ja3036256_si_001.pdf (2.45 MB)
Organelle-Specific Detection of Phosphatase Activities with Two-Photon Fluorogenic Probes in Cells and Tissues
journal contribution
posted on 2012-07-25, 00:00 authored by Lin Li, Jingyan Ge, Hao Wu, Qing-Hua Xu, Shao Q. YaoTwo-photon fluorescence microscopy (TPFM) provides key
advantages over conventional fluorescence imaging techniques, namely,
increased penetration depth, lower tissue autofluorescence and self-absorption,
and reduced photodamage and photobleaching and therefore is particularly
useful for imaging deep tissues and animals. Enzyme-detecting, small
molecule probes provide powerful alternatives over conventional fluorescent
protein (FP)-based methods in bioimaging, primarily due to their favorable
photophysical properties, cell permeability, and chemical tractability.
In this article, we report the first fluorogenic, small molecule reporter
system (Y2/Y1) capable of imaging endogenous phosphatase
activities in both live mammalian cells and Drosophila brains. The
one- and two-photon excited photophysical properties of the system
were thoroughly investigated, thus confirming the system was indeed
a suitable Turn-ON fluorescence pair for TPFM. To our knowledge, this
is the first enzyme reporting two-photon fluorescence bioimaging system
which was designed exclusively from a centrosymmetric dye possessing
desirable two-photon properties. By conjugation of our reporter system
to different cell-penetrating peptides (CPPs), we were able to achieve
organelle- and tumor cell-specific imaging of phosphatase activities
with good spatial and temporal resolution. The diffusion problem typically
associated with most small molecule imaging probes was effectively
abrogated. We further demonstrated this novel two-photon system could
be used for imaging endogenous phosphatase activities in Drosophila
brains with a detection depth of >100 μm.