efficiencies, or in terms of the time dependence of the FRET efficiency,
corresponding to the rate of change between the open and closed molecular 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 molecules 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 14.20 Different 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
9781405124362_4_014.qxd 4/29/08 13:09 Page 312
corresponding to the rate of change between the open and closed molecular 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 molecules 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 14.20 Different 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
9781405124362_4_014.qxd 4/29/08 13:09 Page 312