Statement of Requirement (Rev.1.12.22).pdf

Statement of Requirements (Revised 1/12/22) Digital Holographic Microscope (DHM)

Background The National Institute of Standards and Technology (NIST) requires a transmission digital holographic microscope (DHM) to make measurements of cell morphology and viability, which are quality attributes of cell-based therapeutics. This project is part of the NIST program in regenerative medicine and advanced therapies.

Specifications [1] DHM shall do transmission mode quantitative phase imaging (QPI) via digital holographic microscopy. Holograms shall be produced by interference between a beam transmitted through a sample and a reference beam so that cells do not have the “halo” artifact observed in phase contrast imaging or DIC imaging (differential interference contrast). The holograms shall be processed numerically to reconstruct a 3D optical map of the specimen.

[2] DHM shall be a stand-alone, automated system that is fully integrated with turrets, condensers, controllers, camera, phase optics, fluorescence and stage.

[3] DHM shall have an inverted microscope configuration with objectives below the stage.

[4] DHM shall have a turret with slots for 5 or more objectives.

[5] DHM shall use a standard USA NEMA 5-15 (15A/125V) electric receptacle.

[6] DHM shall include a computer [Win10 or Win11, 64 bit; 16 GB RAM (or more); hard drive

SSD 512 GB + HDD 1 TB (or more); 4GB graphic card (or more)], software, monitor (26” screen or larger), mouse and keyboard to run the microscope.

[7] In addition to QPI, the multimodal microscope shall also have the ability to do brightfield imaging and epifluorescence imaging.

[8] Microscope shall include a set of eight objectives with various magnification and working distance specifications that fall within 25% of the following specifications:

a. 2.5x lens, dry, NA 0.07, WD 11.2 mm, FOV 2640 μm square, lateral resolution 4.90 μm, condenser assembly, no coverglass correction

b. 4x lens, dry, NA 0.10, WD 26.2 mm, FOV 1650 μm square, lateral resolution 2.85 μm, condenser assembly, no coverglass correction

c. 10x lens, dry, NA 0.30, WD 11 mm, FOV 660 μm square, lateral resolution 1.15 μm, condenser assembly, no coverglass correction

d. 10x lens, dry, NA 0.25, WD 17.7 mm, FOV 660 μm square, lateral resolution 1.15 μm, condenser assembly

e. 20x lens, dry, NA 0.55, WD 1.2 mm, FOV 330 μm square, lateral resolution 0.67 μm, condenser assembly, for measuring through coverglass 0.17 mm

f. 40x lens, dry, NA 0.75, WD 0.4 mm, FOV 165 μm square, lateral resolution 0.45 μm, condenser assembly, for measuring through coverglass 0.17 mm

g. 100x lens, oil, NA 1.30, WD 0.13 mm, FOV 66 μm square, condenser assembly, for measuring through coverglass 0.17 mm [9] Quantitative Phase Imaging (QPI): QPI shall have specifications that fall within 20% of the following specifications:

a. QPI shall have dual lasers (666±1 nm and 794±1 nm; bandwidth less than 20 nm;

stability 0.01 nm per degree Celsius): improves image quality, reduces speckling, allows greater flexibility when working with fluorescence and QPI at the same time, allows retrieval of information over thicker samples, allows decoupling of morphology and refractive index, accounts for phase shift of 2π (wrapping).

b. QPI lateral (XY) resolution shall be diffraction limited (down to 300 nm).

c. QPI shall have a CMOS camera that shall meet the following specifications: acquisition rate 190 frames per second (video rate), 1024x1024 pixels.

d. QPI shall have a phase shift measurement accuracy of 1 nm, resolution of 2.0 nm and repeatability of 0.02 nm.

e. QPI shall have a vertical measuring range of 0.5 mm.

f. QPI shall have a sample illumination down to 1 μW/cm2.

g. QPI must not produce a halo or shadow around cells (as is present in phase contrast) and the background (areas between cells) shall be flat.

[10] Fluorescence: QPI shall have a fluorescence module with specifications that fall within

20% of the following specifications:

a. DHM fluorescence module shall have a fluorescence CMOS camera that shall be within

20% of the following specifications: 165 frames per second, 1936 x 1216 pixels, peak quantum efficiency 75% at 500 nm, USB 3.1, 2.3 megapixel, monochrome, compatible with Micro-manager.

b. Fluorescence shall have an LED fluorescence light with specifications that fall within 20% of the following specifications: with 7 or more channels ranging from 365 nm to 740 nm.

c. Fluorescence shall include a motorized filter wheel to accommodate 9 or more emission filters in the emission path.

d. Fluorescence shall include a motorized filter wheel to accommodate 9 or more excitation filters in the excitation path.

[11] Software:

a. DHM shall have an intuitive, software driven interface, that can run on a Win10 or

Win11 computer.

b. The software shall run the DHM to perform automated high-throughput screening as well as time-lapse and dose response 4D tracking at single and multi-sites.

c. Software shall include post-processing capability to re-focus the acquired QPI images after the acquisition (to correct for axial drift and to position samples in 3D and track their trajectories in real time).

d. Software shall have the capacity to export data in non-proprietary formats for images (.tif) and data files (.csv, .xlsx, .txt or ASCII).

e. Software must be able to setup complicated workflows that run over several days to repeatedly image plates of cells with user-defined objectives, channels and timepoints.

f. Software shall include phase and fluorescence image processing capability, such as stitching, cell segmentation, counting cells over time, measuring cell shape and tracking cell health and dynamics.

[12] Automated stage:

a. DHM shall have a motorized stage with specifications that fall within 20% of the following specifications: that can translate at least 114 mm x 76 mm x 38 mm in the XYZ directions with a minimum step size of 40 nm.

b. The stage shall have a joystick and controller.

c. The stage shall be controlled by the software and be precisely integrated with data acquisition so that the user can program the microscope to image entire plates, on multiple channels (QPI, fluorescence, brightfield), with overlapping images.

d. The stage shall have adapters to securely accommodate culture plates (96-well, 48-well, 24-well), 90 mm petri dishes and microscope slides (76 mm x 26 mm).

[13] QPI shall have a Stage-top environmental chamber (EC) with specifications that fall within 20% of the following specifications:

a. Microscope shall have a stage-top environmental chamber (EC) to maintain cell health while imaging for extended periods of time (days).

b. EC shall accommodate multiwell plates, microscope slides and Petri dishes.

c. EC shall include digitally controlled temperature (accuracy 0.3 degrees Celsius), CO2 gas (gas range 0% to 18%, actively controlled, accuracy: ± 0.1%) and humidity (active humidity controller, humidity range 50% to 95%, 1% resolution).

d. EC shall include a closed perfusion chamber for coverslips of diameter range 12 mm to 25 mm.

[14] Quote shall include shipping container and freight costs for delivery to NIST Gaithersburg campus (100 Bureau Drive, Gaithersburg, MD, 20899, USA).

[15] Delivery and Installation [16] Delivery shall be FOB destination. FOB destination delivery is absolutely required and absolutely must be included in bids and pricing. Delivery and installation must result in a fully functional device including imaging, camera, lasers, QPI, fluorescence imaging, brightfield imaging, translation stage, computer, software and cell culture environmental controls. Delivery and installation shall be completed within 24 weeks of the award.

Delivery shall be made to the NIST campus at 100 Bureau Drive, Gaithersburg, MD, 20899, USA. Delivery and installation will occur during normal business hours defined as Monday through Friday, 8:30 am to 5:00 pm, except for federal holidays. Installation will be scheduled in coordination with the Contracting Officers Representative (COR).

Training In-person training for 2 people shall be provided and shall be completed within 90 calendar days of the award. Training shall include operation of the instrument and all features including camera, optics, stage, environmental chamber, basic data analysis and file saving and exporting.

Testing and Acceptance Acceptance shall occur when the DHM is installed, staff have been trained and the DHM is tested using NIST cell samples (Jurkat cells in a culture dish), including operation of imaging, camera, lasers, QPI, fluorescence imaging, brightfield imaging, translation stage, computer, software and environmental chamber (temperature, CO2, humidity).

Warranty

The DHM shall have a 1-year warranty that includes parts, labor and travel. Warranty period shall commence upon NIST acceptance.