scholarly journals Novel PE and APC Tandems: Additional Near-Infrared Fluorochromes for Use in Spectral Flow Cytometry

2020 ◽  
Author(s):  
Yekyung Seong ◽  
Tuan Andrew Nguyen ◽  
Yian Wu ◽  
Denny Nguyen ◽  
Archana Thakur ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Near Infrared ◽  
Biomarker Discovery ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Cell Types ◽  
Color Flow ◽  
Emission Signal ◽  
Avalanche Diodes

ABSTRACTFlow cytometry is an essential technology to identify and quantify cell populations. To support the need for high content analyses, we developed new fluorochromes designed specifically to enhance our flow cytometry capabilities. A standard Cytek Aurora flow cytometer has 3 excitation lasers (405nm, 488nm, and 640nm) and incorporates the latest Avalanche Diodes Photodetector (ADP) technology, which demonstrates significant improvement in sensitivity for fluorescent emission signals longer than 800nm. However, there are no commercially available fluorochromes capable of being excited by the above lasers with peak emission signal beyond 800nm. To address this gap, we engineered 6 new fluorochromes: PE-750, PE-800, PE-830 for the 488nm laser and APC-750, APC-800, APC-830 for the 640nm laser. These novel fluorescent molecules were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Via the principle of fluorescence resonance energy transfer (FRET), these conjugates create unique fluorochromes with the excitation properties of the donor and the emission properties of the acceptor. After the fluorochromes were created and validated, they were conjugated to a test antibody. We demonstrated long-term stability of the conjugated antibodies at −20°C with protein stabilizing cocktails. Most importantly, in order to show the utility of these novel fluorochromes, we created and validated a 34-color flow cytometry panel designed to measure broad human immune function with high sensitivity. This panel will be applied for immunophenotyping of multiple different cell types from dissociated tumors and blood samples. In conclusion, this novel 34-color high-content flow cytometry panel will be able to support enhanced analysis and diagnosis in Immuno-oncology and facilitate innovation in biomarker discovery.

scholarly journals Biological nanoscale fluorescent probes: From structure and performance to bioimaging

2020 ◽  
Vol 39 (1) ◽  
pp. 209-221
Author(s):  
Jiafeng Wan ◽  
Xiaoyuan Zhang ◽  
Kai Zhang ◽  
Zhiqiang Su
Keyword(s):  
Fluorescent Probes ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Biological Imaging ◽  
Fluorescent Materials ◽  
And Performance

Abstract In recent years, nanomaterials have attracted lots of attention from researchers due to their unique properties. Nanometer fluorescent materials, such as organic dyes, semiconductor quantum dots (QDs), metal nano-clusters (MNCs), carbon dots (CDs), etc., are widely used in biological imaging due to their high sensitivity, short response time, and excellent accuracy. Nanometer fluorescent probes can not only perform in vitro imaging of organisms but also achieve in vivo imaging. This provides medical staff with great convenience in cancer treatment. Combined with contemporary medical methods, faster and more effective treatment of cancer is achievable. This article explains the response mechanism of three-nanometer fluorescent probes: the principle of induced electron transfer (PET), the principle of fluorescence resonance energy transfer (FRET), and the principle of intramolecular charge transfer (ICT), showing the semiconductor QDs, precious MNCs, and CDs. The excellent performance of the three kinds of nano fluorescent materials in biological imaging is highlighted, and the application of these three kinds of nano fluorescent probes in targeted biological imaging is also introduced. Nanometer fluorescent materials will show their significance in the field of biomedicine.

scholarly journals pH-Insensitive FRET Voltage Dyes

2007 ◽  
Vol 12 (5) ◽  
pp. 656-667 ◽  
Author(s):  
Michael P. Maher ◽  
Nyan-Tsz Wu ◽  
Hong Ao
Keyword(s):  
Ion Channel ◽  
High Throughput ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Fast Response ◽  
Resting Membrane Potential ◽  
Absolute Calibration ◽  
High Throughput Assay ◽  
Voltage Sensitive Dyes

Many high-throughput ion channel assays require the use of voltage-sensitive dyes to detect channel activity in the presence of test compounds. Dye systems employing Förster resonance energy transfer (FRET) between 2 membrane-bound dyes are advantageous in combining high sensitivity, relatively fast response, and ratiometric output. The most widely used FRET voltage dye system employs a coumarin fluorescence donor whose excitation spectrum is pH dependent. The authors have validated a new class of voltage-sensitive FRET donors based on a pyrene moiety. These dyes are significantly brighter than CC2-DMPE and are not pH sensitive in the physiological range. With the new dye system, the authors demonstrate a new high-throughput assay for the acid-sensing ion channel (ASIC) family. They also introduce a novel method for absolute calibration of voltage-sensitive dyes, simultaneously determining the resting membrane potential of a cell. ( Journal of Biomolecular Screening 2007:656-667)

scholarly journals A Near-Infrared Fluorescent Probe Based on a FRET Rhodamine Donor Linked to a Cyanine Acceptor for Sensitive Detection of Intracellular pH Alternations

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2679 ◽  
Author(s):  
Yibin Zhang ◽  
Jianheng Bi ◽  
Shuai Xia ◽  
Wafa Mazi ◽  
Shulin Wan ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Intracellular Ph ◽  
Near Infrared ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Sensitive Detection ◽  
Solution Ph ◽  
Acidic Conditions ◽  
Ph Level ◽  
Rhodamine Dye

A fluorescence resonance energy transfer (FRET)-based near-infrared fluorescent probe (B+) for double-checked sensitive detection of intracellular pH changes has been synthesized by binding a near-infrared rhodamine donor to a near-infrared cyanine acceptor through robust C-N bonds via a nucleophilic substitution reaction. To demonstrate the double-checked advantages of probe B+, a near-infrared probe (A) was also prepared by modification of a near-infrared rhodamine dye with ethylenediamine to produce a closed spirolactam residue. Under basic conditions, probe B+ shows only weak fluorescence from the cyanine acceptor while probe A displays nonfluorescence due to retention of the closed spirolactam form of the rhodamine moiety. Upon decrease in solution pH level, probe B+ exhibits a gradual fluorescence increase from rhodamine and cyanine constituents at 623 nm and 743 nm respectively, whereas probe A displays fluorescence increase at 623 nm on the rhodamine moiety as acidic conditions leads to the rupture of the probe spirolactam rings. Probes A and B+ have successfully been used to monitor intracellular pH alternations and possess pKa values of 5.15 and 7.80, respectively.

scholarly journals A general method for heterokaryon detection using resonance energy transfer and a fluorescence-activated cell sorter.

1982 ◽  
Vol 30 (12) ◽  
pp. 1297-1300 ◽  
Author(s):  
P E Wanda ◽  
J D Smith
Keyword(s):  
Energy Transfer ◽  
Somatic Cell ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Types ◽  
Parental Cell ◽  
Cell Sorter ◽  
Cellular Replication ◽  
Universal Applicability ◽  
Unambiguous Detection

A simple and general labeling method has been adapted for the rapid detection and isolation of heterokaryons soon after somatic cell fusion. The method involves prelabeling each parental cell type by adding one of two hydrophobic fluorescent probes, F18 or R16, to the culture medium. These probes are nontoxic, nonmutagenic, and do not inhibit either cellular replication or the efficiency of fusion. Following polyethylene glycol (PEG)-induced fusion, heterokaryons are identified on a cell sorter as cells showing fluorescence as a result of nonradiative resonance energy transfer (RET) between the two fluorochromes. Optimal conditions are described for the unambiguous detection of heterokaryons by fusion-induced RET. The value of this method lies in the universal applicability of F18 and R16 as simple and direct membrane labels for any pair of parental cells, even those lacking selectable genetic markers or detectable antigenic differences. This potential for heterokaryon selection in any cell cross should significantly expand the range of cell types amenable to investigation through somatic cell genetics, while the rapidity of the method should facilitate the study of early events following fusion.

Fluorescence resonance energy transfer (FRET) based nanoparticles composed of AIE luminogens and NIR dyes with enhanced three-photon near-infrared emission for in vivo brain angiography

Nanoscale ◽  
2018 ◽  
Vol 10 (21) ◽  
pp. 10025-10032 ◽  
Author(s):  
Wen Liu ◽  
Yalun Wang ◽  
Xiao Han ◽  
Ping Lu ◽  
Liang Zhu ◽  
...  
Keyword(s):  
Near Infrared ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Infrared Emission ◽  
Biological Imaging ◽  
High Contrast ◽  
Nir Dyes ◽  
Nir Fluorescence ◽  
Near Infrared Emission

Near-infrared (NIR) fluorescence is very important for high-contrast biological imaging of high-scattering tissues such as brain tissue.

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