1. Quantification of Membrane Protein Dynamics and Interactions in Plant Cells by Fluorescence Correlation Spectroscopy 
Xiaojuan Li, Jingjing Xing, Zongbo Qiu, Qihua He, Jinxing Lin 
Molecular Plant 
Volume 9, Issue 9, Pages (September 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1 Schematic Presentation of the Principles of FCS and FCCS.
(A) A laser beam is focused by a high-NA objective lens on a fluorescent sample. The epifluorescence is collected by the same objective, reflected by a dichroic mirror, focused by a tube lens, filtered, and passed through a confocal aperture onto the detector. APD, avalanche photodiode; PMT, photomultiplier tube.
(B) FCS in live plant cells. Upper: Confocal laser-scanning image of root cells expressing eGFP and mCherry. Lower: Magnified focal volume (blue) within which the sample particles (black circles) are illuminated (red and green).
(C) The typical fluorescence signal, as a function of time, measured for red and green fluorophore-tagged molecules.
(D) Autocorrelation curve shows the decay of temporal correlation over time in an FCS experiment.
(E) In dual-color FCCS, the binding and colocalization of spectrally distinct fluorophores (such as red and green) produce fluctuating fluorescence signals between the red and the green fluorescent intensities. Each time trace is autocorrelated (red and green curves). The cross-correlation function between the green and red traces is computed (blue curve) providing information on the degree of binding or colocalization.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2
Schematic Diagram of the FCS Technique to Investigate Particle Mobility and Concentration.
(A) Fluorescence fluctuations occur in a small region of the membrane resulting from the fluorophores diffused in and out of the observation area defined by the laser spot (dashed ellipses).
(B) Autocorrelation function curves for diffusion of a given number of particles (N = 8) and increasing diffusion time. The different colors of the curves (red, green, cyan, and purple) represent the passage of diffusion time (from shorter to longer).
(C) Autocorrelation function curves for diffusion of increasing particle numbers with a given diffusion time (τD = 10 μs). The colors (red, green, cyan, and purple) represent the increasing particle numbers (from lower to higher).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3
Schematic Representation of FCS Probing Membrane Organization and Membrane Protein Lateral Diffusion.
(A) Schematic diagram showing membrane organization with free diffusion (left) and microdomain (right, dashed ellipses) with the trajectory drawn for a single particle.
(B) Autocorrelation curves reflect the changes in plasma membrane protein mobility at different locations. Fast decay arises from free diffusion of the fluorescent molecules in the observation volume. The slow decay corresponds to the slow diffusion behavior of molecules in the membrane microdomain.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4 FCCS Approach Used in the Study of Protein Oligomerization.
(A) Schematic showing PM protein dynamic equilibrium of the monomer, predimer, and active dimer.
(B and C) Theoretical FCCS analysis predicts the proportion of monomeric and dimeric PM protein in living cells. The green circles represent the observation volume in the FCCS experiment. (B) For the resting state case, the ratio of GRG(0) (cross-correlation curves shown in black) to GR(0) or GG(0) should theoretically be zero but shows a finite minimum value resulting from the predimers without stimulation by effectors. (C) For the effector stimulation case, the ratio of GRG(0) to GR(0) or GG(0) increases dramatically due to the induced hetero-oligomerization of red and green fluorescently tagged proteins. Thus, the oligomerization or colocalization equilibrium of PM protein is resolved by calculating the minimum and maximum ratios for the set of resting and stimulated experimental conditions, respectively.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions