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ABSTRACT: Advanced light microscopy offers sensitive and non-invasive means to image neural activity and to control signaling with photolysable molecules and, recently, light-gated channels. These approaches require precise and yet flexible light excitation patterns. For synchronous stimulation of subsets of cells, they also require large excitation areas with millisecond and micrometric resolution. We have recently developed a new method for such optical control using a phase holographic modulation of optical wave-fronts, which minimizes power loss, enables rapid switching between excitation patterns, and allows a true 3D sculpting of the excitation volumes. In previous studies we have used holographic photololysis to control glutamate uncaging on single neuronal cells. Here, we extend the use of holographic photolysis for the excitation of multiple neurons and of glial cells.
The system combines a liquid crystal device for holographic patterned photostimulation, high-resolution optical imaging, the HiLo microscopy, to define the stimulated regions and a conventional Ca(2+) imaging system to detect neural activity. By means of electrophysiological recordings and calcium imaging in acute hippocampal slices, we show that the use of excitation patterns precisely tailored to the shape of multiple neuronal somata represents a very efficient way for the simultaneous excitation of a group of neurons. In addition, we demonstrate that fast shaped illumination patterns also induce reliable responses in single glial cells.
We show that the main advantage of holographic illumination is that it allows for an efficient excitation of multiple cells with a spatiotemporal resolution unachievable with other existing approaches. Although this paper focuses on the photoactivation of caged molecules, our approach will surely prove very efficient for other probes, such as light-gated channels, genetically encoded photoactivatable proteins, photoactivatable fluorescent proteins, and voltage-sensitive dyes.
PLoS ONE 01/2010; 5(2):e9431. · 4.09 Impact Factor
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ABSTRACT: Fluorescent protein-based FRET is a powerful method for visualizing protein-protein interactions and biochemical reactions in living cells. It can be difficult, however, to avoid photobleaching when observing fluorescent cells under the microscope, especially those expressing CFP. We compared the sensitivity of two protein-based FRET pairs to light-induced fluorescence changes in the donor, on FRET determination by fluorescence lifetime imaging microscopy (FLIM). Thanks to the very low excitation light levels of the time- and space-correlated single photon counting (TSCSPC) method, FLIM acquisitions were achieved without donor photobleaching. Here, we show that photobleaching of CFP by a mercury lamp under the microscope induced a decrease in the mean fluorescence lifetime, which interfered with FRET determination between CFP and YFP. Importantly, the range of light-induced variation of the mean fluorescence lifetime of CFP was not proportional to the decrease in the steady state fluorescence intensity and varied from cell to cell. The choice of the CFP/YFP pair therefore requires that the cells be observed and analyzed at very low light levels during the whole FRET experiment. In contrast, the GFP/mCherry pair provided an accurate FRET measurement by FLIM, even if some GFP photobleaching took place. We thus demonstrate that CFP can be an unreliable donor for FRET determination in living cells, due to its photosensitivity properties. We demonstrate that the GFP/mCherry pair is better suited for FRET measurement by FLIM in living cells than the CFP/YFP pair.
Microscopy Research and Technique 12/2006; 69(11):933-9. · 1.79 Impact Factor
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ABSTRACT: The goal of this work is to investigate the usefulness of the optical tweezers for biological sample micro-manipulation.We report multiple optical trapping and manipulation of Escherichia coli cells, immersed in growth medium, by means of diffractive optical elements (DOE). The DOEs are implemented on a spatial light modulator to generate movable geometries of traps.We report also an experiment that allows to mimic the mechanical environment of cells in tissues. Micro-meter sized beads are trapped in circular geometry in order to surround living cells. By dynamically varying the geometry of the configuration and the trapping forces, we can reproduce a controlled environment where only mechanical stimuli are present and biological responses can be monitored. These experiments have been implemented in two different setups. In the first, cell manipulation is performed by the use of two-dimensional optical tweezers system generated by dual axis acousto-optical deflectors. In the second setup, trapping configuration is extended to three-dimensional volumes using DOEs.
Microelectronic Engineering. 01/2005; 78-79:575-581.
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ABSTRACT: We present a multi trap optical tweezes system that enables to generate two-dimensional dynamical configurations of focal spot where the trapping force of each element of the pattern can be individually changed. Force gradients in the pN range can be generated on a micrometer scale.
Optics Express 09/2004; 12(17):3906-10. · 3.59 Impact Factor
