LANL's 3D tracking microscope.
Understanding Diseases at the Molecular Level
December 2008 / January 2009
Volume 6 Number 6
Over the past decade, single-molecule measurement techniques have transfigured experimental biophysics. One method that has garnered much attention is single-molecule fluorescence, chiefly because of its potential application in vivo. However, many single-molecule fluorescence experiments are conducted under artificial conditions that differ significantly from the physiological conditions native to the molecular systems under study because they observe targets that are secured to glass surfaces or other anchors. Following the trajectory of single proteins as they perform their biological functions, in vivo represents an exciting method for observing biomolecular systems.
Single-particle tracking has led to a better understanding of bacteria motility, membrane dynamics and motor proteins.
Until recently, single-particle tracking techniques have mostly employed video microscopy. This method allows particles to be located in two dimensions (2D) accurate to better than 50 nanometers with a time resolution of 10 microseconds.
While 2D tracking has provided valuable insights, most aspects of biology are three dimensional (3D) and require 3D techniques to be fully understood. The extension of single-particle tracking to 3D trajectories represents significant progress toward studying systems such as intracellular signaling and protein trafficking.
Numerous techniques have been developed to extend video microscopy to 3D, but these methods are limited in terms of the speed or depth of field they are able to attain. More recently, some 3D tracking techniques based on a confocal geometry have been advanced. However, such methods require relatively large particles or illumination at laser powers of tens of milliwatts, making them difficult to apply to in vivo single-particle tracking.
Several academic teams nationwide, including Enrico Gratton's group at the University of California at Irvine, Hideo Mabuchi's team at Stanford University and Haw Yang's group at the University of California at Berkeley, are currently developing confocal-based 3D tracking geometries. But it was a group of scientists at Los Alamos National Laboratory who, in 2008, successfully pioneered a microscope able to track protein-sized, hard to see particles in three dimensions. The 3D Tracking Microscope, designed and developed by James H. Werner, Guillaume A. Lessard, Nathan Wells and Peter M. Goodwin of LANL's Center for Integrated Nanotechnologies, won a 2008 R&D 100 award.
The team's invention is a unique confocal 3D tracking microscope capable of following the motion of nanometer-sized objects, such as individual molecules, quantum dots, organic fluorophores and single green fluorescent proteins as they zoom through three-dimensional space at rates faster than many intracellular transport processes. The 3D tracking microscope can follow the transport of nanometer-sized particles at micrometer per second rates. This enables researchers to follow individual protein, ribonucleic acid (RNA), or deoxyribonucleic acid (DNA) motion throughout the full three-dimensional volume of a cell to discover the path a particular biomolecule takes, the method it employs to get there and the specific proteins it may be interacting with along the way.
In addition to its ability to track in three dimensions, the LANL apparatus represents a significant improvement over 2D tracking microscopes because it employs tightly focused illumination and reduces photodamage and background noise. It is also more multifunctional than conventional laser scanning confocal microscopes (LSCMs). LSCMs enable three-dimensional sectioning of cells and tissue with resolutions of several hundred nanometers and are able to generate a three-dimensional rendering of cellular structure. As such, they represent valuable tools for academic researchers and pharmaceutical companies—€”as evidenced by an annual market of about $225 million for these instruments.
However, they are not able to follow individual protein motion in three dimensions. The 3D tracking microscope fulfills the functions of a conventional LSCM and many more; it can track single labeled molecules in three dimensions as well as render three-dimensional images with single fluorophore sensitivity.
In addition to applications in molecular spectroscopy and materials research, the 3D tracking microscope is a powerful tool primarily in the fields of cellular biology and biomedical research, Werner said. "The 3D tracking microscope will advance our understanding of the molecular basis and kinetics of many diseases, such as cancer, diabetes, or muscular dystrophy," he said. "We anticipate the microscope will become a valuable weapon in the arsenal of biomedical researchers who are fighting to find cures for cancer, heart disease and other protein or DNA-based diseases."
Single molecule tracking in two dimensions has already led to a better understanding of receptor transport in cellular membranes, gene transcription, and motor protein kinetics. As cells are three-dimensional structures, the 3D tracking microscope has a nearly unlimited, untapped potential in helping to better understand a number of cellular processes and cellular diseases, Werner said. Using the new instrument, biomedical researchers will be able to study the re-organization of molecules that takes place during cell division (mitosis), the transport of receptors or foreign bodies from the outside to the inside of the cell (endocytosis), the transport of cellular receptors from the inside of the cell to the cell membrane (transcytosis), mRNA transport from DNA to the ribosome, following proteins from synthesis at the ribosome to their final destination in the cell, the kinetics of signal transduction cascades, and of the molecular response of a cell to environmental insults (carcinogens), pharmaceutical treatment, and viral invasion.
The LANL team recently tested the sensitivity and speed of the 3D tracking microscope by following the Brownian motion of individual semi-conductor quantum dots in glycerol-water mixtures. This work was reported in Three Dimensional Tracking of Individual Quantum Dots published in Applied Physics Letters Vol. 91 (2007). When tracking, the 3D tracking microscope follows only one molecule at a time. However, the structure immediately surrounding the tracked molecule can be simultaneously visualized by recording a wide-field XY image at the current Z plane with a charge-coupled device (CCD) camera, which the LANL team has recently incorporated into the 3D tracking microscope.
A patent is pending for the 3D tracking microscope. According to Werner, the researchers are currently involved in on-going discussions with companies interested in potentially licensing this technology. Werner said these include makers of key component technologies and manufacturers of complete laser scanning microscope systems.
Tatjana Rosev, is a communications specialist at Los Alamos National Laboratory.
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