Available Technology

COMBINATION LIVE AND FIXED WHOLE-CELL FLUORESCENT STAIN FOR EFFECTIVE CELL IDENTIFICATION AND SEGMENTATION IN FLUORESCENT MICROSCOPY

Cellular imaging technologies rely heavily on the use of fluorescence staining in a wide array of various techniques and assays. Whole-cell staining serves as a practical means of cell tracking, cell counting,  andcell phenotyping for techniques such as High Content Screening (HCS) or quantitative fluorescence microscopy. Overall, whole-cell fluorescent stains typically highlight cell surface or cytoplasmic factors that are common in most cell types and provide contrast for the edge of the cell body. In many scenarios, there is a need to have a fluorescent dye that can be used to permanently label either live or fixed cells for direct comparison between the two types of samples, stain cells brightly, have low background staining, and be non-toxic. The majority of the techniques to permanently fluorescently stain cells rely on having cells fixed and permeabilized to allow the dyes access to the cytoplasmic space. While being standard practice for fluorescent microscopy, both fixation and permeabilization can affect cell shape and protein localization. Additionally, since the cells are dead, those fluorescent dyes cannot track the dynamic changes of live cells.

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY®, Molecular Probes/Invitrogen) dyes offer an alternative to the traditional fluorescent dyes due to their high quantum yield, small stokes shift, high photostability, and their relatively insensitivity to their environment. In additional to those properties, the BODIPY core structure is much more hydrophobic than other fluorophores, which may allow it to be cell permeable. While there are several BODIPY dyes commercially available, NIST has developed a new synthetic strategy for BODIPY-based fluorophore that is easy, inexpensive, and used routinely as a live or fixed whole-cell fluorescent stain without any additional structural modification or assay protocol modifications. This leads to more robust measurements that can transcend the physical status of the cells which are being fluorescently stained for subsequent measurements. Ultimately it can be used as a reagent in cell assay kits (i.e. cell morphology, antibody staining concentration) and sold directly as a reagent to synthesize other fluorescent compounds.

Abstract: 

The invention is a new molecule that can be used for staining cells regardless of the living state of the cell, and it may comprise a class of similar molecules that might also be effective at whole-cell staining. The new molecule was formed by modifying boron­ dipyrromethene (BODIPY), a fluorescent dye, and conjugating it with N-hydroxysuccinimide through an ester linkage. The resultant dye retains the fluorescent brightness of the hydrophobic BODIPY precursor, while also having a greater hydrophilicity than conventional BODIPY dyes that allows it to distribute through a cell' s cytosol and bind to free amine groups within the cell.

Inventors: 
Christopher Arnatt, John Elliott
Patent Number: 
9951271
Technology Type(s): 
Advanced Manufacturing Processes, Analytical Chemistry, Manufacturing, Health Care, Biochemical Science, Chemical Physics, Chemical Sciences, Physical and Chemical Properties,
Internal Laboratory Ref #: 
15-043
Patent Issue Date: 
April 24, 2018
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