Advanced instrumentation
for Optogenetics

Opto-mechanical devices for holographic photostimulation via
spatial light modulation and high speed diffraction limited point scanning,
both integral to VIVO Multiphoton and Marianas imaging platforms.

Phasor

Phasor is the world’s first commercial system for optogenetic photostimulation using digital holography. It offers the ability to deliver significant power to a user-defined pattern (or multiple patterns) at once, and can actually illuminate at different 3D positions simultaneously. Because Phasor uses diffraction to shape the waveform of light entering the specimen, it is far more efficient than systems that use a digital micromirror device (DMD), which can only block undesired light rather than redirect it where it is needed. Further, those systems are restricted to operating in a single plane of focus. In addition to optogenetic photostimulation, Phasor has many other uses in photomanipulation:

•  Fluorescence recovery after photobleaching (FRAP)

•  Photoconversion (e.g., Kaede)

•  Photoactivation (e.g., paGFP)

•  Uncaging (e.g., MNI-glutamate)

Phasor can be used with widefield, spinning disk confocal, and multiphoton systems, and can also be combined with point-scanning photostimulation such as 3i’s Vector™. It is available in visible light and multiphoton versions, and both versions can be used together in the same system. Coupled with 3i’s LaserStack™, more than six different wavelengths can be selected and even alternated in rapid succession, with different patterns generated for each wavelength.

Screen capture of the preBotC network with neurons of interest selected for simultaneous photostimulation via Phasor to elicit a network burst.
Ephys recording where the bottom trace represents the laser pulse and the t op trace is a recording of the hypoglossal nerve (the output of the preBotC network). Arrows are pointing to endogenous bursts, while evoked bursts are indicated by the timing of the laser pulse.

 

Image and data courtesy Dr. Jason Worrell, UCLA

Vector

Vector executing an automatically generated sequence of stimulation events in a grid pattern for brain circuit mapping using glutamate uncaging or ChR2 activation. Order is generated to maximize spatial separation between any two subsequent stimulation locations.

Vector is a high speed galvanometer-based point scanner capable of delivering powerful and finely controlled laser light to user-defined areas in a specimen. Coupled with LaserStack, it can deliver a diffraction limited spot of intense laser light to a prescribed spot, region or multiple regions from a variety of laser wavelengths across the visible spectrum.

Vector is highly effective for uncaging and photobleaching. Coupled with Ablate!™, it can provide powerful pulsed laser ablation and laser-induced injury. Vector is also central to 3i’s VIVO Multiphoton imaging system.

SlideBook’s control of Vector and Phasor allows synchronization of photostimulation with imaging and electrophysiology with microsecond-level timing precision. SlideBook 5.5 introduces numerous enhancements:

•  Direct recording of electrophysiological data for perfect correlation with imaging

•  Ultra-precise, flexible control of optogenetic and caged compound stimulation

•  Automatic grid pattern sequencing for neural circuit analysis

•  Multichannel streaming capture

•  Extended interoperability with MATLAB®

VIVO Multiphoton

VIVO Multiphoton is designed for high-speed, high-sensitivity multiphoton imaging of live animals and tissue. Incorporating the Vector scanning system, it can acquire whole fields, sets of arbitrary regions, curves or lines, all with synchronized piezo Z focus and power modulation. VIVO Multiphoton can collect multiple volumes per second, with laser power optimized for varying depths and eliminating any excitation in unwanted regions.

VIVO Multiphoton offers many detection options, including a proprietary nosepiece that places two GaAsP PMTs close to the objective and a substage unit that places two GaAsP PMTs close to a high NA water condenser automatically tracked to the nosepiece position. There are many ways to integrate photostimulation, using either 3i’s LaserStack or a second multiphoton laser, which can be addressed by the same Vector, a second Vector, or Phasor.

GFP transfected hippocampal neurons acquired by students at the 2012 CSHL course Imaging Structure and Function of the Nervous System

Key differences between a digital micromirror device (DMD), Vector laser-based ROI scanner, and the Phasor digital holography system:

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Intelligent Imaging Innovations, Inc.
3509 Ringsby Ct
Denver, CO 80216, USA
Phone: +1 (303)-607-9429
Fax: +1 (303)-607-9430
Email: info@intelligent-imaging.com

EUROPE

Intelligent Imaging Innovations GmbH
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D-37081 Göttingen, Germany
Phone: +49 (0)551-508-39-266
Fax: +49 (0)551-508-39-268
3ieurope@intelligent-imaging.com

 

Intelligent Imaging Innovations Ltd
20-22 Wenlock Road
London, UK
Phone: +44 (0) 2037 440031
3ieurope@intelligent-imaging.com