Purchase_Description_(Attachment_1).pdf

PURCHASE DESCRIPTION

1. Statement of Need and Purpose: To meet ongoing requirements of NIMH intramural scientists the System Neuroscience Imaging Resource (SNIR) needs to upgrade a Leica SP8 confocal/multiphoton microscope system to add the capability of performing Fluorescence Lifetime Imagaging Microscopy and on-the-fly image deconvolution.

2. Background Information and Objective: Imaging brain sections that contain multiple fluorophores is an essential step for experiments that are being carried out by basic science investigators within the NIMH intramural program. Simultaneous imaging of multiple fluorophores allows the relationship between the proteins or mRNA molecules labeled by the flurophores to be determined at high resolution. The number of species that can be distinguished is generally limited by the number of fluorophores that can be spectrally resolved from each other. It has recently become possible to add Fluorescence Lifetime Imagaging Microscopy (FLIM) to the Leica SP8 system recently purchased for the SNIR. FLIM takes advantage of the property of fluorescent molecules that light is emitted for a characteristic amount of time (fluorescence lifetime, or decay) following a pulse of exciting illumination. Fluorescent molecules that have indistinguishable spectral emissions can have significantly different fluorescent lifetimes.

Thus, for example, the small molecule dye Alexa 488, and the fluorescent protein GFP cannot be distinguished using only spectral imaging, but are easily distinguished using FLIM. The first goal of this acquisition is add the capability to perform simultaneous spectral and FLIM imaging to an existing Leica SP8 system.

In light microscopy the ability to accurately determine the size of objects or distinguish them from adjacent objects is limited by the spread of out of focus light. The contribution to an image from out of focus light can be mathematically reduced by a computational process called deconvolution. This is a computationally intensive procedure that takes a considerable amount of time and computer power. Leica has implemented a solution that integrates with their microscope acquisition software and performs on-the-fly deconvolution. This software is referred to as Lightning and offers the ability to rapidly and efficiently improve imaging resolution. The second goal of this acquisition is to add Leica Lightning software to the existing Leica SP8 system.

3. Generic Name of Product: Upgrade existing Leica SP8 system to include Fluroscence Lifetime Imaging and on-the-fly deconvolution.

4. Purchase Description:

1) TCS SP8 FALCON

TCS SP8 FALCON (FAst Lifetime CONtrast) consists of two

NIHDA201800242 Attachment 1 main parts. 1. Electronics and interface for synchronization of FLIM excitation sources with the detection logic. 2. LAS X FLIM solution for ultrafast acquisition, processing and analysis of FLIM images. The acquisition software, fully integrated with LAS X, enables FLIM with any imaging workflow (time lapse, 3D, lambda scan, combination with mosaic imaging (LAS X Navigator) possible). It is possible to combine multiple detectors to increase the acquisition speed. The software displays the fastFLIM (mean arrival time) image and the photon/pulse histogram in real time. It derives the IRF (instrument response function) and includes Exponential Reconvolution and Exponential Tail fitting with up to 5 components, deadtime correction and incomplete decay fitting algorithm. The analysis can be done on ROIs as well as on series of images (time lapse, zstack, mosaic) which can analyzed as a complete dataset or separated into images. Binning for pixels or for the lifetime scale is enabled as well as timegating. To allow for highest lifetime precision, Photon Filters can be applied. In single pixels the software fits and displays lifetimes and it can separate components according to lifetime, leading to multiple channel display. The software has the display option "free", where any fitted parameters of choice (e.g. lifetimeamplitudes) can be displayed on a rainbow scale. LAS X FLIM includes the FLIM1FRET analyzer. The analysis software provides the FLIMFRET efficiency map, the apparent FRET efficiency image and a binding map. All fitting options (Overall Decay, ROIs, single pixel fit, separation into channels) are also available with the patternmatching algorithm, selecting a pattern by choosing a ROI in the image or in the Decay Diversity Map. In combination with TCS SP8 FCS TCS SP8 FALCON enables FLCS (Fluorescence Lifetime Correlation Spectroscopy) experiments.

2) FLIM excitation DIVE Each MP laser at the instrument requires its own package.

The package is needed to generate a synchronization signal from an MP laser. The synchronization signal is a prerequisite for lifetime based measurements. The package comprises following main constituents:

Trigger diode assembly for generation of the synchronization signal (applicable till 1100 nm) Optical coupling MP excitation Software controlled selection of MP excitation wavelength

3) High speed data link The High Speed Data Link enables the highspeed Data transmission between microscope and PC. It is required for FLIM and FCS.

4) LAS X Lightning Expert Leica DeconvolutionSoftwarePackage for Confocal, STED and multiphoton data including the following:

1) Wizard for online data acquisition 2) Fully automated "OneClick" functionality 3) Additional deconvolution strategies for Low SNR & High SNR 4) Adaptive extraction of image quality and adaptive deconvolution in the automatic mode 5) Elimination of boundary effects 6) 2D Deconvolution 7) Time series 8) Channel independent deconvolution 9) High performance processing 10) Expert settings for manual deconvolution of confocaland multi photon data 11) Lightning Offline deconvolutionSoftwarePackage For confocal, STEDand multi photon data

5) Expert Workstation High Power HPZ840 Workstation or equal, with Windows 10 Professional (64 bit) operating system.

2x Intel 4Core Xeon E52637 V3 3.5 GHz 128 GByte RAM NVIDIA Quadro M4000 8GB high performance GPU 256 GB SATA SSD 2x 1TB SSD RAID 0 5x 3TByte SATA hard disc drive, RAID5 10 GBit Intel X520 Network Adapter Network Cable SFP+, 3 m 9.5mm Slim Bluray Writer 1st ODD 2x 10/100/1000 Ethernet 8x USB 3 / 2x USB 2 2x IEEE 1392 B Keyboard and mouse HP 3/3/3 Warrant The specifications of the PC can change due to technical upgrades.

6) Installation

5. Salient characteristics:

• Adds Fluorescent Lifetime Imaging Microscopy capacity to existing Leica SP8 system, based on use of exsiting Spectra-Physics X3 pulsed IR laser and other system components,

• Fully integrates with existing Leica SP8 system, including spectral and multiphoton imaging using Leica LAS-X software,

• Provides workstation validated to perform FLIM and Spectral image acquisition of image data sets up to 1 TB ,

• Provides on-the-fly deconvolution of images acquired on the SP8 system.

6. Quantity: 1

7. Delivery Date: 90 days following award.

8. Warranty: All items fully warrantied for 1 year. Equipment is expected to be fully functional for greater than 5 years with minor service, as required. Service after one year may be purchased as an annual contract or on an as needed basis.

9. Option(s): N/A