Steps towards spatially resolved single molecule detection in solution

Abstract

Spatially resolved single molecule detection in solution is a prerequisite for molecule tracking and provides new opportunities in single molecule manipulation, for example in a sample flow. A detector with fast timing properties and high spatial resolution is required for these purposes. We introduce a concept for spatially resolved optical single molecule detection in an epi‐illuminated microscope using a novel kind of detector and a new algorithm in configurable hardware for intelligent data processing. The analysis is performed on a parallel hardware interface and includes burst detection. It can be extended to on‐line spatial and temporal data analysis. This detector will be used in combination with a molecular sorter where molecules are sorted in a microstructured flow device made of silicon. In a first step towards such sorting in these microstructures, we show that a reliable detection of single molecules in silicon microstructures is possible in zero‐dimensional detection volumes. The noise structure of the data is analysed in terms of Poissonian statistics and it is shown that in the smallest structure used (depth 20 μm) a signal‐to‐noise ratio of 40 is achieved.

Detection of Flowing Fluorescent Particles in a Microcapillary Using Fluorescence Correlation Spectroscopy

“…The groups of Eigen and Rigler have pioneered [1][2][3][4] high-throughput screening based on sensitive fluorescence detection methods, and an increasing number of applications are currently under development in the pharmaceutical industry. [5][6][7][8][9][10] Several groups reported detection of single molecules in microstructures using confocal detection schemes [11][12][13][14][15][16][17][18] or recently in submicrometer structures. 19 Here, a feasibility study is described to detect flowing, single fluorescent particles and fluorescent microorganisms in a microfluidic setup.…”

Focus

“…At a similar time Harald Mathis and colleagues at the Institut foer Molekulare Biotechnologie, Jena described SMD of Rhodamine 6G in simple silicon/glass microstructures. 19 Although, the authors demonstrated bursts with good signal-tonoise ratio, scattering from the silicon substrate generated extremely high background levels.…”

Single Molecule Fluorescence Spectroscopy at Ambient Temperature

“…Confocal microscopy was used to detect single molecules inside of micromachined channels on silicon. 157 Bursts were detected with good signal-to-noise but the background from the microchannel was large. Again, the dimensions of the detection volume were much smaller than the microchannel dimensions and the detection efficiency was small.…”

“…Confocal microscopy was used to detect single molecules inside of micromachined channels on silicon . Bursts were detected with good signal-to-noise but the background from the microchannel was large.…”