More Like this

1. SensorFRET: A Standardless Approach to Measuring Pixel-based Spectral Bleed-through and FRET Efficiency using Spectral Imaging

   https://doi.org/10.1038/s41598-017-15411-8  

   Arsenovic, Paul T.; Mayer, Carl R.; Conway, Daniel E. (December 2017, Scientific Reports)
2. Assessing drug-cell and drug-tissue interactions through spectral FRET imaging

   https://doi.org/10.1117/12.2583249  

   Leavesley, Silas J.; Annamdevula, Naga A.; Geurts, Aron; Pleshinger, D. J.; Rich, Thomas C. (March 2021, Proc. SPIE 11624, Visualizing and Quantifying Drug Distribution in Tissue V)

   Evans, Conor L.; Chan, Kin Foong (Ed.)
3. Milestones in the development and implementation of FRET-based sensors of intracellular signals: A biological perspective of the history of FRET

   https://doi.org/10.1016/j.cellsig.2020.109769  

   Deal, J.; Pleshinger, D.J.; Johnson, S.C.; Leavesley, S.J.; Rich, T.C. (November 2020, Cellular Signalling)

   null (Ed.)
4. FRET-Based Ca2+ Biosensor Single Cell Imaging Interrogated by High-Frequency Ultrasound

   https://doi.org/10.3390/s20174998  

   Yoon, Sangpil; Pan, Yijia; Shung, Kirk; Wang, Yingxiao (September 2020, Sensors)

   null (Ed.)

   Fluorescence resonance energy transfer (FRET)-based biosensors have advanced live cell imaging by dynamically visualizing molecular events with high temporal resolution. FRET-based biosensors with spectrally distinct fluorophore pairs provide clear contrast between cells during dual FRET live cell imaging. Here, we have developed a new FRET-based Ca2+ biosensor using EGFP and FusionRed fluorophores (FRET-GFPRed). Using different filter settings, the developed biosensor can be differentiated from a typical FRET-based Ca2+ biosensor with ECFP and YPet (YC3.6 FRET Ca2+ biosensor, FRET-CFPYPet). A high-frequency ultrasound (HFU) with a carrier frequency of 150 MHz can target a subcellular region due to its tight focus smaller than 10 µm. Therefore, HFU offers a new single cell stimulations approach for FRET live cell imaging with precise spatial resolution and repeated stimulation for longitudinal studies. Furthermore, the single cell level intracellular delivery of a desired FRET-based biosensor into target cells using HFU enables us to perform dual FRET imaging of a cell pair. We show that a cell pair is defined by sequential intracellular delivery of the developed FRET-GFPRed and FRET-CFPYPet into two target cells using HFU. We demonstrate that a FRET-GFPRed exhibits consistent 10–15% FRET response under typical ionomycin stimulation as well as under a new stimulation strategy with HFU. 

   more » « less
5. Comparison of spectral FRET microscopy approaches for single-cell analysis

   https://doi.org/10.1117/12.2546308  

   Deal, Joshua; Annamdevula, Naga S.; Pleshinger, D J.; Griswold, John Robert; Odom, Aliyah; Tayara, Alia; Lall, Malvika; Browning, Craig M.; Parker, Marina; Rich, Thomas C.; et al (February 2020, Proc. SPIE 11243, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVIII)

   Förster resonance energy transfer (FRET) is a valuable tool for measuring molecular distances and the effects of biological processes such as cyclic nucleotide messenger signaling and protein localization. Most FRET techniques require two fluorescent proteins with overlapping excitation/emission spectral pairing to maximize detection sensitivity and FRET efficiency. FRET microscopy often utilizes differing peak intensities of the selected fluorophores measured through different optical filter sets to estimate the FRET index or efficiency. Microscopy platforms used to make these measurements include wide-field, laser scanning confocal, and fluorescence lifetime imaging. Each platform has associated advantages and disadvantages, such as speed, sensitivity, specificity, out-of-focus fluorescence, and Zresolution. In this study, we report comparisons among multiple microscopy and spectral filtering platforms such as standard 2-filter FRET, emission-scanning hyperspectral imaging, and excitation-scanning hyperspectral imaging. Samples of human embryonic kidney (HEK293) cells were grown on laminin-coated 28 mm round gridded glass coverslips (10816, Ibidi, Fitchburg, Wisconsin) and transfected with adenovirus encoding a cAMP-sensing FRET probe composed of a FRET donor (Turquoise) and acceptor (Venus). Additionally, 3 FRET “controls” with fixed linker lengths between Turquoise and Venus proteins were used for inter-platform validation. Grid locations were logged, recorded with light micrographs, and used to ensure that whole-cell FRET was compared on a cell-by-cell basis among the different microscopy platforms. FRET efficiencies were also calculated and compared for each method. Preliminary results indicate that hyperspectral methods increase the signal-to-noise ratio compared to a standard 2-filter approach. 

   more » « less