Statement of Need.pdf

Statement of Need (SON)

1. Project Title

Nikon AXR-NSPARC-ABLATION-FLIM Microscopy System Purchase

2. Statement of Need and Objectives

The acquisition of the Nikon AXR NSPARC Ablation FLIM microscope is critical to advancing cell biological research at our institute. Our current Nikon A1R confocal microscope, which has served us for over 12 years, is now technologically outdated. It no longer meets the requirements for sensitivity, stability, and resolution, and frequent system malfunctions are increasingly impeding research productivity. Upgrading to the Nikon AXR NSPARC Ablation FLIM microscope will address these limitations and fulfill urgent research needs.

This new microscope provides not only substantial improvements in confocal imaging but also introduces three transformative capabilities that are essential for ongoing and future studies:

1. Enhanced Confocal Imaging Performance: The AXR detectors offer a significantly larger field of view (up to 25 mm diagonal, four times greater than our existing system) and faster scanning speeds, while preserving multifunctional imaging features. This upgrade will enable more comprehensive and efficient data collection than is currently possible.

2. Super Resolution and Single Photon Detection: The NSPARC array detector delivers unprecedented sensitivity and super-resolution imaging, achieving up to 100 nm spatial resolution. This performance exceeds that of any microscope presently available in our core facility, providing researchers with the ability to visualize cellular structures at the nanoscale.

3. Laser Ablation Capability: The integrated laser ablation system allows for precise, non-contact manipulation of cells and tissues. Researchers can selectively damage, remove, or sever specific cells, organelles, or subcellular structures within living specimens. This capability will facilitate studies in mechanobiology, cell-cell interactions, and organelle manipulations, which are unattainable with our current instruments.

4. FLIM Module Integration: The FLIM module supports fluorescence lifetime imaging, offering critical insights into molecular interactions and the cellular nano-environment. Its compatibility with advanced probes, such as the Flipper- TR membrane tension sensor, will directly benefit several ongoing cell biology projects in the NHLBI.

5. Unique Resource for the NIH Campus: The combined features of laser ablation and confocal-FLIM imaging in a single platform are unique on the NIH campus. This acquisition will create an unparalleled resource, supporting innovative research projects and ensuring our institute remains at the forefront of biological discovery.

In summary, this acquisition represents a vital investment in our research infrastructure, ensures that our scientists have access to a state-of-the-art microscope required for pioneering cell biological studies, and maintains the institute’s leadership in innovative research.

3. Product Specifications/Features/Salient Characteristics

The Nikon AXR NSPARC Ablation FLIM Microscopy System must meet the following specifications and include these salient features:

• Nikon Ti2-E Inverted Microscope Body o 25 mm diagonal field of view (FOV) for wide-area imaging o Equipped with 4th generation Perfect Focus System (PFS), ensuring optimal focus stability during long-term imaging o Fully compatible with the PicoQuant FLIM system (feature unique to Nikon)

• AX-R Confocal Duo-Scanner System o Includes both resonant and Galvano scanners o Resonant scanner enables high-speed imaging up to 720 frames per second at

2048 x 2048 pixel resolution o Galvano scanner provides high-resolution imaging up to 8192 x 8192 pixels, supporting Nyquist sampling criteria for enhanced image quality

• NSPARC Detector o 5x5 array of single-photon avalanche diodes (SPADs). This detector is unique to Nikon systems o Achieves lateral resolution of 100 nm and axial resolution of 300 nm with extremely low noise level at single photon detection sensitivity.

• AX-R DUV-B Tunable Detector o Allows for spectral acquisition across up to 70 channels for multiplexed imaging

• Laser Sources o Multiple visible laser lines that provide both continuous wave (CW) and pulsed modes (405, 440, 488, 514, 640 nm) to satisfy both confocal imaging and FLIM imaging in addition to a CW 516 nm line for common confocal imaging.

o The pulsed laser lines has adjustable repetition rate from single shot to up to 80 MHz depending on the wavelength.

o The LUNF laser unit delivers the pulse lasers at 405 nm and 473 nm to provide precise photo stimulation capability.

o VU OMS provides an onboard 355 nm 1KHz pulsed laser for photo ablation.

• PicoQuant Photon Counting FLIM Module o Provides advanced fluorescence lifetime imaging (FLIM) capabilities o Features Pulsed Interleaved Excitation (PIE) laser sources, supporting both diode and multi-photon excitation o Adjustable excitation laser pulse repetition rates from single shot up to 80 MHz o Supports both FLIM (for nanosecond-scale molecular interactions) and PLIM (for microsecond- to millisecond-scale environmental and metabolic measurements) o Incorporates a single-photon sensitive detector and a Time-Correlated Single Photon Counting (TCSPC) unit with 0.65 ns deadtime and five-picosecond time bins for high temporal resolution o Enables rapid and quantitative FLIM and FLIM-FRET analysis via EU patent rapidFLIM fit algorithm, with fast FLIM acquisition up to several frames per second o Allows access to raw experimental data for advanced analysis

• UV Ablation Tool o Provide the capability of simultaneous and sequential imaging together with photoablation and photo-stimulation via the Optimicroscan on a dedicated light path that is separate from a confocal light path.

• Software Integration o All imaging modes (confocal, laser ablation, and FLIM) are fully integrated and controlled through NIS Elements software, providing seamless operation and data acquisition on a single platform o AI tools for confocal scanning optimization (autosignal.ai), shot noise removal (denoise.ai), and out-of-focus blur removal (clarify.ai) with deconvolution module in the system.

These features are essential for supporting advanced live cell and organoid imaging, precise cellular manipulation, and high-resolution quantitative analysis required for current and future research needs at NHLBI.

4. Delivery Information and Location

Light Microscopy Facility National Heart, Lung and Blood Institute 50 South Drive, MSC 8012 Building 50, Room 2318 Bethesda, MD 20892-8012

Point of Contact and Contact Information

Xufeng Wu, PhD Light Microscopy Facility National Heart, Lung and Blood Institute 50 South Drive, MSC 8012 Bethesda, MD 20892-8012 T: 301-402-4187 E: wuxs@nhlbi.nih.gov