1)(2)(3)(4)(5), which is much less perturbing than imaging through a huge tag such as a plastic bead or actin filament (6). Real-time determination of fluorophore orientation (5,7,8) should be particularly useful, because a conformational change necessarily accompanies reorientation of one part against others.…”