Attachment_1__ SON LSM 980.pdf

STATEMENT OF NEED (SON)

1.0 TITLE

Acquisition of a Carl Zeiss LSM 980 Airyscan 2 Laser Scanning Confocal Microscope

2.0 BACKGROUND

The Laboratory of Cancer Biology and Genetics (LCBG). Microscopy Core was established by the NCI in September 1997. The Core Facility provides state-of-the-art confocal and fluorescence microscopy and digital imaging to LCBG investigators, as well as scientists from other laboratories in the NCI. The LCBG Core aims to support high quality clinical and translational research in cancer biology and to train students, residents, and fellows in the field of confocal and light microscopy. The Core Facility also provides services and opportunities for collaborative research to investigators throughout the NCI.

The LCBG Microscopy Core Facility has a need for a new scanning laser confocal microscope to replace the existing Carl Zeiss LSM 780 microscope. The LSM 780 microscope that was purchased in 2013 is obsolete and is no longer supported by the Zeiss service contract. Any breakage or problem with the instrument is potentially not repairable/terminal due to the lack of support and parts availability for the instrument. In order for the Core Facility to remain state-of-the art, it is imperative that we replace the existing system with a confocal microscope that has updated detectors with exceptional sensitivity, improved spectral scanning capability, increased speed, lower phototoxicity, increased resolution, and updated software for more sophisticated experiments.

Over 200 scientists from at least 50 laboratories have used the LCBG Core Facility, and microscopy conducted in the LCBG core has contributed to 155 publications in peer reviewed journals. Confocal microscopy in the LCBG Core has been used for fixed specimen and live cell imaging to observe cellular component trafficking, localization, colocalization, drug effects, stem cell marker distribution, and organoid structure characterization for basic research, translational, and clinical studies. The system is used on average 40 hours per week. The core personnel provide training in the field of confocal microscopy and opportunities for collaborative research to principal investigators, research fellows, residents, and students throughout the NCI. Technology has significantly advanced in the past decade since the Zeiss LSM 780 was introduced to the Core, and the LSM 980 with Airyscan 2 is two generations more advanced than the existing obsolete instrument. The acquisition of this laser scanning confocal microscope will allow the LCBG Microscopy Core Facility to better support the current and future imaging needs of researchers in the NCI/CCR.

3.0 TYPE OF ORDER

This is a Firm Fixed-Price Purchase Order.

4.0 SPECIAL ORDER REQUIREMENTS

4.1 PRODUCT FEATURES/SALIENT CHARACTERISTICS

The LCBG Microscopy Core Facility has identified a need for a new laser scanning confocal microscope to replace the existing LSM 780. To meet the needs of the researchers currently using the Core Facility, the new system will need to possess several unique characteristics NCI requires a brand name Carl Zeiss LSM 980 with Airyscan 2 Confocal Microscope System with two cutting edge detection units that can be used simultaneously or individually to allow for super resolution down to 90nm in XY with an innovative 32 channel GaAsP PMT array concept. Each detection channel must function as a single very small pinhole of 0.2 AU to increase resolution, while the overall detector captures 1.25 AU and increases signal-to-noise by 4-8 times over traditional GaAsP PMT based confocal systems. The unique 32-Channel GaAsP PMT area detector must have a Zoom optic to adapt illumination for different objective lenses and acquisition modes. It must also include a micro lens array that provides a light fill factor of > 95%. The system must include a filter wheel with up to 7 emission filters, each providing dual detection windows for fast multitrack imaging in combination with secondary beam splitters to expand the usability for multicolor samples. The system must also include a Sample Navigator module to perform a low power overview scan with autofocus function using a cooled monochromatic camera to create an overview image for sample navigation and identification of regions of interest.

The following brand-name salient characteristics for the new instrument have been identified:

Inverted research microscope with adaptive focus control and motorized objective revolver, motorized fluorescence axis for 6 filter cubes, motorized shutter, motorized light axis, motorized focus, condenser, lamp mount and confocal mounting kit.

TFT touch panel independent of microscope stand with focus control and access to all microscope motorized functions.

Automated Component recognition for objective and filter sets with environmental control units for heating, cooling, humidity, and CO2 concentration fully integrated into the microscope stand via TFT touchscreen control.

6 position motorized nosepiece for high resolution objectives with the longest working distance possible including 40x/1.2NA water immersion FWD=0.28mm, 10X/0.30 WD=5.2mm, 20x/0.8NA FWD=0.55mm, and 63x/1.4NA Oil Immersion DIC FWD=0.19mm.

Light source with seven switchable LED excitation lines for fluorescence imaging fully controlled through software.

High efficiency hard coated shift free quad band filter for DAPI, GFP, CY3, CY5, and a tri band filter for CFP, YFP and mCherry for standard visual fluorescence imaging

Rear port configuration for future upgrade to Two Photon imaging.

Motorized X,Y scanning stage with Z-Piezo to enable quick and high-resolution sample positioning.

Condenser with an LED array for contrast enhancement and a deflecting mirror and CMOS camera for obtaining an overview image Seven diode laser lines (405nm, 445nm, 488nm, 514nm, 561nm, 594nm, 639nm) with independent power dynamics for Vis lasers. Absolute linear power control using control electronics for AOTF, with direct modulation of 405 laser diode with 1:500 dynamic range (no ND filter for laser attenuation in the lower power range). Lasers must be power regulated with output power of laser constant till end of lifetime.

Scanners with two independent, galvometric scan mirrors with ultrashort line and frame fly-back. Short scanner turnaround times, > 85% of the frame time is effectively used for image acquisition. Absolute linear scanner movement to ensure equal pixel dwell-times as a prerequisite for any quantitative study, including RICS.

Scan resolutions from 32x1 up to 8192 x 8192 pixels, freely adjusted by the user. Line Scan with a maximum frequency of 6830 lines/sec. Spot scan for continuous or time series imaging with minimum dwell time of 1.23 microseconds. Scanning zoom (hardware-zoom) from 0.6x to 40x, digitally adjustable in 0.1 steps for magnifications up to and beyond the optical limits of resolution.

A 4+2-Channel Quasar Detection Unit, consisting of of a calibrated 4 channel multielement GaAsP detector with typical QE >45% (peak) and two flanking single multi-alkali PMT detectors. The 4 channel Quasar GaAsP detector must include micro lenses in front of the individual detector-elements for improved light efficiency. The 4 channel multi-element GaAsP detector must show very low dark noise and allow for spectral binning of individual channels without sacrificing signal/noise ratio.

The main beam splitter must be a TwinGate arrangement to suppress laser light more optimally than a crystal-based acoustic optical beam splitter (AOBS). The TwinGate main beam splitter arrangement in combination with the Quasar detection unit must have laser suppression with an OD typically >7, making it possible to detect emission light emerging from less than 10nm away from the laser. It must be possible to select emission bands for detection, omitting secondary dichroics & emission filters, even over laser lines due to the effective laser blocking.

The microscope, laser, scanning module, accessories, along with data acquisition and synchronization must be managed through real-time electronics utilizing an oversampling read-out logic. Gain calibration and linearization of the Quasar Detection Unit must allow allow for all 6 channels to be used for spectral acquisition and analysis.

The Quasar Detection Unit must use holographic grating rather than a prism. Holographic grating should ensure an even spectral dispersion over the entire visual spectral range (380- 750 nm). A spectral recycling loop should recover non-separated light and redirect it onto the holographic grating for optimum sensitivity.

Acquisition of the entire spectral range should be possible as a lambda stack (370-760 nm).

More than 10 fluorescent dyes should be able to be imaged and separated by spectral unmixing. Reproducible high-resolution spectral data acquisition with 8.9, 4.3 or 2.9 nm steps must be possible through sequential spectral scanning.

Emission Fingerprinting must be performed on data acquired in channel mode. Spectra should be stored in the spectral database and later used for reference-based linear unmixing.

Spectral bandwidth for lambda stack acquisition should be adjustable by the user.

Emission Fingerprinting: Reliable separation of different fluorescent dyes even with highly overlapping emission spectra (e.g. simultaneous detection and separation of FITC, GFP, YFP, and autofluorescence) based upon known, previously recorded reference spectra. The separated dyes (crosstalk free) are displayed in separate dyechannel images along with the option to display a merged image.

Advanced Tools for Emission Fingerprinting/Linear Unmixing including 1) Weighted unmixing: improved unmixing algorithm for better unmixing results and 2) Channel with Residuals: displays the uncertainties of the unmixing result.

Automatic component extraction (ACE) of spectra from a lambda stack for a one- click identification of possible reference spectra.

Free combination of manually defined reference spectra, automatically extracted reference spectra, and reference spectra stored within the database can be used for performing Linear Unmixing on a lambda stack.

Reference spectra can be stored in a database and later recalled. Spectral information is stored automatically with each unmixed image for traceable and reproducible workflow and unmixing experiments

Online Fingerprinting: Image channels are not defined by the traditional detected emission bands but rather by referenced spectra. On-the-fly online unmixing occurs using these referenced spectra to linear unmix the emission bands of overlapping dyes during the image acquisition.

The system needs to have an Axiocam 705 mono R2 high performance CMOS camera which includes driver software 64bit, USB 3.0 PCIe x1 interface. Number of Pixels: 2464 (H) x 2056 (V) = 5.07 Mega Pixels Pixel size: 8.5 mm x 7.1 mm Chip size: 11.3 mm x 7.1 mm, Spectral range: With protection glass app. 350 nm to 1000 nm Quantum Efficiency 72% at 520 nm (including sensor cover slip).

The camera must be supported by Windows 10 x64 Prof./Ultimate SP1 with application support by ZEN 3.6 or higher and interface via a C-Mount Adapter 60N C 1" 1.0x

The system needs to have an Airyscan 2 Super Resolution Detection system consisting of a 32-Channel GaAsP area detector with zoom optic to adapt illumination for different objective lenses and acquisition modes, and a microlens array that provides a light fillfactor of > 95%.

The AiryScan 2 detector needs to have different modes of operation including:

Superresolution (SR and Confocal sampling), and Multiplex modes (4Y, 8Y). The Airyscan detector should be able to function as an additional GaAsP confocal detector in addition to the Quasar detector.

The Superresolution mode must allow to increase the image resolution in all three dimensions. This resolution increase should come with an inherent improvement of the signal to noise ratio.

The system should have a lateral resolution of up to 90 nm and axial resolution of 270 nm at 488 nm excitation for 3D data, and a lateral resolution of up to 120 nm at 488 nm excitation for 2D data.

Super resolution mode should have a 4-8x increased sensitivity (compared to GaAsP-PMT detector of traditional confocal at 1 AU pinhole opening).

The multiplex mode should have speeds up to 47 fps at 512x 512. And be easily accessible via a single button that adjusts sampling and pixel size.

In Multiplex mode 8Y, sampling and therefore achievable resolution should be continuously adjustable from Confocal (1x Nyquist) to Superresolution (2x Nyquist). This allows flexibility to setup the conditions for the experiment, depending on which resolution or acquisition speed is needed. For ease of use, 2 sampling options are easily set with single buttons: Superresoultion and Confocal.

The 4-8x enhanced Signal to Noise must be preserved for high speed acquisition in Multiplex mode.

Excitation with a multiphoton laser and detection with Airyscan 2 in all modes must be possible.

AiryScan 2 must be able to be used for all available multidimensional acquisition setups and the available combinations in ZEN software, e.g.: z-stack, multicolor, multiple positions, tiling, ROI, bleaching and photomanipulation experiments, and time-series. Laser line flexibility from 405 nm to 639 nm must be available for covering the entire visible spectrum utilizing the lasers available from thhe confocal.

The system must be capable of Airyscan Joint Deconvolution (jDCV) to push Airyscan resolutions even further. XY resolutions of 90nm and Z resolutions as low as 270nm with processing.

Each workstation should be a Microscopy Workstation Premium model: the HP Z6 G4 Rev.

2 (O). These workstations run on Windows 10 LTSC 2019 LSM 9 (O) System and are powered by an Intel C622 Chipset. The workstations have a maximum memory capacity of 192 GB RAM, and the memory can be expanded via six DDR4, RDIMM, ECC, 2933 MHz Modules slots.

For connectivity, each workstation includes two PCI Express Generation 3 x16 connectors, one PCI Express Generation 3 x8 connector, and three PCI Express Generation 3 x4 connectors. Storage is provided by a 512 GB M.2 NVMe SSD and two 6 TB SATA 7200 rpm hard drives, which are configured as a 6 TB RAID 1 hard drive.

These workstations are designed to control the system and allow for offline (but in-facility) streaming, Airyscan Joint Deconvolution, and offsite Airyscan Joint Deconvolution and analysis. The workstations should have ZEN 3.8 software that should includes a number of features including a System Self-Maintenance Tool, an easy-to-use software along with a calibration objective for maintaining optimal system performance by automated calibration routines.

ZEN should have a Smart Setup for easy hardware control that adapts the system configuration according to chosen dyes from a large database of fluorochromes. Different acquisition modes are suggested (e.g. Fastest or Best Signal).

ZEN should run on a 64-Bit operating system for efficient data handling and memory management of large datasets.

ZEN software must have programmability and automation with control over all image acquisition functions via high level computer language (VBA 6.4).

The software must have the ability to reproduce experiments in multi-user environments via the REUSE function which allows reactivation of all acquisition parameters necessary to reproduce an experiment, stored within each image.

User-specific Workspace setups should be able to be stored within the ZEN software according to the user’s needs and applications.

Lambda Scanning Mode and Linear Unmixing must be fully integrated into ZEN standard software.

The Windows user login must define the user-specific LSM environment. Each user should be able to have individual settings and will not disturb the other users. Easy administration via Windows.

The system must have a number of software modules including: 1) integrated 3D module to permit innovative 3D renderings of the data, 2) Experiment Designer (MTS Experiments Macro) module to permit heterogeneous complex, combined Time Series with changing acquisition configurations, autofocus, and bleach functions in any order, 3) Tiles & Positions module to allow for advanced tile scan options and neighboring, overlapping image stacks in reflection and fluorescent mode should be able to be acquired and stitched together. The imaging of up to 100x100 tiles should be possible, 4) Processing of single images, Z-Stacks, Time Series, and Tiled images with the Shading Correction tool should available, 5) Advanced processing via segmentation analysis, 6) Built in Deconvolution, 7) Direct processing of data for on the fly, deconvolution and Airyscan processing, 8) Airyscan 2 Multiplex Plus software for parallelisation, 9) an additional floating software license key for

Airyscan and Airyscan joint Deconvolution offline processing 10) An additional floating licence for

System must include a 30x36 Table, RP,I325 for microscope stability All adaptors and hardware necessary for integration of the specified components One year warranty at no additional cost

4.2 WARRANTY

In addition to the Implied warranty contained in FAR Clause 52.212-4, quoters are encouraged to provide express warranties. At a minimum, the Contractor should provide a 12-month warranty or longer from date of final installation on all equipment and associated items provided under, or as a part of this purchase order. The warranty shall include repair and or replacement of parts, including travel, labor, and materials, at no additional cost to the Government. The warranty shall not exclude or limit the Governments rights for an implied warranty as established in the terms and conditions of clause 52.212-4.

4.3 DELIVERY / INSTALLATION

Delivery of all items quoted shall have an estimated delivery date of Onehundred and twenty (120) calendar days after receipt of order. All shipping/handling shall be FOB Destination, and delivery/installation fees shall be included in the quote. Upon delivery, the Contractor shall notify the respective NCI Technical Point of Contacts (TPOC), TBD at award, to schedule installation dates and times. Installation shall occur within five (5) business days of delivery, and shall be performed by, or under the direct supervision of, an Original Equipment Manufacturer (OEM) certified technician.

Equipment shall be delivered to and installed at the following address:

Laboratory of Cancer Biology and Genetics 37 Convent Drive Building 37, Rm 1066 Bethesda, MD 20892

4.3.1 DELIVERY GUIDEANCE

The Contractors shall communicate and follow instructions from the Technical Point of Contact (TPOC) to coordinate delivery, installation, and removal of trade-in equipment.

The Contractor shall direct all courier and delivery services – including those not operated by the Contractor (e.g. FedEx, USPS, UPS) – to follow the instructions in this section.

All companies delivering to the NIH main campus must comply with Commercial Vehicle Inspection Facility (CVIF) access procedures. Upon arrival, the CVIF will provide a list of buildings and loading docks to delivery personnel.

Deliveries will be logged in at the loading dock, and the loading dock contractor or Government personnel will attempt to contact the TPOC listed on the package. In the event that the TPOC is unable to accept the package, the delivery shall not be left at the loading dock station. The delivery must be returned to the Contractor, and the Contractor must coordinate with the TPOC to deliver on another date at no additional cost.

Deliveries can only be made between 8:00 a.m. and 4:00 p.m. EST, Monday – Friday, excluding federal holidays.

4.3 TRAINING

The Contractor shall provide one (1) three (3) day training sessions for NCI staff per session.

Training shall be on-site and shall take place within seven (7) days of equipment installation.

Training shall be facilitated by a factory-trained product specialist. Training will include an overview of setup approaches, maintaining instrument settings, establishing operational voltages, and software usage for custom instrument configurations, as well as a demonstration of the performance. All training expenses, including materials and/or travel expenses, shall be included in the quoted price.

5.0 INSPECTION AND ACCEPTANCE

Pursuant to FAR Clause 52.212-4, all items, services, materials, or supplies to be provided by the Contractor in relation to this Purchase Order are subject to final inspection and acceptance by an authorized representative of the Government. The authorized representative of the Government is the Technical Point of Contact (TPOC).

6.0 TECHNICAL POINT OF CONTACT (TPOC)

The Technical Point of Contact (TPOC) is responsible for inspection and acceptance of all work in relation to this requirement and will serve as the Governments primary point of contact throughout the duration of the requirement. All efforts must be coordinated with this individual. The TPOC for this requirement is:

T.B.D. (upon award)

Note: The TPOC has limited authority as defined within this Statement of Need. They do not have the authority to commit or obligate Government funds or make changes to the terms and conditions of the purchase order. As such, any changes to the purchase order must be performed by a warranted Government Contracting Officer with appropriate delegated authority to do so.