BioPHYSICAL chemistry

efficiencies, or in terms of the time dependence of the FRET efficiency,

corresponding to the rate of change between the open and closed mole-

cular configurations.

There are several different fluorescent markers that can be attached to

proteins for these studies. The marker may be a simple dye molecule such

as tetramethyl rhodamine functionalized with malemide, the relatively

stable dye cy3-malemide, dyes such as bis-[(N-iodacetyl)piperazinyl]

sulforhodamine that have two attachment groups, or a label that can be

incorporated genetically, for example green fluorescent protein (Chapter 19).

Special optical configurations must be employed to detect single mole-

cules in a very small area. The sample concentration is kept very low so

that, at any given time, only one molecule is being probed by the beam.

Several optical configurations have been used to measure fluorescence

from single molecules involving different microscopic configurations,

including total internal reflection (Figure 14.20a, b) and epifluorescence

(Figure 14.20c).

Holliday junctions

Genetic diversity is required for long-term survival of a species. Exchange

of genetic material between homologous chromosomes provides a means

of generating diversity. In homologous recombination the parent DNA aligns

Figure 14.20Different configurations for single-molecule measurements: (a) total internal
reflection through the objective, (b) prism-based total internal reflection, and (c) epifluorescence
illumination. Modified from Peterman et al. (2004).

312 PART 2 QUANTUM MECHANICS AND SPECTROSCOPY

Slide with sample

(a) (b) (c)

Objective

Excitation

Fluorescence filter

Dichroic mirror

Prism

Total internal reflector fluorescence EPI fluorescence

Detector