Structure–Function Relationships in Fluorescent Marker Proteins of the Green Fluorescent Protein Family - Fluorescent Proteins: From Understanding to Design - page 251

precise localization of individual fluorophores that yields an image resolution of
typically 20-50 nm.
Because the tetrameric nature of IrisFP may create problems in fusion protein
applications, we have developed an advanced, monomeric variant denoted as
mIrisFP, which displays excellent properties as a genetically encoded fluorescence
marker [ 43 ]. Compared with mEosFP, mIrisFP contains four additional mutations:
Ala69Val, Phe173Ser, Lys145Ile, and Tyr189Ala.
In a pulse-chase PALM experiment, green mIrisFP is fused to a protein of interest
and expressed by a living cell. Subsequently, many (typically a few thousand) CCD
camera frames of ~50 ms exposure time are collected. These images are taken with
473-nm light for both excitation and off-switching of the green form of mIrisFP.
In each individual frame, only s small number (~100) of mIrisFP molecules are
detected that have thermally reverted to their fluorescent state (Fig. 8a ). Conse-
quently, their point-spread functions do not overlap, and they can be localized to
within 20-50 nm. The super-resolution PALM image is finally reconstructed from
all the molecular locations in all CCD frames (Fig. 8e ). Later, a sub-ensemble of
mIrisFP molecules is photoconverted to the red form by targeted irradiation of a
selected cell region (marked by the violet rectangles) with a pulse of 405-nm light
(Fig. 8b, f ). Migration of the tagged proteins (Fig. 8c ) out of the conversion region
into other regions of the cell can be observed by super-resolution PALM imaging,
now exploiting the photoswitching capability of the red species (Fig. 8d, g ). Individ-
ual frames of the red form are collected with 561-nm light for excitation and
off-switching, and weak 473-nm light is also applied to enhance on-switching of a
few molecules to the red-fluorescent state within each individual camera frame.
Again, all individual frames are combined to yield the super-resolution PALM
image (Fig. 8g ).

4 Conclusions

In this chapter, we have discussed structure-dynamics-function relationships in FP
marker proteins. This knowledge forms the basis for the continuing rational devel-
opment of these important tools for the life sciences. Further extension of the
color palette toward the near-infrared region would be highly desirable for many
biomedical applications, especially for deep tissue and whole organism imaging.
Additional bathochromic shifts by extension of the conjugated p -electron system

are limited by the size of the -can. Additional red shifts may, however, still

be possible by modification of the chromophore environment, for example, by
p

b

-stacking of aromatic amino acids. Advances will also be welcome in the area
of photoactivatable proteins. The applicability of green-to-red photoconvertible
FPs is limited by the nonreversible nature of photoconversion, and reversibly
photoswitching FPs are not visible in their off state. New highlighters such as
IrisFP, which combine the two modes of photoactivation, may alleviate these
drawbacks. The capability of performing multiple phototransformations will