Rational Design of Fluorophores for In Vivo Applications - Progress in Molecular Biology and Translational Science - page 93

longer wavelengths. 244,245 In an alternative strategy, the luciferase/luciferin
pair is used as an energy-transfer donor in bioluminescence resonance energy
transfer (BRET) and transfers the excitation energy to the acceptor emitting
at longer wavelength. In one of the first BRET systems developed, luciferase
was fused with green fluorescent proteins and this system was used to
monitor intracellular molecular events. 242,243 The luciferase-GFP (green
fluorescent protein) pair has been also used in vivo , 246 but the relatively
short wavelength of emission of GFP prompted scientists to seek a BRET
acceptor with a longer wavelength of emission.
Recently, a BRET system containing red fluorescent proteins emitting
at 635 nm as acceptors has been designed and used for in vivo imaging of
protein-protein interactions. 247 Alternatively, luciferase has been conju-
gated with organic near-IR-emitting fluorophores: Alexa Fluors (AF680
and AF750) 245 or cyanine 248 and both exhibit efficient BRET and emission
in the red or the near-IR region.
Rao and coworkers have developed a self-illuminating BRET system suit-
able for in vivo application, which consists of the luciferase/coelenterazine pair
as donor and quantum dots as acceptors. 249,250 In their system, quantum dots
are conjugated to the eight copies of mutated Renilla reniformis luciferase
(called there Luc8) and, in the presence of coelenterazine, show quantum
dot emission, due to the BRET, together with a much weaker emission at
480 from coelenterazine. This system has been examined in vitro and in vivo
in mouse. Luc8 has been subsequently conjugated to quantum dots
emitting at different wavelengths, 605, 655, 705, and 800 nm, respectively,
and used for in vivo spectral multiplexing. 249 A BRET system utilizing the
luciferase-quantum dot pair has been also used for in vivo cancer detection. 251
Although chemiluminescence, that is, luminescence occurring upon a
chemical, nonenzymatic reaction, is a well-known phenomenon, 252 its
application for in vivo imaging has been neglected until recently. 253,254
The main reasons are that most of the chemiluminescent reactions
typically emit short-wavelength light, utilize unstable, highly reactive
compounds (such as peroxides), or require reagents that are toxic or
harmful (e.g., hydrogen peroxide). 252 Chemiluminescent molecular
probes suitable for in vivo applications, where chemiluminescence is
activated by temperature, have been developed by Smith and
coworkers. 254 They found that squaraine rotaxanes (see Section 3 ) having
an anthracene core in their macrocyclic tetralactam components react
with singlet oxygen in cycloaddition reaction to form an adduct, the so-
called endoperoxide. This peroxide, upon warming up to the body