Attachment 1 Purchase Description.pdf

1. Statement of Need and Purpose:

The National Institute of Neurological Disorders and Stroke (NINDS) is establishing a new laboratory: Spatial Navigation and Memory Unit. The laboratory will use cutting-edge imaging and behavioral approaches to investigate the neural basis of spatial navigation and memory at the circuit, synaptic, molecular and cellular levels. The new laboratory requires two multiphoton microscopes to perform the research.

2. Background Information and Objective:

the Spatial Navigation and Memory Unit (SNMU) aims to investigate the neural basis pf spatial navigation and memory. Specifically, the experiments of the SNMU will largely use in vivo two-photon optical approach to measure calcium dynamics of large number of neurons, observe their structural and molecular properties at the subcellular level, and modulate the activity of individual neurons. The laboratory will require two multiphoton microscopes, which will be capable to perform the above experiments and to perform sequential full field photoactivation with LEDs and scanning. The collected data using the microscopes will be used to understand the functional mechanism of the medial entorhinal cortex in spatial navigation.

3. Generic Name of Product:

Multiphoton fluorescence microscopes

4. Purchase Description:

Two Multiphoton Fluorescence Microscopes will be required. One microscope (M1) will be used for in vivo two-photon imaging and another microscope (M2) will be used for in vivo simultaneous two-photon imaging and two- photon optogenetics.

a. The microscope can accommodate a virtual reality environment customized with the following components: a Styrofoam toroidal display, a projector, mirrors, and a translational stage. Translation along X and Y axes, up to 1.5”, in addition to the standard Z axis 1.25”. This allows use of simple, non-motorized, experiment-specific stages for in vivo experiments where the specimen is held stationary while the scope is moved around (such as a head-fixed mouse running on a ball or a customized tread

PURCHASE DESCRIPTION

Multiphoton Fluorescence Microscopes - Dr. Yi Gu Spatial Navigation and

Memory Unit (SNMU), NINDS mill). This also enables Atlas Imaging, a software feature for visualizing a montage and picking out ROIs from a large sample area.

b. M1 and M2 will share a dual-output tunable laser. One output of the laser will be shared by the two microscopes for two-photon imaging and the laser intensity after the objective must be > 150 mW on both microscopes. The second output will be used by M2 to perform two-photon optogenetic stimulation.

c. In order to focus the sample to the tilted objective, the software must control the movement of the microscope X, Y, Z motors together to move the objective along its optical axis and perpendicular to the sample surface.

d. M1 and M2 must be placed on the same optical table (5 ft × 10 ft) and leave enough space for a dual-output tunable laser (~ 450mm × 820mm) and two virtual reality setups. The maximal required space of one virtual reality setup is 840mm × 760mm while the main microscope body can be placed within the setup.

e. In addition to the imaging galvos, M2 should contain the second set of matched

6mm Cambridge galvanometers in the same scan head to provide high-precision two-photon optogenetic manipulation.

4. Salient characteristics

a. The microscope can be used with awake, immobile mice running on an air-floating ball. While ensuring the stability of the microscope, the distance between the bottom of the objective and the optical table should be about 350 mm. The microscope shall be specifically designed for in vivo work. It shall allow for plenty of room underneath the objective to accommodate diverse experimental needs. In addition, the design shall allow plenty of room around the scope to bring in behavioral equipment close to the animal without spatial constraints. There shall be adequate space between objective and the scope body to accommodate cases where the animal’s orientation must be changed for behavioral stimulation. The microscope shall be able to perform off-axis imaging without affecting the efficiency of two-photon excitation and signal collection. The desired imaging/optogenetics angle shall be ranged from 15o to 35o in both clockwise and counterclockwise directions. To ensure the stability of the microscope during the experiments, the off-axis imaging must be achieved by only rotating the objective mounted on a nosepiece without rotating the entire microscope body.

b. The microscope shall contain 8kHz resonant galvanometer for high-speed scanning at 30 frames per second (512 × 512 pixels) with user-definable resolution and region of interest. Resonant scanner must be relayed to standard XY imaging galvos to provide flexibility to image in galvo mode or resonant mode and to re-center the resonant scan for dynamic ROIs (Region of Interest) in resonant mode.

Frame rates greater than 1000 fps; 790fps for 512 x 16 ROI or 1319fps for 512 x 8 ROI. High temporal resolution.

c. The microscope must achieve a 1.4mm × 1.4mm field of view with a 16 × objective in resonant imaging mode and galvo imaging mode (Field Number 28.3).

d. The microscope shall have at least two GaAsP detectors combined with appropriate filter sets (changeable by users) for high sensitivity simultaneous detection of green and red fluorescence signals. GaAsP detectors must have built-in high-speed shutters to modulate with optogenetic stimulation and avoid exposure to ambient light. Photoactivation must be able to be performed rapidly during imaging when high speed shutters are used. Must include high sensitivity GaAsP detectors for QE > 45%. High-speed shutters must be integrated into the detector mounts to protect the PMTs from exposure to any Photoactivation light or ambient light when not in use for imaging.

e. The microscope must allow the excitation of the sample at different wavelengths, such as 920nm, 1040nm, and 1100nm. Optics can be configured for 700-1700nm to support 3P excitation with no compromise to imaging/PSF quality.

f. M1 must contain a Z-piezo with proprietary focus algorithms for high-speed Z-series

(0.1 µm step size, 250 µm range)

g. M2 must contain electrically tunable lens (ETL) for simultaneous stimulation and 3D imaging, with stimulation Z position decoupled from imaging path. ETL must meet the following specifications:

1. 480um range

2. <15ms step response

3. Magnification corrected at each limit of range to 1.1x

4. Spherical aberration corrected with custom aspheric lens

5. Axial PSF measured 5 – 5.5um throughout range (16x NA 0.8 objective)

h. M2 must be upgradable for using a Spatial Light Modulator to enable multipoint holographic simultaneous activation. SLM pathway must be decoupled in Z from the imaging pathway.

i. To maintain the center position of the mouse in virtual reality during off-axis imaging, the microscope body should be able to laterally translate for at least 30 mm.

j. The function of the microscope shall be controlled by a software supported by the vendor. The software must allow users to determine specific scan parameters, such as straight, freehand and circular line scan patterns, pixels per line and lines per scan (1- 2048), laser intensity, scan zoom (up to 128 ×), and scan rotation (0 to 360o). The software of M2 should allow the user to determine parameters for two-photon optogenetic stimulation, such as scanning pattern (e.g. raster or spiral scan), laser intensity, region of interest, and synchronization of the stimulation relative to the time of the imaging. The software should also allow users to customize functions according to the needs of the experiments. Provide a highly intuitive user interface to capture high resolution images of large samples (whole brains in 3D). Provide a preview mode which allows users to setup the acquisition, and the software combined with high precision stages collect the images in “tiles” to construct the whole image in 3D. Data shall be made available to an external program so that the data can be analyzed in real time if required. The software shall provide accurate timing information about scans using pixel clock, line clock, and frame clock signals (TTL). These signals shall be able to trigger external devices. In addition, the software shall provide the ability to produce up to 8 analog outputs and record from 16 analog inputs synchronized with photo-stimulation experiments.

k. Major functions of the microscope, shall include two-photon imaging and two photon optogenetics, must be demonstrated in multiple publications.

5. Other Requirements:

a. The contractor shall provide software support for life of the instrument and one year of warranty covering parts and labor.

b. The contractor shall provide an Original Equipment Manufacturer (OEM) certified service technician to perform setup, installation, and an on-site 2-day training session for staff members. The contractor shall also provide one day of follow up training.

6. Quantity:

Two systems

7. Delivery:

The Contractor shall deliver and install the equipment within 90 days after receipt of order. Delivery must be Freight on Board (FOB) Destination and include inside delivery. On-site training must take place within two (2) weeks of equipment installation. The equipment shall be delivered and installed in coordination with the Contracting Officer’s Representative (COR).