Spectral Versatility of Fluorescent Proteins Observed on the Single Molecule Level - Fluorescent Proteins: From Understanding to Design - page 217

Fig. 3 Typical spectral series recorded from a single EYFP with integration time of 1 s per
spectrum. A transition to a dark state and recurrence of emission is evident, the spectra before and
after the dark period show only minor differences (Reprinted from [ 36 ], with permission from
Elsevier)
to yield insights into the appearance and dynamics of spectral forms and the
possibility to analyze the chromophore-nanoenvironment interaction make the
method a powerful tool to analyze the complex emission behavior of VFPs, as
will be discussed below.
However, the gain in information by recording emission spectra is accompanied
by a loss of time resolution and a substantial increase in experimental complexity
compared to spectrally integrated detection. To record single molecule emission
spectra without wasting any photons by scanning the detected wavelength, the
emitted fluorescence is dispersed by a spectrometer and imaged onto a highly
sensitive, usually cooled, CCD camera. In this way, emission spectra can be recorded
with integration times in the order of hundreds of milliseconds to seconds - depend-
ing on the experimental conditions used, on the excitation powers, on the overall
sensitivity of the setup, and on the extinction coefficient and quantum efficiency
characteristic of the molecule being studied.
3.4 Single VFP Emission Spectra for Target Molecule
Identification
Single emitter fluorescence spectra are especially rich in information about com-
plex emitting systems showing spectral diversity such as VFPs. However, to record
emission spectra of high spectral resolution with acceptable signal to noise ratio
rather large numbers of photons need to be collected. VFPs are known to be
exquisitely sensitive emitters and their photostability is often far worse than that
of many synthetic fluorescent dyes [ 72 ], which makes the recording of emission
spectra or even series of emission spectra from single VFPs an especially challeng-
ing task.
These experimental challenges long hampered the systematic use of spectrally
resolved single molecule spectroscopy to analyze VFP properties. The first single
molecule emission spectrum recorded from VFPs was hence not used to analyze the