Attachment 1 Purchase Description.pdf

Purchase Description 75N95021Q00064

Background In the central nervous system, oligodendrocytes are cells responsible for producing myelin, the insulation around electrically active axons of neurons. Myelin facilitates fast electrical conductance in an analogous way as rubber insulation around electrical wiring. Proper development and maintenance of oligodendrocytes has important implications on demyelinating disease, including multiple sclerosis, a disease in which immune cells attack and destroy myelin, and leukodystrophies, childhood diseases in which myelin fails to form properly leading to progressive disability. In addition, recent studies also support a role for oligodendrocytes in psychiatric disease and in memory, including in Alzheimer’s disease.

The Glial Cell Biology Unit in NINDS (National Institute of Neurological Disorders and Stroke) focuses on understanding the cell biology and building blocks of the oligodendrocyte, and how they contribute to normal brain function and disease. The oligodendrocyte is a cell that is shaped like a tree, with many branches that terminate to form myelin sheaths, which wrap concentrically around the axon in many layers, much like a long paper towel roll or a bolt of fabric. The questions primarily underly the structure of these branches – microtubules that form the roads of these cells – and the cargos that travel along them, including organelles and mRNA genetic messages. Studying the oligodendrocyte has many technical challenges and we approach our experiments from many levels: 1) in vivo at the level of whole brains using rodent models, 2) in vitro at the cellular level using primary cell culture of cells purified from rodent brains and grown on either a 2D flat substrate or on a 3D substrate, 3) at the molecular level using recombinant proteins.

Statement of Need and Purpose This proposed equipment purchase allows us to address our mission-critical experiments using one microscopy system that combines that functions of 3-4 microscopes. Combining multiple functions in one system not only saves on space, but also saves money.

This system combines the following techniques and applications:

1. Spinning-disk confocal microscopy used to image living cells with high speed and resolution,

2. Epifluorescence microscopy used to image rodent and human brain for histology,

3. TIRF (total internal reflection fluorescence) microscopy used for cell-free biophysics assays for in vitro reconstitution of cytoskeletal dynamics and transport,

4. Photostimulation/photobleaching to manipulate and record cellular and protein dynamics,

5. Semiautomated image acquisition for efficient workflows and high-throughput screening.

Generic Name of Product:

Multi-module custom microscopy system with spinning-disk confocal microscopy, epifluorescence microscopy, TIRF (total internal reflection fluorescence) microscopy, photostimulation/photobleaching unit, and semi-automated image acquisition.

Minimum Salient Characteristics The technical specifications of this customized system will contain the following functions/modalities:

1. Epifluorescence imaging of stained brains.

The following components will be used for optimized imaging of brain slices that have been stained with fluorescent antibodies:

a. Separate LED light source (Solid state white light source with 365-680nm wavelength range) These light sources minimize bleaching and are not as intense as laser light sources needed for confocal and TIRF microscopy.

b. Lower magnification objectives.

4-5X and 10-20X objectives are ideal for imaging brain sections.

c. Motorized XY stage.

d. Additional dichroic for 350nm (DAPI)

e. Customized workflow and artificial intelligence aided imaging.

Each brain section will have different sizes and contours. In order to capture the full brain section, stitching capabilities are needed. Software with automated contour detection will save time by not needing to capture more images than necessary or align / find the center of the brain section. higher magnification images of certain brain regions will be taken, and an automated manner will be needed. This not only saves times, but ensures images are not acquired in a biased manner (e.g., cherry picking).

2. Rapid live-cell imaging using a spinning-disk confocal.

The following components will be used for imaging both flat 2D-cultured cells and thick 3D-cultured cells. In order to capture by video the dynamics of microtubules and their cargos, it will needed to be optimize for both speed and thickness. The following components are necessary for these purposes:

a. Spinning disk.

This device modulates laser light passing through small pinholes and is very important for speed of imaging.

b. Separate camera (back-lit scMOS).

A camera that is separate from the TIRF modality ensures that less light is lost. The new generation of back-lit scMOS cameras developed in the last 1-3 years have higher sensitivity (95% quantum efficiency) and higher spatial resolution (~6-7 um pixels).

These technical specifications are very important for imaging mRNA cargos, which are a challenge to video because they are both very dim and very fast.

c. Piezo stage.

This motorized stage allows fast captures across the z-dimension (e.g. up and down).

This is especially important when taking movies of cargos moving in thick 3D-cultured cells that are 200 microns or thicker; in other words, these movies need to be captured with fast frame rates and across all z-stacks.

d. Drift-correction hardware.

As samples and/or equipment heat up or in response to structural vibrations or forces (e.g., a nearby door closing), minute drift can occur in the sample, but this is amplified on the microscope, leading to a blurry image. Drift correction hardware uses an internal light source to calculate the refractive index and automatically makes adjustments in the z-direction (e.g., up and down) to prevent a blurry movie. This is especially important in long-term movies (e.g., hours, days) of the type to take to study myelin sheath elongation.

e. Multiple colored lasers (405, 488, 561, 640).

This allows to image/monitor different proteins in the same cell.

f. Environmental chamber.

In order to image living cells that are unstressed, the chamber needs to modulate and maintain constant: 1) temperature, 2) CO2 gas content, 3) humidity. In addition, it should eliminate background from ambient lighting (e.g., overhead lights); this is usually done by using dark materials to make the chamber.

g. Analysis modules.

Software features needed during acquisition include intensity measurements and kymograph generation.

h. Customized workflow and artificial intelligence aided imaging.

The cells will be cultured on a variety of substrates and plates (e.g., 35-mm imaging dishes, 12-well, 96-well plates). For high number of samples, like 96-well plates, it is required to automate that imaging for: 1) speed and 2) non-biased imaging/screening.

In addition, it is needed to image multiple cells in 35-mm plates; for example, in long-term imaging experiments, it will be necessary to image 20+ cells across many hours or days and it will be needed to automate this workflow. This function requires that the software memorize the location of each cell and integrate it with the drift correction hardware.

3. Manipulation of fluorescent proteins in cells by photobleaching/photostimulation.

This function will allow to perform two types of experiments. First, bleach fluorescent proteins and watch their recovery to understand their dynamicity (i.e., do they form rigid, stable structures or labile, dynamic structures?). Second, stimulate fluorescent proteins and cause them to change colors; this allows to segregate one protein into different populations and separately monitor their movements and lifetimes (i.e., how long-lived are they?). The following components are needed for these experiments:

a. Photostimulation device.

Two different wavelengths (488/561, 405) are needed for the specific fluorophores we intend to use.

b. Simple, fast, and effective calibration.

In order to target specific regions of the cell, the laser needs to hit the sample at the correct location. Calibration needed for location accuracy and should be able to be performed by non-expert users prior to each experiment.

c. Analysis modules.

For photobleaching experiments, we will need to measure recovery time.

d. Customized workflow and artificial intelligence aided imaging.

For photobleaching experiments, it is not known when to stop imaging (e.g., 1 minute or 5 minutes?). Logic based workflows can automate this (e.g. stop imaging once the intensity has reached a certain value). This is valuable for: 1) saving the experimenter’s time by being able to walk away while the system images dozens of cells, 2) increases the number of cells that can be healthily imaged per sample/dish, which increases rigorousness / robustness of data.

4. Protein dynamics imaging using TIRF (total internal reflection fluorescence) microscopy.

TIRF is a special type of microscopy in which light is bounced off the imaging surface at an angle (rather than through the entire sample) to more clearly capture the activity of individual proteins or of cellular activity near the membrane edge. TIRF will be used for recombinant protein assays in which we look at: 1) the formation and dynamicity of microtubules as well as 2) cargo motility assays to characterize the minimal elements necessary to activate transport.

a. Motorized TIRF and software controlling the angle.

It is important for reproducibility to have a motorized TIRF controls (rather than manual controls) that adjust the angle of light so that experiments can be repeated under the same conditions.

b. Separate camera (back-lit scMOS).

A camera that is separate from the confocal modality ensures that less light is lost. The higher sensitivity (minimum 95% quantum efficiency) and higher spatial resolution (minimum~6-7 um pixels) is required.

5. A computer and monitor A computer and monitor are needed for processing images and visualizing the field of view. The following specifications will optimize speed of acquisition and visualization of images:

a. 30+ inch monitor with 4K resolution

b. 3.7 GHz processor

c. 64 GB RAM

d. 8 GB video card

e. 512 GB SSD O/S drive, 1TB SSD data drive

f. 12 TB data storage

g. Mouse and keyboard

h. 3-years warranty for parts, labor and service

6. Seamless and User-Friendly Integration

The integration of these components and modalities is important. This integration occurs at both the hardware and software level. At the hardware level, it is important to conserve light so that as much of the signal is captured and in a non-noisy way. At the software level, it is important to have a user-friendly interface (intuitive controls, all modalities controlled by one software, takes less than 1 day to learn).

Quantity

One (1) System

Delivery, Installation and Training

The Contractor shall deliver and install the equipment to the NIH in North Bethesda, Maryland within 90 days after receipt of order. The equipment shall be delivered and installed between the hours of 8:00am and 5:00pm EST, Monday through Friday. Delivery must be Freight on Board (FOB) Destination and include inside delivery. The equipment shall be delivered and installed in coordination with the Contracting Officer’s Representative (COR) at the time the equipment is ready to be shipped.

Contractor shall provide on-site training within 30 days weeks of equipment installation.

Contractor shall deliver, install, and provide training on the system within 120 days after receipt of order.

Other Requirements

The Contractor shall provide an Original Equipment Manufacturer (OEM) certified service technician to perform setup and installation for staff members.

Warranty - The Contractor shall warrant that all computer equipment (computer, monitor) will be free from defects for a period of at least three (3) years from the date of installation, inspection by the Government, and acceptance. The Contractor shall further warrant that all optical and mechanical components will be free from defects for a period of at least five (5) years from the date of installation, inspection by the Government and acceptance. Warranty service shall include trouble-shooting capabilities based on complete knowledge of the entire system. A rapid response (within one business day) to service requests is required. The Contractor shall demonstrate that it is capable of providing this level and speed of service. Warranty shall not include routine or periodic maintenance services.