Optical methods for single molecule detection and analysis

Abstract

As analytical chemists, the highest resolution measurement one can make is at the single molecule level; it just does not get any better than that. To determine the concentration of a molecule in solution, the best way is to count the number of molecules in a given volume. As long as the volume contains a statistically large enough number of molecules and is above the Poisson noise limit, molecular counting is the most accurate way to make a measurement. Molecular counting is the method of the future and is beginning to be performed today.

Figure 1

Top: Analog measurements give increasing intensity as the concentration increases. Bottom: In contrast, digital measurements are independent of intensity and simply rely on a signal/no signal readout.

Figure 2

a, b, Immunoprecipitated protein complexes are visualized using TIRF microscopy using fluorophore-labelled antibody (a) or fluorescent protein (FP) tags (b). TIR, total internal reflection. c, Multi-colour colocalization can distinguish between subcomplexes (for example, AB + AC versus ABC). d, Photobleaching analysis can provide stoichiometric information. A simulated photobleaching trajectory for a trimeric protein. e, TIRF images for YFP pulled down from cells expressing His₆–YFP (YFP) and control cells (Con) using His-tag or a control (Flag-tag) antibody. Minus sign indicates no antibody or sample. IP, immunoprecipitate. Scale bar, 5 µm. f, Average number of fluorescent molecules per imaging area, N_f. Error bars denote standard deviation (s.d.) (n > 20). Reprinted by permission from Macmillan Publishers Ltd: NATURE 2011, 473, p484.

Figure 3

Proteins are captured on antibody coated beads. One or zero molecules of analyte are captured on each bead. A labeled detection antibody creates a sandwich complex . When the beads are loaded into microwells along with substrate and sealed, enzyme-labeled beads covert the substrate into a fluorescent product that can be easily detected. Reprinted by permission from Macmillan Publishers Ltd: NATURE BIOTECHNOLOGY, 2010, 28, p596.