Holographic photolysis of caged neurotransmitters

Abstract

Stimulation of light-sensitive chemical probes has become a powerful tool for the study of dynamic signaling processes in living tissue. Classically, this approach has been constrained by limitations of lens-based and point-scanning illumination systems. Here we describe a microscope configuration that incorporates a nematic liquid-crystal spatial light modulator to generate holographic patterns of illumination. This microscope can produce illumination spots of variable size and number, and in patterns shaped to precisely match user-defined elements in a specimen. Using holographic illumination to photolyze caged glutamate in brain slices, we show that shaped excitation on segments of neuronal dendrites and simultaneous, multispot excitation of different dendrites enables precise spatial and rapid temporal control of glutamate receptor activation. By allowing the excitation volume shape to be tailored precisely, the holographic microscope provides an extremely flexible method for activation of various photosensitive proteins and small molecules.

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Figure 1: Optical setup and spot generation in a layer of coumarin.
Figure 2: Shaping of an excitation spot to match fine cellular processes.
Figure 3: Axial propagation of a holographic beam.
Figure 4: Varying the shape of illumination for photolysis influences the time course of glutamate clearance in cerebellar slices.
Figure 5: Using increasing lengths of shaped illumination to uncage glutamate along a dendrite preserves the time course of PECs in hippocampal slices.
Figure 6: Simultaneous uncaging of glutamate at two spots on granule-cell dendritic claws in acute cerebellar slices.

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Acknowledgements

We thank D. Cojoc, E. Ferrari and E. di Fabrizio for collaborating in the development of the software; E. Papagiakoumou for helping to set up the double microscope system; D. Oron (Weizmann Institute of Science) for stimulating discussions and providing the coumarin samples; D. Ogden for stimulating discussions, comments on the manuscript and suggesting the use of 405-nm laser as a source; and A. Silver and A. Marty for critical reading of the manuscript. This work was supported by grants from the European Science Foundation and the Centre Nationale de la Recherche Scientifique through the European Young Investigators program (to V.E.), the European Commission FP6 Specific Targeted Project “Photolysis” LSHM-CT-2007-037765 (to V.E., S.C. and D.A.D.), Fondation Fyssen, the Actions Thématiques et Incitatives sur Programme Jeune Chercheurs, Federation pour la Recherche sur le Cerveau (to D.A.D.), Foundation Bettencourt-Schueller (to V.E. and S.C.), US National Institutes of Health grant NS49238 (to T.S.O.), the Network of European Science Institutes (to D.A.D. and V.E.) and the Alexander von Humboldt Foundation (to C.L.).

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Contributions

C.L. set up the holographic optical microscope and performed the theoretical simulation for axial beam propagation; C.L., T.S.O. and D.A.D. performed and analyzed the cerebellar brain slice experiments; C.L. and S.C. performed and analyzed the hippocampal brain slice experiments; T.S.O., S.C. and D.A.D. contributed to writing the manuscript. V.D. developed the software. V.E. conceived and supervised the project, and prepared the manuscript.

Corresponding author

Correspondence to Valentina Emiliani.

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Supplementary Notes 1–3, Supplementary Methods (PDF 274 kb)

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Lutz, C., Otis, T., DeSars, V. et al. Holographic photolysis of caged neurotransmitters. Nat Methods 5, 821–827 (2008). https://doi.org/10.1038/nmeth.1241

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