Margaret W. Bushee, email@example.com
BUFFALO, N.Y. – Studying apoptosis, or programmed cell death, is imperative for understanding
normal physiological functions as well as disorders such as cancer and neurodegenerative
and autoimmune diseases. Being able to recognize the intracellular configurations
of macromolecules that indicate apoptosis is essential for advancing biomedical
science and applied medicine.
Until now, however, it has not been possible to get an in-depth
view of the apoptotic process. Only limited glimpses have been seen of the proteins,
RNA, DNA and lipids that comprise the four major types of subcellular macromolecules.
Imaging techniques have been unable to show all of these at the same time, so their
spatial relationships and structural transformations during programmed cell death
have been undetectable.
To achieve real-time monitoring of apoptosis, a multidisciplinary
team at the University at Buffalo developed a multiplex nonlinear optical imaging
method that uses three types of complementary microscopies: coherent anti-Stokes
Raman scattering (CARS), two-photon excitation fluorescence (TPEF) and fluorescence
recovery after photobleaching (FRAP). The first two modes, operating in tandem,
scan multiple pictures at almost exactly the same time, allowing all four types
of macromolecules to be viewed in relation to each other. The third method, which
quantifies two-dimensional lateral diffusion, characterizes the motion of nuclear
proteins. The study, led by Paras N. Prasad, was published in the July 20, 2010,
issue of Proceedings of the National Academy of Sciences.
The four panels of this CARS/TPEF image demonstrate the redistribution of macromolecules during apoptosis over a 24-h period. Proteins are red, RNA is green, DNA is blue, and lipids are
gray. Researchers at the Institute for Lasers, Photonics and Biophotonics at the
University at Buffalo in New York have developed a multiplex biophotonic imaging
platform that permits real-time imaging of dying cells. Courtesy of Paras N. Prasad.
The CARS technique, a form of nonlinear optical microscopy that
uses multiple photons to sense vibrational frequencies of specific chemical bonds,
detected proteins and lipids at their characteristic vibrations of 2928 and 2890
cm—1, respectively. TPEF was required to sense the nucleic acids, selectively stained
by acridine orange, in the green (DNA) and red (RNA) spectral channels.
“This multiplex imaging is a step forward from traditional
microscopy and provides direct information on the presence of nonlabeled molecules
in the cell interior,” co-author Andrey N. Kuzmin said.
Using this multiplex biophotonic platform permitted the researchers
to see that, before apoptosis, while the cell is still proliferating, proteins in
the nucleus are distributed nearly uniformly, although with accumulation in several
subnuclear structures. However, at the onset of apoptosis, this pattern changes
abruptly, with proteins forming progressively irregular clusters. The FRAP technique
revealed that, during apoptosis, the diffusion of nuclear proteins gradually slows
The most exciting potential applications of this multiplex imaging
method include in vivo monitoring of infectious diseases and other cell function
abnormalities, along with therapeutic drug administration, according to co-author
Artem M. Pliss. “The ability to quantitatively characterize and monitor in
real time biomolecular distribution and kinetics during drug-cell interactions would
be in high demand for clinical administration and therapeutic drug development.”
As for the future, the team’s plan is to concentrate on
the “most essential and socially important biomedical challenges, such as
disease diagnostics and monitoring and therapeutic drug interaction studies,”
co-author Aliaksandr V. Kachynski said.