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readily observed at the single molecule level, which suggests a high quantum
efficiency. Additionally, experiments at cryogenic temperatures revealed that
reversible photoconversion between a super-red form and other spectral forms
is possible [ 47 ], which contradicts the proposed mechanism since photoinduced
decarboxylation is usually irreversible. Considering all the information gathered
from ensemble spectroscopy, cryogenic spectroscopy, and single molecule spec-
troscopy, it appears likely that two different super-red-emitting forms exist, one
with high fluorescence quantum efficiency, which reversibly photoconverts to
other forms, and which is observed at the single molecule level and in the
experiments at cryogenic temperatures. The second super-red form would then
be of low quantum efficiency and is potentially formed by irreversible decarbox-
ylation as suggested [ 46 ].
The histograms of single molecule emission maximum positions revealed
emission spectra with maximum positions between 530 and 570 nm that were
foundforDsRedandallthevariantssampled(Fig. 10 ). It was impossible to
assign these spectra (Fig. 11a ) to any known form of the green- or the red-emitting
chromophore. The percentage of tetramers showing this rare form emission is
generally low but not negligible (DsRed ~3%, Fluorescent Timer ~1%, DsRed2
~6%, AG4 ~2% and DsRed_N42H ~2%). Spectral series showed transitions from
the known main forms to the rare forms and thus demonstrated that these rare
forms are connected to the main forms. (Fig. 11b ). It is also striking that these
forms appeared at similar wavelengths as the rare forms found for proteins from the
GFP group of proteins.
As demonstrated before, it is often helpful to combine results obtained from the
single molecule experiments with results obtained from conventional ensemble
spectroscopy to understand the details of complex emitting systems. Comparing
the emission observed from the rare forms of DsRed and its variants (Fig. 11 ) to the
dominant emission from the protein zFP538 from coral Zoanthus sp. and mOrange
reveals clear similarities. The emission maximum of zFP538 is at 538 nm, the
emission maximum of mOrange is at 562 nm, which is in the same wavelength
region as the observed DsRed rare forms. zFP538 and mOrange embody a chromo-
phore that resembles a truncated red-emitting DsRed chromophore [ 41 , 42 ]. This
similarity is suggestive of an analogous, possibly photoactivated, modification of
the red-emitting chromophore from an acylimine to an imine resulting in the
truncated chromophore. In spectral series only transitions from the main forms to
the rare spectral forms were observed, but never transitions back from rare forms to
one of the main forms. This observation supports the hypothesis of a modification
of the chromophore since the creation of the imine is not likely to be reversible. As
discussed above, not only the chemical structure of the chromophore determines the
spectral properties, but also the chromophore nanoenvironment contributes to the
exact emission position and distribution of the emission maximum positions
observed at the single molecule level. It is likely that these differences in the
chromophore embedding between the DsRed variants and zFP538 and mOrange
accounts for the variances in the emission maximum position of the DsRed rare
forms compared to zFP538 and mOrange.
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