Scope:
The overall project requires advanced imaging infrastructure used by a team of highly collaborative scientists with complementary and synergistic expertise to: 1) study synaptic and dendritic integrative function in neurons maintained in acute brain slices; 2) identify dendritic, cellular and network strategies in cortex during decision-making tasks in head-fixed awake animals; 3) dissecting the functional roles and connectivity of local inhibitory circuits within the motor cortex during reward-based motor skill learning; 4) deciphering the spatiotemporal relationship of synaptic plasticity with dendritic inhibition during motor skill learning; 5) visualizing and perturbing distinct neuronal ensembles during behavior.
This research will highlight basic aspects of strategies used by the brain to learn. It will have a significant impact on the development of therapeutic strategies for counteracting learning and memory deficits as well as motor circuit-related dysfunction associated with stroke, neurodegenerative disease, and traumatic brain injury.
General Description:
The requested solution needs to allow researchers to optically monitor and manipulate large ensembles of synaptic and neuronal ensembles simultaneously across different cortical layers in acute brain slices and in in vivo awake, head-restrained animals engaged in sophisticated decision-making tasks. The requested system is a microscope allowing for multiphoton imaging and manipulation of synaptic and cellular activity. The intended research requires ultra-high speed cellular imaging and photostimulation of live neuronal structures (i.e., cell bodies, axon terminals, synapses) with sub-cellular resolution from rodent brains.
The ability of controlling light in space and time afforded by Acousto-Optic Deflectors (AODs) constitutes the preferred technological basis for the intended imaging system. For imaging, the system needs to be able to acquire at high speed and be able to sample from a large number of points of variable size (from synapses to individual neurons in a 3D volume). The system must also be flexible to allow for fast selection of 3D region of interests (ROIs) with various shapes and sizes to detect activities all the way from axonal boutons and dendritic spines, to neuronal cell bodies. In addition, the system must allow for flexible photostimulation of complex spatial (in 3D) and temporal patterns, simultaneously with imaging, at different wavelengths. Lastly, the system needs to satisfactorily deal with motion artefact vessel pulsation, respiration or limb movements from the head-fixed awake animals in real time.
Mandatory Requirements:
1. Sample handling and size
1.1 Must have a motorized platform for positioning experimental rodents or brain slice holding apparatus in the x-y plane. Said capability must have an accuracy of 0.1 um, or better.
1.2 The motorized platform in 1.1 must be amenable to accommodate platforms for either in vivo imaging of awake head-restrained animals or in vitro work in acute slices using Differential Interference Contrast (DIC) optics (or equivalent accepted by the University). There must be a clearance of at least 30 cm between the bottom of the objective and the top surface of the platform. The bottom photon detector (PMT), condenser and DIC optics (or equivalent accepted by the University) must be removable for swaping between in vivo and vitro setups.
1.3 The objective must be motorized in the z-dimension by way of motoric movement, PIEZO elements or deflection methods.
1.4 Objectives for in vivo imaging:
- Must be capable of 16x or 20x magnification.
- Numerical Aperture (NA) must be 0.9 or better (high numerical aperture capabilities)
- Water immersion must allow for a working distance of ≥ 3mm1.5Objective for electrophysiology: Must have a long-working distance (≥ 2.0 mm) and high (NA ≥ 0.8) water-dipping objective for electrophysiology, DIC (or equivalent accepted by the University) and fluorescence imaging.
1.5. Objective for electrophysiology: Must have a long-working distance (≥ 2.0 mm) and high (NA ≥ 0.8) water-dipping objective for electrophysiology, DIC (or equivalent accepted by the University) and fluorescence imaging.
1.6. The system must be compatible with Gradient-Index (GRIN) lens imaging for in vivo imaging of head-fixed awake mice.
2. AOD Microscope
2.1 Must be based on Acousto-Optical Deflector technology (or equivalent accepted by the University).
2.2 The microscope must include a light path with camera to orient the specimen. The system must come equipped with Infrared Differential Interference Contrast (IR-DIC) optics (or equivalent accepted by the University) for brain slice visualisation while using the camera.
2.3 The microscope must be driven by two lasers: One for imaging and one for photostimulation (i.e., uncaging of caged molecules or optogenetics) at two different wavelenghts. The coupling must allow for two different wavelengths to be used for imaging or photostimulation. Photostimulation and imaging must occur quasi-simultaneously (in the milisecond range), with optical cross allignement of imaging planes in the two modalities of the x, y and z planes.
2.4 Overall sensitivity as well as image repetition rates must be capable of volumetric imaging of genetic (e.g., GCaMP), organic dye (e.g., Fluo4FF) indicators and multi-point scanning of voltage indicators in user-defined regions of interest (ROIs) at arbitrary points. For 2D imaging of a 510 x 510 pixel frame, for example, the system must acquire a minimum of 40 fps, or greater. The size of the 2D ROIs cannot be fixed and must be user-defined. The imaging plane during acquisition must be capable of being rotated arbitrarily in 3D.
2.5 The system must allow for imaging of multiple user-defined ROIs in a 3D volume
2.6 Beam Stabilization and Dispersion Compensation must be integrated across the 750 – 1050 nm wavelength range.
2.7 Must have non-descanned (NDD) GaAsP detectors with quantum efficiencies at ≥ 40%
2.8 The system will be used to image fine structures such as neurites (e.g., dendritic spine or axonal boutons) in head-fixed awake animals. Motion artifacts are notoriously problematic in this approach. The system must therefore have user-defined three-dimensional real-time motion correction capabilities (or equivalent accepted by the University); allowing the images to track the desired structure in real time.
2.9 The system must have the capability for targeted optogenetic and photostimulation of user-defined ROIs of different sizes (e.g., cell bodies or dendritic spines) in 3D. These stimulations must be able to be performed concurrently while imaging user-defined parameters.
2.10 The system must accommodate the following excitation wavelength pairs for simultaneous stimulation/imaging experiments: 720-750/810-830 nm; 720-750/900-950 nm; 1020-1040 nm/810-830; and 1020-1040 nm/900-950 nm
2.11 The system will be driven by a pair of existing lasers. As such, the proposed system must be compatible with the following make/models: One INSIGHT X3+ DA DeepSee laser, and ONE Mai Tai DeepSee Laser; both from Spectra Physics, inc.
2.12 The system, including the two existing lasers (identified in 2.11), must fit onto an existing optical table that is 150 cm x 360 cm, and in a room of 300 x 610 cm. The clearance between the optical table and the ceiling is 170cm. As such, the instrument proposed must be installed and fully functional within this allocated space.
3. Acquisition Software and workstation
3.1 The system must include software to run image acquisition and photomanipulation experiments. The software offered must enable, via digital hardware (TTL) or software (API) interfaces, the ability to control and exchange experimental parameters and image data in real time. In particular, the software must be able to be used either as master or slave during open and closed-looped behavioral experiments. Imaging or photomanipulation sequences must be triggered by TTL pulses with 2 ms (or less) temporal precision. The software must be compatible with Windows 11.
3.2 The proposed solution must include a Desktop PC that is capable of running the necessary software; as well as any and all required peripherals.
4. Other Requirements
4.1 Installation and training must be included.
4.2 Must include a minimum of one (1) year of warranty on parts and labor.
4.3 Must be capable of providing up to a total of 5 years of warranty
4.4 If needed, the instrument must be cooled by way of an air-cooled chiller, or a closed-loop water chiller.
4.5 The instrument must meet North American (NA) voltage requirements. If a transformer is required to achieve this, one must be included in the proposal.
