University at Buffalo (USA) scientists have developed a new imaging approach capable of monitoring in real-time the transformations that cellular macromolecules undergo during programmed cell death.
In this composite image from the University at Buffalo, proteins are shown in red, RNA in green, DNA in blue, and lipids in gray.
The work could help realize the potential of customized molecular medicine, such as chemotherapy treatments that precisely target cellular changes exhibited by individual patients. It can also be a valuable drug-development tool for detailed screening of drug-biomolecular interaction.
“This new ability provides us with a dynamic mapping of the transformations occurring in the cell at the molecular level,” says SPIE Fellow Paras N. Prasad, executive director of the UB Institute for Lasers, Photonics and Biophotonics (ILPB) and a co-author of the study. “It provides us with a very clear visual picture of the dynamics of proteins, DNA, RNA, and lipids during the cell’s disintegration.”
Solving a mystery
This programmed cell death, apoptosis, is essential to normal development, healthy immune system function, and cancer prevention. The process dramatically transforms cellular structures, but the limitations of conventional microscopy methods have kept much about this structural reorganization a mystery.
Prasad notes that molecular medicine, in which treatments or preventive measures can be tailored to cellular properties exhibited by individual patients, depends on much better methods of visualizing what’s happening during critical cellular processes.
The new research, reported in the Proceedings of the National Academy of Sciences in July, can help improve our understanding of cellular events at the molecular level, Prasad says. “If we know that specific molecular changes constitute an early signature of a disease, or what changes may predispose a patient to that disease, then we can take steps to target treatment or even prevent the disease from developing in the first place.”
To capture the cellular images, a team of biologists, chemists, and physicists at UB, led by Prasad, utilized an approach that combines three imaging techniques: a nonlinear, optical imaging system (CARS or coherent anti-Stokes Raman scattering), TPEF (two-photon excited fluorescence), which images living tissue and cells at deep penetration, and fluorescence recovery after photobleaching to measure dynamics of proteins.
“For the first time, this approach allows us to monitor in a single scan, four different types of images, characterizing the distribution of proteins, DNA, RNA, and lipids in the cell,” says Aliaksandr V. Kachynski, research associate professor at the ILPB and co-author. The resulting composite image (above) integrates in one picture the information on all four types of biomolecules, with each type of molecule represented by a different color: proteins in red, RNA in green, DNA in blue, and lipids in gray.
Visualizing final transformation
Multiplex imaging provided new information on the rate at which proteins diffuse through the cell nucleus, the UB scientists say in “Biophotonic probing of macromolecular transformations during apoptosis.”
Before apoptosis was induced, the distribution of proteins was relatively uniform, but once apoptosis develops, nuclear structures disintegrate, the proteins become irregularly distributed, and their diffusion rate slows down, says Artem Pliss, another co-author on the paper from the ILPB.
“This research gives us the unique ability to study and improve our understanding of individual subcellular structures and the transformations they go through,” Pliss says.
Such precise information will be especially useful for monitoring how specific cancer drugs affect individual cells.
“For example, say drug therapy is being administered to a cancer patient; this system will allow for the monitoring of cellular changes throughout the treatment process,” Kachynski notes. “Clinicians will be able to determine the optimal conditions to kill a cancer cell for the particular type of disease. An improved understanding of the drug-biomolecule interactions will help discover the optimal treatment doses so as to minimize side effects.”
Andrey Kuzmin, the fourth co-author, adds that a related study by the UB team images the distribution of protein, DNA, RNA, and lipid macromolecules throughout the cell cycle, not just at death. See page 17.
The work on both studies was supported by a grant from the John R. Oishei Foundation of Buffalo.
Why detect apoptosis?
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A 2006 article by Rostylav Bilyy discussed the use of biophotonics to study programmed cell death and its importance in medical diagnostics:
“During apoptosis a cell performs suicide. It enzymatically slices the intercellular content, including DNA and proteins. As a result, the cell shrinks and is fragmented into membrane-coated vesicles, called apoptotic bodies, which are then engulfed by cells of the immune system.
“Apoptosis removes elderly and impaired cells so they can be replaced with fresh cells. This huge turnover makes programmed cell death an important player in the game called ‘Life.’
“We will not notice anything when things are going right, but if a mismatch arises between cell production and death, we suffer from a range of diseases. For this reason, control and detection of apoptosis is of great importance for clinical and biomedical diagnostics.”
Researcher at BiOS
University at Buffalo scientist and SPIE Fellow Paras N. Prasad is a member of the program committee for the Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications conference at SPIE Photonics West 2011.
He will discuss multiplex imaging at the BiOS Hot Topics session 22 January and in the Multiphoton Microscopy in the Biomedical Sciences conference.
He is also scheduled to teach an SPIE course in biophotonics in San Francisco.
See the 2009 SPIE Newsroom video interview with Prasad discussing nanophotonics’ applications in solar and medicine.
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