Microflow time-resolved cytometry for FRET and fluorescent protein development

用于 FRET 和荧光蛋白开发的微流时间分辨细胞术

• 批准号: 10388738
• 负责人: Jessica Perea Houston
• 金额: $ 20万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10388738
• 关键词: Acoustics; Applications Grants; Cell Separation; Cells; Cytometry; Data; Development; Event; Faculty; Flow Cytometry; Fluorescence; Fluorescence Resonance Energy Transfer; Frequencies; Future; Hispanic-serving Institution; Image; Investigation; Kinetics; Mammalian Cell; Measurement; Measures; Microfluidics; Microscope; Movement; New Mexico; Optics; Parents; Proteins; Request for Applications; Resolution; Sorting - Cell Movement; Students; System; Time; Training; Universities; Validation; Work; base; cellular imaging; design; design and construction; fluorescence lifetime imaging; fluorophore; instrument; microchip; microscopic imaging; novel; screening; tool

Summary This application requests supplemental support for the purchase of a fluorescence lifetime imaging microscope (FLIM). A new microscope of this type will significantly advance the aims of the project: R01GM129859 Microflow time-resolved Flow Cytometry for FRET and Fluorescence Protein Development. FLIM systems have unique abilities to image cells whereby the image is a rendering of the fluorescence lifetimes measured across cells at a high resolution. The fluorescence lifetime is the average time a fluorophore spends in the excited state; it carries a significant amount of information about the fluorophore such as the microenvironment, near-neighbor proximity, and movement of intracellular molecules. This will support the parent R01 project because the objective for this work is to design and adapt fluorescence lifetime flow cytometry onto a microchip platform for cell screening and eventual sorting. The aims will develop a novel microflow cytometer that will, when completely vetted, incorporate many unique features such as acoustic focusing of cells through microfluidic channels, multi-frequency measurements that give rise to multiple- fluorescence lifetime values per cell, and electro-optical design for eventual cell sorting based on fluorescence lifetime measurements. Concurrently with the design and construction of the microflow cytometer two major applications of this tool are planned/underway. These are 1) the quantification of Förster resonance energy transfer (FRET) events inside of mammalian cells, and 2) the enrichment of near-infrared fluorescent proteins based on photo-kinetic data. Therefore a microscope (FLIM) with the unique ability to measure fluorescence decay kinetics is essential for 4 reasons: 1) it will enable us to independently cross-validate our cytometry measurements thereby providing resolution and precision that are orders of magnitude greater for reliable optimization; 2) it will significantly enhance the collaborative applications that we are exploring with our novel cytometer and current aims; 3) it will greatly expand the applicability of our cytometer for other fields thus broadening the biomedical impact while supporting future grant applications; and 4) it will become a tool for training and development of students and collaborating faculty at New Mexico State University, a Hispanic Serving Institution.

总结

项目成果

Toward the Development of an On-Chip Acoustic Focusing Fluorescence Lifetime Flow Cytometer.

致力于开发片上声聚焦荧光寿命流式细胞仪。

• DOI: 10.3389/fphy.2021.647985
• 发表时间: 2021
• 期刊: Frontiers in physics
• 影响因子: 3.1
• 作者: SambranoJr,Jesus;Rodriguez,Felicia;Martin,John;Houston,JessicaP
• 通讯作者: Houston,JessicaP

Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time-resolved flow cytometry.

• DOI: 10.1002/cyto.a.23606
• 发表时间: 2019-01
• 期刊: Cytometry. Part A : the journal of the International Society for Analytical Cytology
• 作者: Alturkistany F;Nichani K;Houston KD;Houston JP
• 通讯作者: Houston JP

Evaluation of Caspase-3 Activity During Apoptosis with Fluorescence Lifetime-Based Cytometry Measurements and Phasor Analyses.

• DOI: 10.1002/cyto.a.24207
• 发表时间: 2020-12
• 期刊: Cytometry. Part A : the journal of the International Society for Analytical Cytology
• 作者: Nichani K;Li J;Suzuki M;Houston JP
• 通讯作者: Houston JP

Investigating differences between tamoxifen resistant and sensitive breast cancer cells with flow cytometry.

• DOI: 10.1002/cyto.a.24306
• 发表时间: 2021-03
• 期刊: Cytometry. Part A : the journal of the International Society for Analytical Cytology
• 作者: Bitton A;Zheng Y;Houston JP;Houston KD
• 通讯作者: Houston KD