Highly Efficient Single Molecule Detection in Different Micro and submicrometer Channels with cw-excitation

Abstract

In this article we describe a detailed comparison between single molecule detection in three different types of microchannels. The overall detection efficiencies and the signal-to-noise ratios are compared. A new data evaluation technique that is used is explained in detail. The average number of photons per molecule is calculated from the measurements and the detection properties of each channel with respect to the diffusional characteristics of the dye molecules are investigated. From the detection efficiencies advantages and drawbacks of the individual methods are elucidated as well as possible applications of the techniques are illustrated.

Quantitative Counting of Single Fluorescent Molecules by Combined Electrochemical Adsorption Accumulation and Total Internal Reflection Fluorescence Microscopy

“…Recently, much attention has been focused on single-molecule detection (SMD) in solutions. Fluorescence detection techniques coupled with hydrodynamically focused flows, – microdroplets, – capillary or microchannels, – nanometer-scale pores, confocal fluorescence microscopy, ,– total internal reflection fluorescence microscopy (TIRFM), – and epifluorescence microscopy , are the most popular techniques in SMD. Of these SMD studies with fluorescence detection, only a few reports are concerned with quantitative analysis.…”

Quantitative Counting of Single Fluorescent Molecules by Combined Electrochemical Adsorption Accumulation and Total Internal Reflection Fluorescence Microscopy

“…Recently, much attention has been focused on single-molecule detection (SMD) in solutions. Fluorescence detection techniques coupled with hydrodynamically focused flows, – microdroplets, – capillary or microchannels, – nanometer-scale pores, confocal fluorescence microscopy, ,– total internal reflection fluorescence microscopy (TIRFM), – and epifluorescence microscopy , are the most popular techniques in SMD. Of these SMD studies with fluorescence detection, only a few reports are concerned with quantitative analysis.…”

Focal Volume Confinement by Submicrometer-Sized Fluidic Channels

“…The use of microfabricated channels for single-fluorophore detection with high-throughput has been demonstrated previously, and various channel or capillary geometries with dimensions of one to several micrometers diameter have been evaluated. ,− The width of the focal volume was either expanded to span the entire channel width, or only a fraction of the channel was illuminated, and thus, not all molecules flowing through the channel were detected. The upper limit in concentration to avoid simultaneous passage of two or more molecules through the observation volume was therefore similar or worse compared to diffraction-limited confocal optics.…”

Single-molecule fluorescence spectroscopy of biomolecular folding

“…Another modification of the free diffusion experiment is achieved by flowing molecules in capillaries [50], or in the channels of microfluidic devices [51]. Flowing can improve the sampling rate of single molecules in solution.…”

Quantitative Counting of Single Fluorescent Molecules by Combined Electrochemical Adsorption Accumulation and Total Internal Reflection Fluorescence Microscopy

“…Recently, much attention has been focused on single-molecule detection (SMD) in solutions. Fluorescence detection techniques coupled with hydrodynamically focused flows, – microdroplets, – capillary or microchannels, – nanometer-scale pores, confocal fluorescence microscopy, ,– total internal reflection fluorescence microscopy (TIRFM), – and epifluorescence microscopy , are the most popular techniques in SMD. Of these SMD studies with fluorescence detection, only a few reports are concerned with quantitative analysis.…”

Focal Volume Confinement by Submicrometer-Sized Fluidic Channels

“…The use of microfabricated channels for single-fluorophore detection with high-throughput has been demonstrated previously, and various channel or capillary geometries with dimensions of one to several micrometers diameter have been evaluated. ,− The width of the focal volume was either expanded to span the entire channel width, or only a fraction of the channel was illuminated, and thus, not all molecules flowing through the channel were detected. The upper limit in concentration to avoid simultaneous passage of two or more molecules through the observation volume was therefore similar or worse compared to diffraction-limited confocal optics.…”

Single-molecule fluorescence spectroscopy of biomolecular folding

“…Another modification of the free diffusion experiment is achieved by flowing molecules in capillaries [50], or in the channels of microfluidic devices [51]. Flowing can improve the sampling rate of single molecules in solution.…”

Quantitative Counting of Single Fluorescent Molecules by Combined Electrochemical Adsorption Accumulation and Total Internal Reflection Fluorescence Microscopy

“…Recently, much attention has been focused on single-molecule detection (SMD) in solutions. Fluorescence detection techniques coupled with hydrodynamically focused flows, – microdroplets, – capillary or microchannels, – nanometer-scale pores, confocal fluorescence microscopy, ,– total internal reflection fluorescence microscopy (TIRFM), – and epifluorescence microscopy , are the most popular techniques in SMD. Of these SMD studies with fluorescence detection, only a few reports are concerned with quantitative analysis.…”

Focal Volume Confinement by Submicrometer-Sized Fluidic Channels

“…The use of microfabricated channels for single-fluorophore detection with high-throughput has been demonstrated previously, and various channel or capillary geometries with dimensions of one to several micrometers diameter have been evaluated. ,− The width of the focal volume was either expanded to span the entire channel width, or only a fraction of the channel was illuminated, and thus, not all molecules flowing through the channel were detected. The upper limit in concentration to avoid simultaneous passage of two or more molecules through the observation volume was therefore similar or worse compared to diffraction-limited confocal optics.…”

Single-molecule fluorescence spectroscopy of biomolecular folding

“…Another modification of the free diffusion experiment is achieved by flowing molecules in capillaries [50], or in the channels of microfluidic devices [51]. Flowing can improve the sampling rate of single molecules in solution.…”