Chapter 14
Using Genetically Encoded Fluorescent Biosensors
for Quantitative In Vivo Imaging
Akira Yoshinari, Jacob Moe-Lange, Thomas J. Kleist,
Heather N. Cartwright, David A. Quint, David W. Ehrhardt,
Wolf B. Frommer, and Masayoshi Nakamura
Abstract
Fluorescent biosensors are powerful tools for tracking analytes or cellular processes in live organisms and
allowing visualization of the spatial and temporal dynamics of cellular regulators. Fluorescent protein (FP)based biosensors are extensively employed due to their high selectivity and low invasiveness. A variety of
FP-based biosensors have been engineered and applied in plant research to visualize dynamic changes in
pH, redox state, concentration of molecules (ions, sugars, peptides, ATP, reactive oxygen species, and
phytohormones), and activity of transporters. In this chapter, we briefly summarize reported uses of
FP-based biosensors in planta and show simple methods to monitor the dynamics of intracellular Ca
2+ in
Arabidopsis thaliana using a ratiometric genetically encoded Ca
2+ indicator, MatryoshCaMP6s.
Key words Fluorescence microscopy, FRET , cpFP, Genetically encoded fluorescent biosensors,
Arabidopsis root perfusion chamber
1 Introduction
Fluorescent biosensors have proven to be important tools in plant
sciences for visualization and measurement of the concentration of
various metabolites, ions, and phytohormones, of the state of biochemical parameters such as pH or redox, and of cellular and
subcellular signaling networks. Fluorescent biosensors are organic
compounds or proteins engineered to respond to fluctuations in
analyte concentration or biochemical/biophysical parameters with
changes in fluorescence excitation, emission, or lifetime. In particular, genetically encoded fluorescent biosensors enable the monitoring of analyte concentrations in a variety of plant tissues in a
noninvasive way that maintains structures and compartments
(Fig. 1 and Table 1) and facilitates spatiotemporal analysis of
biological activities altered by environmental cues. Below, we
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
303
Using Genetically Encoded Fluorescent Biosensors
for Quantitative In Vivo Imaging
Akira Yoshinari, Jacob Moe-Lange, Thomas J. Kleist,
Heather N. Cartwright, David A. Quint, David W. Ehrhardt,
Wolf B. Frommer, and Masayoshi Nakamura
Abstract
Fluorescent biosensors are powerful tools for tracking analytes or cellular processes in live organisms and
allowing visualization of the spatial and temporal dynamics of cellular regulators. Fluorescent protein (FP)based biosensors are extensively employed due to their high selectivity and low invasiveness. A variety of
FP-based biosensors have been engineered and applied in plant research to visualize dynamic changes in
pH, redox state, concentration of molecules (ions, sugars, peptides, ATP, reactive oxygen species, and
phytohormones), and activity of transporters. In this chapter, we briefly summarize reported uses of
FP-based biosensors in planta and show simple methods to monitor the dynamics of intracellular Ca
2+ in
Arabidopsis thaliana using a ratiometric genetically encoded Ca
2+ indicator, MatryoshCaMP6s.
Key words Fluorescence microscopy, FRET , cpFP, Genetically encoded fluorescent biosensors,
Arabidopsis root perfusion chamber
1 Introduction
Fluorescent biosensors have proven to be important tools in plant
sciences for visualization and measurement of the concentration of
various metabolites, ions, and phytohormones, of the state of biochemical parameters such as pH or redox, and of cellular and
subcellular signaling networks. Fluorescent biosensors are organic
compounds or proteins engineered to respond to fluctuations in
analyte concentration or biochemical/biophysical parameters with
changes in fluorescence excitation, emission, or lifetime. In particular, genetically encoded fluorescent biosensors enable the monitoring of analyte concentrations in a variety of plant tissues in a
noninvasive way that maintains structures and compartments
(Fig. 1 and Table 1) and facilitates spatiotemporal analysis of
biological activities altered by environmental cues. Below, we
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
303