Expanding NIST Calibration of Fluorescent Microspheres for Flow Cytometry to More Fluorescence Channels and Smaller Particles

Abstract

The National Institute of Standards and Technology (NIST), the National Institutes of Health (NIH) and other industry stakeholders have been working together to enable fluorescence intensities of flow cytometer calibration beads to be assigned quantitative equivalent reference fluorophore (ERF) values with high accuracy and precision. The ultimate goal of this effort is to accurately quantify the number of antibodies bound to individual living cells. The expansion of this effort to assign ERF values to more than 50 fluorescence channels and particles with diameters ranging from 10 μm down to 80 nm is reported here.

Fire Burn and Cauldron Bubble: What Is in Your Genome Editing Brew?

“…Measure the volume of the editing solution and the number of editing molecules delivered into a single cell (Figure C). Technology requirement: The ability to quantify the volume of a solution and the quantity of biomolecules delivered to a cell. If using a visual readout such as fluorescent reporters, this may require a calibration standard to convert a measured fluorescence intensity into known quantities of volumes and editing biomolecules delivered to the cell. Possible innovations of existing technologies: Microfluidic flow measurement analysis, calibration slide with photostable fluorescent material, microspheres with assigned equivalent reference fluorophore units (ERFs), , and adoption of ASTM F3294-18 that provides a guide of standards for using microscopy-based methods to make quantitative fluorescence intensity measurements in cell-based assays …”

“…However, for those research applications in which it could be advantageous or informative to have control over dose delivery or high-resolution data on the distribution of genomic and phenotypic outcomes with relatively few cells, we ask those readers to consider the following ideas for future needs and technological advancements that would allow precision manipulation to be coupled with precision measurement: Deliver “treatment” and “control” conditions side by side to cells in the same environment (Figure A). Technology requirement: The ability to identify, index, and sort cells, so that each condition delivered to cells is distinguishable and can be observed or assayed separately. Possible innovations on existing technologies: CRISPR/Cas9 optogenetic variants (recently reviewed in refs and ) coupled with photostimulation instrumentation that delivers a controlled illumination pattern to part of a field of view; , direct microinjection. , Reproducible controlled delivery of a solution to a single cell, and the capability to shift the dose delivered in controlled increments (Figure B). Technology requirement: Ability to control and produce parameters reproducibly (i.e., for electroporation, N pulses, voltage, pulse duration; for injection, dimensions of microinjection needle, aperture area, pressure, and duration). Possible innovations in existing technologies: Microinjection with pressure and duration control to deliver editing reagents, electroporation coupled with single-cell electroporation in microchannels, nanostraws, − electrowetting, injections using microfluidically formed droplets, and injections with a cantilever connected to a microfluidic reservoir. , Measure the volume of the editing solution and the number of editing molecules delivered into a single cell (Figure C). Technology requirement: The ability to quantify the volume of a solution and the quantity of biomolecules delivered to a cell. If using a visual readout such as fluorescent reporters, this may require a calibration standard to convert a measured fluorescence intensity into known quantities of volumes and editing biomolecules delivered to the cell. Possible innovations of existing technologies: Microfluidic flow measurement analysis, calibration slide with photostable fluorescent material, microspheres with assigned equivalent reference fluorophore units (ERFs), , and adoption of ASTM F3294-18 that provides a guide of standards for using microscopy-based methods to make quantitative fluorescence intensity measurements in cell-based assays …”

“…Microfluidic flow measurement analysis, calibration slide with photostable fluorescent material, 78 microspheres with assigned equivalent reference fluorophore units (ERFs), 79,80 and adoption of ASTM F3294-18 that provides a guide of standards for using microscopy-based methods to make quantitative fluorescence intensity measurements in cell-based assays. 81 There is broader significance to the biochemistry measurement concepts discussed in this paper.…”

Automated analysis of bacterial flow cytometry data with FlowGateNIST

“…As with the FlowCal and TASBE Flow Analytics software packages, FlowGateNIST includes functions for calibration of fluorescence data using fluorescent beads. A variety of fluorescence calibration beads are now available, and some of them are NIST traceable [15,16]. For bacterial work, we use Sphereotech Rainbow Calibration Particles, catalog number RCP-30-5A, because they have fluorescence intensities comparable to that found in bacteria engineered to express fluorescent proteins.…”

Automated Analysis of Bacterial Flow Cytometry Data with FlowGateNIST

“…As with the FlowCal and TASBE Flow Analytics software packages, FlowGateNIST includes functions for calibration of fluorescence data using fluorescent beads. A variety of fluorescence calibration beads are now available, and some of them are NIST traceable [14,15]. For bacterial work, we use Sphereotech Rainbow Calibration Particles, catalog number RCP-30-5A, because they have fluorescence intensities comparable to that found in bacteria engineered to express fluorescent proteins.…”