How the Molecule Number Is Correctly Quantified in Two-Color Fluorescence Cross-Correlation Spectroscopy: Corrections for Cross-Talk and Quenching in Experiments

2005 ◽  
Vol 6 (6) ◽  
pp. 437-444 ◽  
Author(s):  
Zeno Foldes-Papp
Keyword(s):  
Cross Talk ◽  
Cross Correlation ◽  
Correlation Spectroscopy ◽  
Molecule Number

scholarly journals Cross-Talk-Free Dual-Color Fluorescence Cross-Correlation Spectroscopy for the Study of Enzyme Activity

2010 ◽  
Vol 82 (4) ◽  
pp. 1401-1410 ◽  
Author(s):  
Wonbae Lee ◽  
Yong-Ill Lee ◽  
Jeonghoon Lee ◽  
Lloyd M. Davis ◽  
Prescott Deininger ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cross Talk ◽  
Cross Correlation ◽  
Correlation Spectroscopy ◽  
Dual Color

scholarly journals Cross-talk-free Fluorescence Cross-Correlation Spectroscopy by the Switching Method

2008 ◽  
Vol 33 (1) ◽  
pp. 143-150 ◽  
Author(s):  
Yasuo Takahashi ◽  
Junichi Nishimura ◽  
Akemi Suzuki ◽  
Kiyochika Ishibashi ◽  
Masataka Kinjo ◽  
...  
Keyword(s):  
Cross Talk ◽  
Cross Correlation ◽  
Correlation Spectroscopy ◽  
Switching Method

scholarly journals Cross Talk Free Fluorescence Cross Correlation Spectroscopy in Live Cells

2005 ◽  
Vol 89 (3) ◽  
pp. 2069-2076 ◽  
Author(s):  
Elmar Thews ◽  
Margarita Gerken ◽  
Reiner Eckert ◽  
Johannes Zäpfel ◽  
Carsten Tietz ◽  
...  
Keyword(s):  
Cross Talk ◽  
Cross Correlation ◽  
Correlation Spectroscopy ◽  
Live Cells

scholarly journals Correcting for Spectral Cross-Talk in Dual-Color Fluorescence Cross-Correlation Spectroscopy

ChemPhysChem ◽  
2012 ◽  
Vol 13 (5) ◽  
pp. 1221-1231 ◽  
Author(s):  
Kirsten Bacia ◽  
Zdeněk Petrášek ◽  
Petra Schwille
Keyword(s):  
Cross Talk ◽  
Cross Correlation ◽  
Correlation Spectroscopy ◽  
Dual Color

scholarly journals Single-color Fluorescence Lifetime Cross-Correlation Spectroscopy in vivo

2020 ◽  
Author(s):  
M. Štefl ◽  
K. Herbst ◽  
M. Rübsam ◽  
A. Benda ◽  
M. Knop
Keyword(s):  
Fluorescence Lifetime ◽  
Cross Talk ◽  
Protein Interactions ◽  
Cross Correlation ◽  
Fluorescent Dyes ◽  
Fluorescent Proteins ◽  
Correlation Spectroscopy ◽  
Biological Origin ◽  
Complex Processes

ABSTRACTThe ability to quantify protein concentrations and to measure protein interactions in vivo is key information needed for the understanding of complex processes inside cells, but the acquisition of such information from living cells is still demanding. Fluorescence based methods like two-color fluorescence cross-correlation spectroscopy can provide this information but measurement precision is hampered by various sources of errors caused by instrumental or optical limitations such as imperfect overlap of detection volumes or detector cross-talk. Furthermore, the nature and properties of used fluorescent proteins or fluorescent dyes, such as labeling efficiency, fluorescent protein maturation, photo-stability, bleaching and fluorescence brightness can have an impact.Here we take advantage of lifetime differences as a mean to discriminate fluorescent proteins with similar spectral properties and to use them for single-color fluorescence lifetime cross-correlation spectroscopy (sc-FLCCS). By using only one excitation and one detection wavelength, this setup avoids all sources of errors resulting from chromatic aberrations and detector cross-talk. To establish sc-FLCCS we first engineered and tested multiple GFP-like fluorescent proteins for their suitability. This identified a novel GFP variant termed slmGFP (short lifetime monomeric GFP) with the so-far shortest lifetime. Monte-Carlo simulations were employed to explore the suitability of different combinations of GFP variants. Two GFPs, Envy and slmGFP were predicted to constitute the best performing couple for sc-FLCCS measurements. We demonstrated application of this GFP pair for measuring protein interactions between the proteasome and interacting proteins and for measuring protein interactions between three partners when combined with a red florescent protein. Together, our findings establish sc-FLCCS as a valid alternative for conventional dual-color(dc)-FCCS measurements.STATEMENT OF SIGNIFICANCEThe quantification of protein concentrations and protein-protein interactions in vivo is a crucial information needed for the understanding of complex processes inside cells. Determination of such information is unfortunately still challenging. Fluorescence-based method like fluorescence cross-correlation spectroscopy (FCCS) is the only method which provides this information in vivo and almost in the real time, however it suffers from limitations caused by experimental setup and biological origin of fluorescent proteins. We present single-color fluorescence lifetime cross-correlation spectroscopy as an alternative to FCCS, which uses the information of fluorescence lifetime to overcome some of these limitations. We challenged the method and determined its advantages and limitations and demonstrated the applicability of the method on the proteins of yeast proteasome.

Cross-talk-free fluorescence cross-correlation spectroscopy by the switching method

2007 ◽  
Author(s):  
Junichi Nishimura ◽  
Yasuo Takahashi ◽  
Masataka Kinjo ◽  
Atsushi Miyawaki
Keyword(s):  
Cross Talk ◽  
Cross Correlation ◽  
Correlation Spectroscopy ◽  
Switching Method

scholarly journals 2P328 Cross talk free Fluorescence Cross Correlation Spectroscopy using switching method

2005 ◽  
Vol 45 (supplement) ◽  
pp. S201
Author(s):  
J. Nishimura ◽  
T. Sugiyama ◽  
Y. Takahashi ◽  
M. Knjo ◽  
A. Miyawaki
Keyword(s):  
Cross Talk ◽  
Cross Correlation ◽  
Correlation Spectroscopy ◽  
Switching Method

scholarly journals Fluorescence Cross-Correlation Spectroscopy Yields True Affinity and Binding Kinetics of Plasmodium Lactate Transport Inhibitors

2021 ◽  
Vol 14 (8) ◽  
pp. 757
Author(s):  
Iga Jakobowska ◽  
Frank Becker ◽  
Stefano Minguzzi ◽  
Kerrin Hansen ◽  
Björn Henke ◽  
...  
Keyword(s):  
Rate Constants ◽  
Cross Correlation ◽  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Confocal Imaging ◽  
Binding Kinetics ◽  
Acceptor Site ◽  
Lactate Transport ◽  
Entry Site ◽  
Hydrogen Bond Acceptor

Blocking lactate export in the parasitic protozoan Plasmodium falciparum is a novel strategy to combat malaria. We discovered small drug-like molecules that inhibit the sole plasmodial lactate transporter, PfFNT, and kill parasites in culture. The pentafluoro-3-hydroxy-pent-2-en-1-one BH296 blocks PfFNT with nanomolar efficiency but an in vitro selected PfFNT G107S mutation confers resistance against the drug. We circumvented the mutation by introducing a nitrogen atom as a hydrogen bond acceptor site into the aromatic ring of the inhibitor yielding BH267.meta. The current PfFNT inhibitor efficiency values were derived from yeast-based lactate transport assays, yet direct affinity and binding kinetics data are missing. Here, we expressed PfFNT fused with a green fluorescent protein in human embryonic kidney cells and generated fluorescent derivatives of the inhibitors, BH296 and BH267.meta. Using confocal imaging, we confirmed the location of the proposed binding site at the cytosolic transporter entry site. We then carried out fluorescence cross-correlation spectroscopy measurements to assign true Ki-values, as well as kon and koff rate constants for inhibitor binding to PfFNT wildtype and the G107S mutant. BH296 and BH267.meta gave similar rate constants for binding to PfFNT wildtype. BH296 was inactive on PfFNT G107S, whereas BH267.meta bound the mutant protein albeit with weaker affinity than to PfFNT wildtype. Eventually, using a set of PfFNT inhibitor compounds, we found a robust correlation of the results from the biophysical FCCS binding assay to inhibition data of the functional transport assay.

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