Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points

Summary

Digital micromirror devices (DMD) can generate complex patterns in time and space with which to control neuronal excitability. Issues relevant to the design, construction, and operation of DMD systems are discussed. Such a system enabled the demonstration of non-linear integration across distal dendritic branch points.

Abstract

Light is a versatile and precise means to control neuronal excitability. The recent introduction of light sensitive effectors such as channel-rhodopsin and caged neurotransmitters have led to interests in developing better means to control patterns of light in space and time that are useful for experimental neuroscience. One conventional strategy, employed in confocal and 2-photon microscopy, is to focus light to a diffraction limited spot and then scan that single spot sequentially over the region of interest. This approach becomes problematic if large areas have to be stimulated within a brief time window, a problem more applicable to photostimulation than for imaging. An alternate strategy is to project the complete spatial pattern on the target with the aid of a digital micromirror device (DMD). The DMD approach is appealing because the hardware components are relatively inexpensive and is supported by commercial interests. Because such a system is not available for upright microscopes, we will discuss the critical issues in the construction and operations of such a DMD system. Even though we will be primarily describing the construction of the system for UV photolysis, the modifications for building the much simpler visible light system for optogenetic experiments will also be provided. The UV photolysis system was used to carryout experiments to study a fundamental question in neuroscience, how are spatially distributed inputs integrated across distal dendritic branch points. The results suggest that integration can be non-linear across branch points and the supralinearity is largely mediated by NMDA receptors.

Protocol

General design considerations

If the wavelength of photo stimulation is in the visible range, such as for optogenetic experiments, the layout of the system will be considerably simpler than for UV photolysis experiments. One only needs to purchase a dual camera port module that is available from all of the microscope companies. The DMD plane can be positioned at the conjugate image plane of either one of the two camera ports. But in UV photolysis the light originating from the DMD must be brought in through the epi-illumination path (Fig. 3A), because the imaging tube lens leading to the camera is not corrected for UV wavelength in any of the....

Discussion

The advantage of DMD based photostimulation approach is most apparent for situations where the target occupies a relatively large area. If the target of interest is very small, such as a few dendritic spines, sequential scanning confocal and 2-photon systems are likely to be the better approach. One significant weakness of the DMD approach is its inefficient use of the available light. The bulk of the available light is necessarily directed to the OFF mirrors and not used.

The DMD based syste.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Modern upright fluorescent microscope
CCD camera and image acquisition software
Computer and data acquisition/interface system
DLP Discovery Developer Kit
ALP3 USB interface
S2 + Optics w/LED
Dual camera port unit
355nm frequency tripled NdVO4 laser (~1 W)	DPSS Laser Inc.
Laser shutter Model LS6	Uniblitz
Multimode optical fiber and fiber stretcher Model# 915	Canadian Instrument and Research, Ltd		100 um core multimode fiber
Multimode Fiber launcher	Oz Optics
Signal generator			up to 50 kHz
Beam collimator	Olympus Corporation	DApo20UV340
UV relay lens	Special Optics	#: 54-25-60-355