Spatiotemporal control of neuronal activity by holographic photoactivation patterns

This activity is divided into two parts:

1. One-photon (1P) scanningless holographic microscopy
2. Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses

1- One-photon (1P) scanningless holographic microscopy

In recent years the use of advanced optical techniques has generated a growing interest in the field of neurobiology not only for visualizing neuronal structures and signaling processes, but also for controlling neuronal activity. This has been possible thanks to a growing list of existing photosensitive tools that upon illumination can modify their chemical or conformational structures. For example, caged-compounds are commonly used photoactivable compounds that are inert when the active compound is covalently bound to a protecting group (cage). Upon shining light the covalent bond is broken and the physiologically active molecule (Ca2+, cAMP, or neurotransmitters such as glutamate) is released at the location of illumination. The use of these compounds in conjunction with spatiotemporally resolved photo-stimulation techniques allows a precise control of neuronal activity and to date represents an extremely promising alternative to electrode stimulation.

To reproduce a variety of intra- and extracellular signaling patterns, it is desirable to have as much flexibility as possible in the generation of excitation patterns. To this end, we have recently presented the first one-photon (1P) scanningless holographic microscope, where the use of a liquid crystal spatial light modulator (LC-SLM) in the excitation path allows reproducing multiple localized synaptic inputs by the generation of 2D and 3D multiple diffraction-limited spots. Meanwhile, the release of active molecules along extended dendritic segments can be mimicked by generating excitation spots that perfectly match the shape of specific sub-cellular compartments (Fig.1) (C. Lutz et al. Nature Methods (2008)).

Using holographic illumination to photolyse caged glutamate in brain slices, we demonstrated, in collaboration with David Di Gregorio (Laboratory of Brain Physiology, UMR 8118), Tom Otis (University of California Los Angeles, USA) and Serge Charpak, that shaped excitation on segments of neuronal dendrites and simultaneous multi-spot excitation of different dendrites enable precise spatial and rapid temporal control of glutamate receptor activation (C. Lutz et al. Nature Methods (2008)).

Fig. 1: Holographic patterned light. A defined region of a Purkinje cell (a) is selectively photoexcited by a holographic illumination pattern (b) generated with the phase hologram reported in (c).

2- Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses

To further improve the performances of the microscope (penetration depth and axial resolution), we have very recently implemented the system for two photon (2P) excitation. In this case, control and localization of illumination patterns in the axial direction have been achieved by combining holographic illumination with the technique of temporal focusing (TF) (E. Papagiakoumou et al. Optics Express (2008)). In temporally focused microscopy, depth resolution results from the fact that the excitation pulse is temporally stretched by dispersion of geometrical origin outside the focal plane. Therefore, the laser pulse is compressed as it propagates through the sample, reaches its peak value at the focal plane and stretches again as it propagates beyond it. The temporal focus works by spatially separating the frequency components of an ultrashort pulse with a grating (Fig.2) at the back focal plane of a telescope, which uses the objective as the second lens [D. Oron et al. Opt. Express 13, 1468 (2005)].

With this approach we could demonstrate that spatiotemporal shaping of ultra-short pulsed allows an almost 3D control of the excitation volume. In agreement with the theoretical predictions we proved that, despite the rapidly varying spatial phase of an excitation beam generated by digital holography, it is possible to create, with 140fs pulses and a NA=0.9 objective, arbitrary excitation patterns with a pattern-independent depth resolution of ~5μm (Fig.2), i.e only slightly larger than that achievable with wide-field temporally focused microscopy with flat phase excitation (E. Papagiakoumou et al. Optics Express (2008), E. Papagiakoumou et al. Optics Express (2009))

Fig.2: a) Layout of the experimental setup for the generation of holographic two-photon temporally focused excitation spots. b) Images of 10, 20 and 30µm diameter spots and a dendritic shape obtained by two-photon excitation of a ~0.9?m thick spin-coated fluorescent layer and c) respective measured axial profiles by implementing temporal focusing.

References

[1] C. Lutz, T. Otis, V. De Sars, S. Charpak, D. DiGregorio, and V. Emiliani, "Holographic photolysis of caged neurotransmitters," Nature Methods (2008).
[2] E. Papagiakoumou, V. de Sars, D. Oron, and V. Emiliani, "Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses," Opt. Express 16, 22039-22047 (2008).
[3] E. Papagiakoumou, V. de Sars, V. Emiliani, and D. Oron, "Temporal focusing with spatially modulated excitation," Opt. Express 17, 5391-5401 (2009) .