Attachment_C_-_Brand_Name_Only_Justification.pdf

Justification and Approval for Brand-Name Only Items (J&A)

Justification and Approval for Sole Source

“Source Selection Information – see FAR 2.101 and 3.104”

1. Identification of the agency and contracting activity.

a. Federal agency and contracting activity. HHS/NIH/NEI/OGVFB

b. Sponsoring organization. NIH/NEI/OGVFB

Project Officer Information:

• Project Officer name: Dr. Tiziana Cogliati

• Mailing address. 10 Center Drive, Bldg 10; Rm 10B11B, Bethesda MD 20892

• E-mail address. cogliatitp@mail.nih.gov

• Telephone number. 301-451-5954

2. Nature and/or description of the action being approved.

Acquisition purpose and objectives. The purpose of this acquisition is to procure the following Carl Zeiss microscope products:

- Apotome Trade Up

- AxioCam Trade Up for Apotome

- High Speed Deconvolution Upgrade for Apotome

- LSM 800 confocal microscope Upgrades on the existing Apotome and the new LSM 800 confocal microscope will provide state-of-the-art 3D microscopy capability for high content screening using novel absorbance imaging techniques and long-term deep tissue confocal microscopy on live cells/animals across multiple days. This acquisition will serve multiple labs within the OGVFB.

a. Project background. The OGVFB relies on microscopy imaging for a large part of its research activities. With the proposed upgrade of the existing Apotome, high content 3-dimensional screening of 384- and 1536 well plates will be possible. This screening will be used in conjunction with novel software developed in the Bharti lab, specifically for the Apotome microscope, to quantitatively assess melanin content in RPE cells in the investigation of albinism and hyperpigmentation diseases of the eye. The proposed platform has been specifically optimized to reduce image acquisition times due to the large number (~100K) of images acquired on live samples per plate. This specific requirement is not currently met by the NEI core and it is best addressed with a custom microscope such as the one proposed. Use of live imaging across multiple days to image deep within developing zebrafish, mouse, and iPSC derived organoids is the proposed application of the new Carl Zeiss LSM 800 microscope.

Currently, constant live confocal imaging of embryos across multi-day experiments is not easily enabled in the core due to logistics of scheduling microscopes for prolonged periods of time and lack of customized stage components. This microscope will be used with custom designed hardware for multi-embrio imaging across multiple days. Both microscopes are envisioned to become part of a dedicated imaging suite accessible to all labs within OGVFB.

3. Description of the supplies or services required to meet the agency’s needs (including the estimated value).

a. Project title. High-throughput screening 3-dimensional fluorescent and live imaging deep tissue confocal microscope set up

b. Project description.

OGVFB will establish a microscopy suite with high-throughput screening 3-dimensional fluorescent microscopy capability (upgrade of existing Apotome) and live imaging deep tissue confocal microscopy capability (new LSM 800). The laboratory already owns an AxioObserver Z1 microscope and AxioVision Software license. The existing microscope stand and existing software license will be used for a upgrade for the addition of high speed image acquisition hardware and software which is only available from Carl Zeiss microscopy for the upgrade of this microscope. All existing optics will be used in this upgrade. The LSM 800 microscope will complement the existing Apotome, benefitting from compatibility of the two systems, common objectives and comparable software.

c. Requirement type x Research & development (R&D) R & D support services Support services (non-R&D) Supplies/equipment Information technology (IT) Construction Architect-engineer (A & E) services Design-build Other (specify): ____________________

• Type of action. :

New requirement Follow-on Other (specify): ______________________

• Proposed contract/order type. :

Firm-fixed-price Other fixed-price (specify, e.g., fixed-price award-fee, fixed-price incentive-fee): __________ Cost-plus-fixed-fee Other cost reimbursement (specify, e.g., cost-plus-award-fee, cost-plus-incentive-fee): ___________ Time and materials Indefinite delivery (specify whether indefinite quantity, definite quantity, or requirements): ____Indefinite Quantity - Requirements___ Other (specify): ___________ Completion Form Term form

• Acquisition identification number. N/A

d. Total estimated dollar value and performance/delivery period.

$ 346,205.76/ delivery to be agreed upon receipt of order

4. Identification of the statutory authority permitting other than full and open competition.

This acquisition is conducted under the authority of 41 United States Code (U.S.C.) 253(c)(5) as set forth in Federal Acquisition Regulation (FAR) 6.302- (1)(c).

This acquisition is conducted under the authority of section 4202 of the Clinger-Cohen Act of 1996.

This acquisition is conducted under the authority of the Services

Acquisition Reform Act of 2003 (41 U.S.C. 428a).

5. Demonstration that the proposed contractor(s) unique qualifications or the nature of the acquisition requires use of the authority cited.

a. Name and address of the proposed contractor(s).

Carl Zeiss Microscopy, LLC One Zeiss Drive

Thornwood, NY 10594

(800) 233-2343

Fax (800) 488-6351 Email: roxanne.moosher@zeiss.com

b. Nature of the acquisition and proposed unique qualifications of the contractor(s).

OGVFB will use the existing AxioObserver Z1 microscope stand and AxioVision Software license for an upgrade and the addition of high speed image acquisition hardware and software which are only available from Carl Zeiss microscopy. OGVFB will further acquire the modular laser scanning microscope – LSM 800, 2 GaAsP PMT System with future upgrade option for AiryScan Super Resolution Detector from Carl Zeiss to complement the upgraded AxioObserver Z1 microscope. The system specifications are listed below with the critical technical characteristics highlighted in yellow.

System Specifications for Upgrade to Existing Zeiss AxioObserver Z1 microscope

• Addition of Colibri 7 LED light source with UV: 385 / 30nm, V: 423 / 44nm, B: 469 / 38nm, C: 511 / 44nm, G: 555 / 30nm, Y: 590 / 27nm, R: 631 / 33nm.

Automatic instrument calibration to guarantee reproducible output power levels for each line. Trigger control for microsecond switching of wavelengths.

• Integrated high speed dual turret with 6 beam splitter positions and 8 emission filters. Proprietary upgrade for AxioObserver Z1 microscope.

• Apotome 2 Structured Illumination proprietary for existing AxioObserver microscope: Three optimized grid patterns selected for optimal optically sectioning with matched Zeiss objectives. Grid patterns are motorized and automatically selected by ZEN software. Optionally, the user has the option to choose between the grids within one second. Two ApoTome slider positions with a hardware and software detected click stop allowing switching between conventional imaging and optical sectioning without removing the ApoTome from the microscope light path. Acquisition of images in a RAW data mode allowing the user to select between conventional fluorescence, optically sectioned or deconvolved for the final image product. The ApoTome.2 includes an integrated and dedicated and optimized deconvolution algorithm that can be applied to acquired images.

• X,Y,Z piezo stage for high speed, high accuracy x,y,and z positioning

• GPU deconvolution

• High speed 6 MP CCD camera with 4.54 um pixel size and 1” sensor.

• Upgrade of existing AxioVision software license to Zen software license

(successor software).

• Software programmability and automation with control over all image acquisition functions via high level computer language (Iron Python).

• High end computer for rapid computation of deconvolved images.

System Specifications for the Modular Laser Scanning Microscope – LSM 800, 2 GaAsP PMT System with future upgrade option for AiryScan Super Resolution Detector from Carl Zeiss

Microscope Features o Axio Observer 7

• Unique Features

• Fully integrated environmental control units into microscope stand control via TFT

• Optional Thermally isolated Objectives for Live Cell Imaging

• General Features

• Inverted microscope stand with fully motorized 6 position compatible objective nosepiece and 6 position push and click motorized fluorescence filter turret

• Motorized integrated Z focus with a step size of 10nm and travel range of 25mm

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

• Computer controlled metal halide fluorescence illuminator with variable intensity output.

• High efficiency hard coated shift free filters for GFP, CY3, DAPI for standard visual fluorescence Imaging.

• High resolution scanning stage with joystick and integrated computer control including universal specimen holder suitable for variety of slide and round dishes up to 60mm with 5µ reproducibility over the full travel range.

• Integrated contrast manager to preset all light and microscope conditions

• Definite focus 2 for automatic focus maintenance for time lapse studies

• Complete incubation system for temperature, humidity and CO2 control for live cell imaging.

• Objectives 10x/0.45, Long working distance 25x/0.8 live cell imaging objective, 63x/1.4. The microscope is compatible with a large variety of existing Zeiss microscope objectives already owned by the lab.

LSM 800 System o 2 Channel System

• Unique Features

• The separation of light between detectors is achieved by the utilization of the Variable Secondary Dichroic (VSD) which allows the splitting of emission light from 420 to 620 nm in 1 nm steps within a detection range of 400 to 700 nm between the two channels. The implementation of the VSD allows two distinct modes of spectral image acquisition;

• Flexible multi-channel configuration in which the user can specify the emission light to be separated between channels at a precision of 1nm with an overall range of 420 to 620nm

• Sequential high resolution spectral scanning using the movement of the VSD across the spectrum with user defined spectral resolution. The utilization of the VSD ensures that all emission light is incident on either detector 1 or detector 2.

o Unique Feature for future upgrade

• Future upgrade option to Proprietary Airyscan detection unit consists of a calibrated 32-element Gallium Arsenide Phosphide (GaAsP) PMT with typical QE >45 % (peak) and fixed zoom optics (designed for either 40x or 63x objectives).

The 32-element proprietary area detector is arranged in a compound eye fashion where the central element is adjusted to lie on the optical axis. The Airyscan detector is operated in two different modes: superresolution and standard confocal. The fixed optics always ensure that a total of 1.25 airy units is projected onto the detector allowing that each detector element to represents 0.2 Airyunits worth of light.

• In superresolution mode the Airyscan detector system achieves images with spatial resolutions of 140nm in x-y and 400nm in z when utilizing 488nm excitation and a 63x 1.4 NA Plan-Apochromat objective.

• In confocal mode a traditional pinhole aperture is used in addition to the detectors 32 GaAsP detection elements. All elements are then summed together to yield a traditional confocal image that can be combined with the two other PMT detectors of the system.

• Electronically switchable illumination and detection module (ESID) for transmitted light illumination and detection in one component. No mechanical parts are moved to switch between modes o General Features

• The LSM 800 is a confocal laser scanning microscope with up to 3 calibrated and linearized PMT (Photomultiplier tube) detectors. It is designed for biomedical (fluorescence), material (reflection) or combined biomedical and material applications.

o 2x Gallium Arsenide Phosphide (GaAsP) PMT detectors

• The primary dichroics provide a laser suppression of 6-7 OD through the utilization of low angle (~15°) dichroics – as opposed to the conventional 45° orientation.

• Multitrack imaging, for the prevention of cross-talk, by linewise or framewise switching of the laser excitation line, essential for multi-fluorescence applications and co-localization studies.

• Digital quantitative gain: noise-free contrast enhancement before bit depth conversion.

• Digital, quantitative Offset; units of offset control slider always correspond to the image grey level values.

• The pinhole opening diameter is freely variable and adjustable through ZEN software.

• Freely rotatable scan field (0-360 degree in 0.1° steps) and free xy offset (crop functionality).

• Use up to 4 laser lines simultaneously.

• 16 different scan speed levels (32 levels when including bi-directional scans).

• Scan Speeds:

o 512 x 512 pixels at max. 8 frames/sec o 256 x 256 pixels at max. 16 frames/sec o 512 x 16 pixels at max. 250 frames/sec

• Absolute linear scanner movement to ensure equal pixel dwell-times as a prerequisite for any quantitative study.

• OSCiscan, Online Scanner Calibration, for artifact-free, fast and reliable bidirectional scanning without the need of manual adjustment.

• Variable scan field size (hardware-zoom) from 0.5 to 40 in 0.1 steps for magnifications up to and beyond the optical limits of resolution, with a scan field of 18 mm diagonally in the intermediate image plane.

• Image format from 4x1 to 6000x6000 pixels with up to 16 bit depth (65536 gray values) in all PMT channels.

• Multidimensional image acquisition with easy combinations of Z-Stack, Time Series, Multi-positions (including software based autofocus options), and Tile Scan experiments.

• Time Series with automatic bleach control to design the entire experimental protocol. All scan strategies can be applied (line, spline, frame, z-line, z-spline, Z-Stack, ROIs, z-ROIs) in the Time Series.

• Multiple (up to 99) freely definable regions of interest (ROIs). Pixel precise control of the laser intensity and signal detection. Prevention of photo damage outside the ROIs boundaries necessary when work on living cells, FRET, FRAP, uncaging, photoactivation and photoconversion experiments.

• Possible to select different scan speeds and laser attenuation for bleach ROIs and image frames.

• Possible to move location of bleach ROI within image FOV during time series acquisition

• The acquisition and analysis of spectral data is fully integrated in the basic software (ZEN).

• Gain calibration and linearization of the two PMT detectors is foundation for reliable spectral acquisition and analysis.

• The acquisition of spectral data by homogeneously or heterogeneously defined steps, down to 1 nm, is easily and flexibly done by moving the VSD in the emission beampath, therefore creating a series of images (lambda stacks) on both detectors.

• High efficient spectral imaging is achieved by using both detectors, capturing all emission light of the sample at every scan.

• Spectral data can be stored in the spectral database and later used for reference-based linear unmixing.

• 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 on known, previously recorded reference spectra. Display of the unmixed dyes (crosstalk free) into separate dye-channel images and overlay images.

• 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.

• Lasers with wavelengths from 405nm to 640nm can be combined. All lasers are solid state laser which are directly modulated and controlled by the standard software.

• Laser Attenuation Range of 1:10,000 (Standard for GaAsP-PMT configurations)

• Available Lasers (all power specifications reference out of fiber measurements) :

o 405 nm, solid state laser, 5 mW o 488 nm, solid state laser, 10 mW o 561 nm, solid state laser, 10 mW o 640 nm, solid state laser, 5 mW

Software Features o Unique Features- Zen Software

• Smart Setup: Easy hardware control - adapts system configuration according to chosen dyes from a large database of fluorochromes. Different acquisition modes are suggested (e.g. Fastest or Best Signal).

• Zen software platform already runs numerous scopes in the laboratory which the lab members are already familiar with. The use of Zen software will minimize training time and will enable the use of instrumentation for important experiments quickly.

• 64-Bit operating system for efficient data handling and memory management of large datasets.

• Innovative concept for reproducing 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 can be stored within the ZEN software according to the user’s needs and applications.

• Lambda Scanning Mode and Linear Unmixing are fully integrated into ZEN standard software.

• The Windows user login defines the user-specific LSM environment. Each user can have individual settings and will not disturb the other users. Easy administration via Windows.

o Available Modules

• Quantitative Physiology module for acquisition of ion indicator dyes and display and analysis of ion concentrations including online and offline ratio for ratiometric dyes, online and offline F/F0 and DF/F for single wavelength dyes, calibration for single-wavelength and ratiometric dyes in-situ and in-vitro including background correction (after titration and with various curve fits or after Grynkiewicz). Includes “mean of ROI” measurement, displaying intensity changes over time.

• Quantitative FRET plus module for acquisition and analysis of sensitized emission, acceptor photobleaching, and Emission Fingerprinting based FRET using Youvan, Gordon, and Xia analysis methods. Includes “mean of ROI” measurement, displaying intensity changes over time.

• FRAP Efficiency Analysis module for analysis of original FRAP curve and fitted curve according to the parameters provided by the original curve. With tabulated output of fitting parameters (rate constant for recovery). Includes “mean of ROI” measurement, displaying intensity changes over time.

• The 3D VisArt module permits innovative 3D and 4D renderings of the data. Up to three clipping planes allow different rendering modes within the same sample (e.g. transparent combined with surface renderings). In 3D, measurements of distances, lines, and angles are possible. Furthermore, a fly-through mode (using the PC mouse) is implemented. All view modes can easily be used to create innovative movies in 3D.

• ROI-HDR. High dynamic range imaging module with intelligent local improvement of signal dynamics, free choice for gain or laser power modulation, the latter resulting in quantifiable images.

• The 3D Analysis module allows evaluation and display of 3D image data with various measurement tools.

• The Experiment Designer module permits heterogeneous complex, combined Time Series with changing acquisition configurations, autofocus, and bleach functions in any order.

• The Visual Macro Editor (VME) module allows for symbol driven design of automated complex imaging strategies by using the full functionality of the ZEN software, which is represented as action blocks in a graphical interface. It includes autofocus functionality as well as a variety of feedback imaging tasks.

• The module Visual Basic Macro, VBA, allows dedicated visual basic programming to create macros and includes a macro recorder function.

• Deconvolution module for improvement of 3D image stacks with 4 different deconvolution algorithms possible. Advanced correction methods possible for bleaching, background, and spherical aberration. An interactively definable region is possible for a quick check of various set deconvolution parameters.

• The Topo module is a dedicated tool to analyze surfaces (not only) in material microscopy. E.g. measurements of multiple roughness parameters, volumes, and surface profiles can be easily performed.

With Tiles & Positions module advanced tile scan options are available and neighboring, overlapping image stacks in reflection and fluorescent mode can be acquired and stitched together. Imaging up to 100x100 tiles is possible. Processing of single images, Z-Stacks, Time Series, and Tiled images with the Shading Correction tool is available.

6. Description of the efforts made to ensure that offers are solicited from as many potential sources as practicable. Indicate whether a FedBizOpps notice was or will be publicized as required by FAR Subpart 5.2 and, if not, which exception under FAR 5.202 applies.

We have evaluated and obtained quotations for similar competitive models from other companies and we have established that the proposed solution is the most suitable for OGVFB experimental needs and the most cost effective, especially in consideration of the fact that one microscope will be upgraded.

7. Determination by the Contracting Officer that the anticipated cost/price to the Government will be fair and reasonable.

Carl Zeiss is a publicly held provider of life science imaging tools. The company's technology enables a wide variety of basic research and translational medicine applications. Trade Up Promotions and New Investigator discounts have been applied.

Based on the above information the price has been determined fair and reasonable to the government.

8. Description of the market research conducted (see FAR Part 10) and the results, or a statement of the reasons market research was not conducted.

Based on our own experience, that of the NEI Imaging Core and of other NIH investigators, Carl Zeiss products are of the highest quality and performance, and therefore, we decided for the proposed company and products.

9. Any other facts supporting the use of other than full and open competition.

OGVFB has been highly satisfied with the use of the existing Zeiss AxioObserver Z1 microscope as well as with other Zeiss confocal miscroscopes of the LSM series also available in the NEI Core.

10. Listing of sources, if any, that expressed, in writing, an interest in the acquisition.

No other vendors have instruments that will completely match the specifications required by our current research.

11. Statement of the actions, if any, the agency may take to remove or overcome any barriers to competition before any subsequent acquisition for the required supplies or services.

N/A

12. Program office certification.

This is to certify that the portions of this justification that have been developed by the undersigned program office personnel, including supporting information and/or data verifying the Government’s minimum needs, schedule requirements and other rationale for other than full and open competition, are accurate and complete.

Official Name & Title Signature Date

Project Officer Dr. Tiziana Cogliati

Deputy Branch Chief, OGVFB.

Project Officer’s

Immediate Supervisor

Dr. Brian Brooks

Chief, OGVFB

Head of the Sponsoring

Program Office

13. Contracting Officer Certification

This is to certify that the justification for the proposed acquisition has been reviewed and that to the best of my knowledge and belief the information and/or data provided to support the rationale and recommendation for approval is accurate and complete.

Official Name & Title Signature Date

Contracting Officer

14. Chief of the Contracting Office and Head of the Contracting Activity signature(s).

Official Name & Title Signature Date

Chief of the

Office

Activity

15. Competition Advocate signature.

Official Name & Title Signature Date

Competition Advocate

Justification and Approval for Brand-Name Only Items (J&A)

Justification and Approval for Sole Source

“Source Selection Information – see FAR 2.101 and 3.104”

1. Identification of the agency and contracting activity.

a. Federal agency and contracting activity. HHS/NIH/NEI/OGVFB

b. Sponsoring organization. NIH/NEI/OGVFB

Project Officer Information:

• Project Officer name: Dr. Tiziana Cogliati

• Mailing address. 10 Center Drive, Bldg 10; Rm 10B11B, Bethesda MD 20892

• E-mail address. cogliatitp@mail.nih.gov

• Telephone number. 301-451-5954

2. Nature and/or description of the action being approved.

Acquisition purpose and objectives. The purpose of this acquisition is to procure the following Carl Zeiss microscope products:

- Apotome Trade Up

- AxioCam Trade Up for Apotome

- High Speed Deconvolution Upgrade for Apotome

- LSM 800 confocal microscope Upgrades on the existing Apotome and the new LSM 800 confocal microscope will provide state-of-the-art 3D microscopy capability for high content screening using novel absorbance imaging techniques and long-term deep tissue confocal microscopy on live cells/animals across multiple days. This acquisition will serve multiple labs within the OGVFB.

a. Project background. The OGVFB relies on microscopy imaging for a large part of its research activities. With the proposed upgrade of the existing Apotome, high content 3-dimensional screening of 384- and 1536 well plates will be possible. This screening will be used in conjunction with novel software developed in the Bharti lab, specifically for the Apotome microscope, to quantitatively assess melanin content in RPE cells in the investigation of albinism and hyperpigmentation diseases of the eye. The proposed platform has been specifically optimized to reduce image acquisition times due to the large number (~100K) of images acquired on live samples per plate. This specific requirement is not currently met by the NEI core and it is best addressed with a custom microscope such as the one proposed. Use of live imaging across multiple days to image deep within developing zebrafish, mouse, and iPSC derived organoids is the proposed application of the new Carl Zeiss LSM 800 microscope.

Currently, constant live confocal imaging of embryos across multi-day experiments is not easily enabled in the core due to logistics of scheduling microscopes for prolonged periods of time and lack of customized stage components. This microscope will be used with custom designed hardware for multi-embrio imaging across multiple days. Both microscopes are envisioned to become part of a dedicated imaging suite accessible to all labs within OGVFB.

3. Description of the supplies or services required to meet the agency’s needs (including the estimated value).

a. Project title. High-throughput screening 3-dimensional fluorescent and live imaging deep tissue confocal microscope set up

b. Project description.

OGVFB will establish a microscopy suite with high-throughput screening 3-dimensional fluorescent microscopy capability (upgrade of existing Apotome) and live imaging deep tissue confocal microscopy capability (new LSM 800). The laboratory already owns an AxioObserver Z1 microscope and AxioVision Software license. The existing microscope stand and existing software license will be used for a upgrade for the addition of high speed image acquisition hardware and software which is only available from Carl Zeiss microscopy for the upgrade of this microscope. All existing optics will be used in this upgrade. The LSM 800 microscope will complement the existing Apotome, benefitting from compatibility of the two systems, common objectives and comparable software.

c. Requirement type x Research & development (R&D) R & D support services Support services (non-R&D) Supplies/equipment Information technology (IT) Construction Architect-engineer (A & E) services Design-build Other (specify): ____________________

• Type of action. :

New requirement Follow-on Other (specify): ______________________

• Proposed contract/order type. :

Firm-fixed-price Other fixed-price (specify, e.g., fixed-price award-fee, fixed-price incentive-fee): __________ Cost-plus-fixed-fee Other cost reimbursement (specify, e.g., cost-plus-award-fee, cost-plus-incentive-fee): ___________ Time and materials Indefinite delivery (specify whether indefinite quantity, definite quantity, or requirements): ____Indefinite Quantity - Requirements___ Other (specify): ___________ Completion Form Term form

• Acquisition identification number. N/A

d. Total estimated dollar value and performance/delivery period.

$ 346,205.76/ delivery to be agreed upon receipt of order

4. Identification of the statutory authority permitting other than full and open competition.

This acquisition is conducted under the authority of 41 United States Code (U.S.C.) 253(c)(5) as set forth in Federal Acquisition Regulation (FAR) 6.302- (1)(c).

This acquisition is conducted under the authority of section 4202 of the Clinger-Cohen Act of 1996.

This acquisition is conducted under the authority of the Services

Acquisition Reform Act of 2003 (41 U.S.C. 428a).

5. Demonstration that the proposed contractor(s) unique qualifications or the nature of the acquisition requires use of the authority cited.

a. Name and address of the proposed contractor(s).

Carl Zeiss Microscopy, LLC One Zeiss Drive

Thornwood, NY 10594

(800) 233-2343

Fax (800) 488-6351 Email: roxanne.moosher@zeiss.com

b. Nature of the acquisition and proposed unique qualifications of the contractor(s).

OGVFB will use the existing AxioObserver Z1 microscope stand and AxioVision Software license for an upgrade and the addition of high speed image acquisition hardware and software which are only available from Carl Zeiss microscopy. OGVFB will further acquire the modular laser scanning microscope – LSM 800, 2 GaAsP PMT System with future upgrade option for AiryScan Super Resolution Detector from Carl Zeiss to complement the upgraded AxioObserver Z1 microscope. The system specifications are listed below with the critical technical characteristics highlighted in yellow.

System Specifications for Upgrade to Existing Zeiss AxioObserver Z1 microscope

• Addition of Colibri 7 LED light source with UV: 385 / 30nm, V: 423 / 44nm, B: 469 / 38nm, C: 511 / 44nm, G: 555 / 30nm, Y: 590 / 27nm, R: 631 / 33nm.

Automatic instrument calibration to guarantee reproducible output power levels for each line. Trigger control for microsecond switching of wavelengths.

• Integrated high speed dual turret with 6 beam splitter positions and 8 emission filters. Proprietary upgrade for AxioObserver Z1 microscope.

• Apotome 2 Structured Illumination proprietary for existing AxioObserver microscope: Three optimized grid patterns selected for optimal optically sectioning with matched Zeiss objectives. Grid patterns are motorized and automatically selected by ZEN software. Optionally, the user has the option to choose between the grids within one second. Two ApoTome slider positions with a hardware and software detected click stop allowing switching between conventional imaging and optical sectioning without removing the ApoTome from the microscope light path. Acquisition of images in a RAW data mode allowing the user to select between conventional fluorescence, optically sectioned or deconvolved for the final image product. The ApoTome.2 includes an integrated and dedicated and optimized deconvolution algorithm that can be applied to acquired images.

• X,Y,Z piezo stage for high speed, high accuracy x,y,and z positioning

• GPU deconvolution

• High speed 6 MP CCD camera with 4.54 um pixel size and 1” sensor.

• Upgrade of existing AxioVision software license to Zen software license

(successor software).

• Software programmability and automation with control over all image acquisition functions via high level computer language (Iron Python).

• High end computer for rapid computation of deconvolved images.

System Specifications for the Modular Laser Scanning Microscope – LSM 800, 2 GaAsP PMT System with future upgrade option for AiryScan Super Resolution Detector from Carl Zeiss

Microscope Features o Axio Observer 7

• Unique Features

• Fully integrated environmental control units into microscope stand control via TFT

• Optional Thermally isolated Objectives for Live Cell Imaging

• General Features

• Inverted microscope stand with fully motorized 6 position compatible objective nosepiece and 6 position push and click motorized fluorescence filter turret

• Motorized integrated Z focus with a step size of 10nm and travel range of 25mm

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

• Computer controlled metal halide fluorescence illuminator with variable intensity output.

• High efficiency hard coated shift free filters for GFP, CY3, DAPI for standard visual fluorescence Imaging.

• High resolution scanning stage with joystick and integrated computer control including universal specimen holder suitable for variety of slide and round dishes up to 60mm with 5µ reproducibility over the full travel range.

• Integrated contrast manager to preset all light and microscope conditions

• Definite focus 2 for automatic focus maintenance for time lapse studies

• Complete incubation system for temperature, humidity and CO2 control for live cell imaging.

• Objectives 10x/0.45, Long working distance 25x/0.8 live cell imaging objective, 63x/1.4. The microscope is compatible with a large variety of existing Zeiss microscope objectives already owned by the lab.

LSM 800 System o 2 Channel System

• Unique Features

• The separation of light between detectors is achieved by the utilization of the Variable Secondary Dichroic (VSD) which allows the splitting of emission light from 420 to 620 nm in 1 nm steps within a detection range of 400 to 700 nm between the two channels. The implementation of the VSD allows two distinct modes of spectral image acquisition;

• Flexible multi-channel configuration in which the user can specify the emission light to be separated between channels at a precision of 1nm with an overall range of 420 to 620nm

• Sequential high resolution spectral scanning using the movement of the VSD across the spectrum with user defined spectral resolution. The utilization of the VSD ensures that all emission light is incident on either detector 1 or detector 2.

o Unique Feature for future upgrade

• Future upgrade option to Proprietary Airyscan detection unit consists of a calibrated 32-element Gallium Arsenide Phosphide (GaAsP) PMT with typical QE >45 % (peak) and fixed zoom optics (designed for either 40x or 63x objectives).

The 32-element proprietary area detector is arranged in a compound eye fashion where the central element is adjusted to lie on the optical axis. The Airyscan detector is operated in two different modes: superresolution and standard confocal. The fixed optics always ensure that a total of 1.25 airy units is projected onto the detector allowing that each detector element to represents 0.2 Airyunits worth of light.

• In superresolution mode the Airyscan detector system achieves images with spatial resolutions of 140nm in x-y and 400nm in z when utilizing 488nm excitation and a 63x 1.4 NA Plan-Apochromat objective.

• In confocal mode a traditional pinhole aperture is used in addition to the detectors 32 GaAsP detection elements. All elements are then summed together to yield a traditional confocal image that can be combined with the two other PMT detectors of the system.

• Electronically switchable illumination and detection module (ESID) for transmitted light illumination and detection in one component. No mechanical parts are moved to switch between modes

• The LSM 800 is a confocal laser scanning microscope with up to 3 calibrated and linearized PMT (Photomultiplier tube) detectors. It is designed for biomedical (fluorescence), material (reflection) or combined biomedical and material applications.

o 2x Gallium Arsenide Phosphide (GaAsP) PMT detectors

• The primary dichroics provide a laser suppression of 6-7 OD through the utilization of low angle (~15°) dichroics – as opposed to the conventional 45° orientation.

• Multitrack imaging, for the prevention of cross-talk, by linewise or framewise switching of the laser excitation line, essential for multi-fluorescence applications and co-localization studies.

• Digital quantitative gain: noise-free contrast enhancement before bit depth conversion.

• Digital, quantitative Offset; units of offset control slider always correspond to the image grey level values.

• The pinhole opening diameter is freely variable and adjustable through ZEN software.

• Freely rotatable scan field (0-360 degree in 0.1° steps) and free xy offset (crop functionality).

• Use up to 4 laser lines simultaneously.

• 16 different scan speed levels (32 levels when including bi-directional scans).

• Scan Speeds:

o 512 x 512 pixels at max. 8 frames/sec o 256 x 256 pixels at max. 16 frames/sec o 512 x 16 pixels at max. 250 frames/sec

• Absolute linear scanner movement to ensure equal pixel dwell-times as a prerequisite for any quantitative study.

• OSCiscan, Online Scanner Calibration, for artifact-free, fast and reliable bidirectional scanning without the need of manual adjustment.

• Variable scan field size (hardware-zoom) from 0.5 to 40 in 0.1 steps for magnifications up to and beyond the optical limits of resolution, with a scan field of 18 mm diagonally in the intermediate image plane.

• Image format from 4x1 to 6000x6000 pixels with up to 16 bit depth (65536 gray values) in all PMT channels.

• Multidimensional image acquisition with easy combinations of Z-Stack, Time Series, Multi-positions (including software based autofocus options), and Tile Scan experiments.

• Time Series with automatic bleach control to design the entire experimental protocol. All scan strategies can be applied (line, spline, frame, z-line, z-spline, Z-Stack, ROIs, z-ROIs) in the Time Series.

• Multiple (up to 99) freely definable regions of interest (ROIs). Pixel precise control of the laser intensity and signal detection. Prevention of photo damage outside the ROIs boundaries necessary when work on living cells, FRET, FRAP, uncaging, photoactivation and photoconversion experiments.

• Possible to select different scan speeds and laser attenuation for bleach ROIs and image frames.

• Possible to move location of bleach ROI within image FOV during time series acquisition

• The acquisition and analysis of spectral data is fully integrated in the basic software (ZEN).

• Gain calibration and linearization of the two PMT detectors is foundation for reliable spectral acquisition and analysis.

• The acquisition of spectral data by homogeneously or heterogeneously defined steps, down to 1 nm, is easily and flexibly done by moving the VSD in the emission beampath, therefore creating a series of images (lambda stacks) on both detectors.

• High efficient spectral imaging is achieved by using both detectors, capturing all emission light of the sample at every scan.

• Spectral data can be stored in the spectral database and later used for reference-based linear unmixing.

• 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 on known, previously recorded reference spectra. Display of the unmixed dyes (crosstalk free) into separate dye-channel images and overlay images.

• 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.

• Lasers with wavelengths from 405nm to 640nm can be combined. All lasers are solid state laser which are directly modulated and controlled by the standard software.

• Laser Attenuation Range of 1:10,000 (Standard for GaAsP-PMT configurations)

• Available Lasers (all power specifications reference out of fiber measurements) :

o 405 nm, solid state laser, 5 mW o 488 nm, solid state laser, 10 mW o 561 nm, solid state laser, 10 mW o 640 nm, solid state laser, 5 mW

Software Features o Unique Features- Zen Software

• Smart Setup: Easy hardware control - adapts system configuration according to chosen dyes from a large database of fluorochromes. Different acquisition modes are suggested (e.g. Fastest or Best Signal).

• Zen software platform already runs numerous scopes in the laboratory which the lab members are already familiar with. The use of Zen software will minimize training time and will enable the use of instrumentation for important experiments quickly.

• 64-Bit operating system for efficient data handling and memory management of large datasets.

• Innovative concept for reproducing 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 can be stored within the ZEN software according to the user’s needs and applications.

• Lambda Scanning Mode and Linear Unmixing are fully integrated into ZEN standard software.

• The Windows user login defines the user-specific LSM environment. Each user can have individual settings and will not disturb the other users. Easy administration via Windows.

o Available Modules

• Quantitative Physiology module for acquisition of ion indicator dyes and display and analysis of ion concentrations including online and offline ratio for ratiometric dyes, online and offline F/F0 and DF/F for single wavelength dyes, calibration for single-wavelength and ratiometric dyes in-situ and in-vitro including background correction (after titration and with various curve fits or after Grynkiewicz). Includes “mean of ROI” measurement, displaying intensity changes over time.

• Quantitative FRET plus module for acquisition and analysis of sensitized emission, acceptor photobleaching, and Emission Fingerprinting based FRET using Youvan, Gordon, and Xia analysis methods. Includes “mean of ROI” measurement, displaying intensity changes over time.

• FRAP Efficiency Analysis module for analysis of original FRAP curve and fitted curve according to the parameters provided by the original curve. With tabulated output of fitting parameters (rate constant for recovery). Includes “mean of ROI” measurement, displaying intensity changes over time.

• The 3D VisArt module permits innovative 3D and 4D renderings of the data. Up to three clipping planes allow different rendering modes within the same sample (e.g. transparent combined with surface renderings). In 3D, measurements of distances, lines, and angles are possible. Furthermore, a fly-through mode (using the PC mouse) is implemented. All view modes can easily be used to create innovative movies in 3D.

• ROI-HDR. High dynamic range imaging module with intelligent local improvement of signal dynamics, free choice for gain or laser power modulation, the latter resulting in quantifiable images.

• The 3D Analysis module allows evaluation and display of 3D image data with various measurement tools.

• The Experiment Designer module permits heterogeneous complex, combined Time Series with changing acquisition configurations, autofocus, and bleach functions in any order.

• The Visual Macro Editor (VME) module allows for symbol driven design of automated complex imaging strategies by using the full functionality of the ZEN software, which is represented as action blocks in a graphical interface. It includes autofocus functionality as well as a variety of feedback imaging tasks.

• The module Visual Basic Macro, VBA, allows dedicated visual basic programming to create macros and includes a macro recorder function.

• Deconvolution module for improvement of 3D image stacks with 4 different deconvolution algorithms possible. Advanced correction methods possible for bleaching, background, and spherical aberration. An interactively definable region is possible for a quick check of various set deconvolution parameters.

• The Topo module is a dedicated tool to analyze surfaces (not only) in material microscopy. E.g. measurements of multiple roughness parameters, volumes, and surface profiles can be easily performed.

With Tiles & Positions module advanced tile scan options are available and neighboring, overlapping image stacks in reflection and fluorescent mode can be acquired and stitched together. Imaging up to 100x100 tiles is possible. Processing of single images, Z-Stacks, Time Series, and Tiled images with the Shading Correction tool is available.

6. Description of the efforts made to ensure that offers are solicited from as many potential sources as practicable. Indicate whether a FedBizOpps notice was or will be publicized as required by FAR Subpart 5.2 and, if not, which exception under FAR 5.202 applies.

We have evaluated and obtained quotations for similar competitive models from other companies and we have established that the proposed solution is the most suitable for OGVFB experimental needs and the most cost effective, especially in consideration of the fact that one microscope will be upgraded.

7. Determination by the Contracting Officer that the anticipated cost/price to the Government will be fair and reasonable.

Carl Zeiss is a publicly held provider of life science imaging tools. The company's technology enables a wide variety of basic research and translational medicine applications. Trade Up Promotions and New Investigator discounts have been applied.

Based on the above information the price has been determined fair and reasonable to the government.

8. Description of the market research conducted (see FAR Part 10) and the results, or a statement of the reasons market research was not conducted.

Based on our own experience, that of the NEI Imaging Core and of other NIH investigators, Carl Zeiss products are of the highest quality and performance, and therefore, we decided for the proposed company and products.

9. Any other facts supporting the use of other than full and open competition.

OGVFB has been highly satisfied with the use of the existing Zeiss AxioObserver Z1 microscope as well as with other Zeiss confocal miscroscopes of the LSM series also available in the NEI Core.

10. Listing of sources, if any, that expressed, in writing, an interest in the acquisition.

No other vendors have instruments that will completely match the specifications required by our current research.

11. Statement of the actions, if any, the agency may take to remove or overcome any barriers to competition before any subsequent acquisition for the required supplies or services.

N/A

12. Program office certification.

This is to certify that the portions of this justification that have been developed by the undersigned program office personnel, including supporting information and/or data verifying the Government’s minimum needs, schedule requirements and other rationale for other than full and open competition, are accurate and complete.

Official Name & Title Signature Date

Project Officer Dr. Tiziana Cogliati

Deputy Branch Chief, OGVFB.

Project Officer’s

Immediate Supervisor

Sponsoring

Program Office

13. Contracting Officer Certification

This is to certify that the justification for the proposed acquisition has been reviewed and that to the best of my knowledge and belief the information and/or data provided to support the rationale and recommendation for approval is accurate and complete.

Official Name & Title Signature Date

Officer

14. Chief of the Contracting Office and Head of the Contracting Activity signature(s).

Official Name & Title Signature Date

Chief of the

Office

Activity

15. Competition Advocate signature.

Official Name & Title Signature Date

Competition Advocate