Multifocal array at sample
JUSTIN E. JURELLER, HEE Y. KIM, AND
NORBERT F. SCHERER
Ti:S laser
60x
F DOE Multifocal array L3 L4
luorescence microscopy is L1 L2 a workhorse of biophysics and cell biology. 1 Imaging Galvanometer scanner drive signal EMCCD of fluorescence from endogenous or exogenous fluorophores is used to FIGURE 1. In the SS-MMM setup, a multifocal array created
UNIVERSITY OF CHICAGO
reveal structural localization, binding, expression, or con- by a diffractive optical element (DOE) is scanned by a two- formational state of proteins or other objects of interest at mirror galvanometer. Separate waveforms stochastically drive submicron length scales. However, live biological systems each axis. The inset shows a multiphoton-fluorescence im- such as cells are dynamic, with processes occurring on all age of the array ( 6 µm spot spacing) generated in a solution of
Rhodamine 6G at the sample focal plane.
time and length scales. Rapid high-resolution imaging or correlation measurements are needed to resolve processes (for example, signaling Stochastic scanning multi- or intracellular transport of vesicles and proteins) that occur on the order of milli- photon multifocal microscopy seconds or faster. 2
The need for such dynamic imaging combines a diffractive-optic- has driven the development of high- speed multiphoton and confocal-scan- generated array of spots with ning fluorescence microscopies. 3 Cur- random-access scanning to rent challenges include improving spatial resolution “beyond the diffraction limit” achieve rapid, high-resolution and increasing imaging speed beyond video rates. 4 These dual goals allow see-
JUS TIN F. JURELLER is technical director of the NanoBiology Facility in the Institute for Biophysical Dynamics, University of Chicago; HEE Y. KIM is a graduate student in the Department of Chemistry at the University of Chicago; and NORBERT F. SCHERER is a professor in the Department of Chemistry, the James Franck Institute, and the Institute for Biophysical Dynamics at the University of Chicago, 5735 S. Ellis Avenue, Chicago, IL 60637; e-mail: nfschere@uchicago.edu.
ing finer detail while understanding cellular processes on native timescales. Unfortunately, extending traditional scanning microscopies to higher speeds introduces spatiotemporal artifacts. This means the complexity of instruments with the required performance to capture fast dynamics without artifacts has
grown concurrently. The high cost and “black-box” design of commercial instruments create a large barrier that impedes widespread adoption and user development of new dynamic imaging techniques. To address these issues, we have developed stochastic scanning multiphoton multi-
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