Microscopy approaches to investigate protein dynamics and lipid organization

Figures & data

Table 1. Summary of microscopy techniques.

Method	Principle	Advantage	Limitation	Applications	Reference
Phasor analysis to fluorescent lifetime microscopy (FLIM)	FLIM detects changes in the lifetime of a fluorescent dye or protein in each pixel of an image and the phasor approach transforms it into a fit free 2D coordinate system.	• Does not require a priori knowledge of the number of components that need to be unmixed • Applicable to live cell imaging	• Slow acquisition speed • Does not provided information on domain size and geometry	• Membrane order measurements to detect lipid phases • Useful for FRET measurements	(Digman et al., 2008) (Owen et al., 2012b) (Golfetto et al., 2013)
Fluorescent resonance energy transfer (FRET)	A donor fluorophore non-radiatively transfers energy to an acceptor fluorophore by means of intermolecular long-range dipole-dipole coupling.	• Can detect interactions <10 nm • Suitable for live cell measurements • Applicable to confocal microscopy • Can be combined with FLIM	• Photobeaching • Probe orientation • Dipole moment • Must incorporate the distance of spacers	• Measures protein-protein interactions • Protein association with domains • Receptor oligomerization	(Clegg et al., 2005) (Varma & Mayor, 1998) (Kenworthy & Edidin, 1998) (Kenworthy et al., 2000) (Siegel, 2000)
Fluorescent recovery after photobleaching (FRAP)	Fluorescent molecules are irreversibly photobleached by a high power laser in the region of interest of the cell and the recovery of fluorescence from surrounding non-bleached fluorescence molecules monitored.	• Easy to perform • Live cell measurements • Applicable to confocal microscopy	• Ensemble measurement • Cannot reveal different diffusion modes	• Monitoring the mobility of proteins and fluorescent lipid analogues	(Ishikawa-Ankerhold et al., 2012)
Fluorescent correlation spectroscopy (FCS)	Fluctuations in fluorescence intensity caused by the diffusion of fluorescent molecules through a small focal volume are observed. The corresponding autocorrelation function can be fitted to quantify diffusion coefficients and molecule concentrations within the focal volume.	• Single molecule technique (nM fluorophores concentrations) • Small observation volume (<10 µl) • High temporal resolution with large range (µsec-sec) • Live cell measurements • Can be applied to a single point measurement or line scan	• Does not provide spatial information • Long acquisition times • Data fitting and interpretation is complex	• Measures: • Protein–protein interactions • Diffusion mode and coefficient • Chemical kinetics and photo-physical properties	(Benda et al., 2003) (Hinde et al., 2013) (Clegg et al., 2005) (Enderlein & Gregor, 2005) (Bohmer, 2002) (Benda et al., 2005) (Benes et al., 2004)
Raster image correlation spectroscopy (RICS)	A time series of frames is acquired using a raster scan, where pixels in each image are measured at different times and therefore the spatial correlation of the whole image contains details about dynamic processes occurring on different time scales.	• Relative simple method for diffusion measurements • Can differentiate slow diffusion from binding events • Large temporal range (µsec-sec) • Live cell measurements • Applicable to confocal microscopy	• Averaging over pixels, lines and frames within one measurement limits spatial resolution	• Measures protein behaviour such as formation of protein complexes and binding dynamics to an immobile or mobile structure.	(Digman et al., 2005) (Digman et al., 2009)
Single particle tracking (SPT)	Position of sparsely labelled single molecules is precisely mapped in time and space	• High spatial and temporal resolution • Live cell measurements	• Labelling of individual molecules is required • Small number of molecules is measured • Requires complex analysis	• Molecular mobility and detection of transient confinement • Hop diffusion (requires ultrafast cameras)	(Kusumi et al., 2011)
Stimulation emission depletion (STED)	Sample is scanned with two overlapping lasers. Molecules located in the outer part of the diffraction-limited excitation area are depleted by stimulated emission (doughnut shape beam) so that only fluorophores from the central region of the excitation spot are able to emit fluorescence.	• Super-resolution technique with 30–150 nm resolution (depends on STED laser power) • Built on laser-scanning microscopy • Can be combined with FCS	• Limited choice of fluorescent probes • Photo-bleaching due to high laser paper • Specialized microscope set-up	• Imaging with spatial super-resolution	(Geumann et al., 2010) (Halemani et al., 2010) (Singh et al., 2012) (Wurm et al., 2011)
STED-FCS	Combines spatial resolution of STED with temporal resolution of FCS.	• High spatial resolution of 30–150 nm (due to reduced excitation volume) • High temporal resolution (<µsec) • Live cell measurements	• Limited choice of fluorescent probes • Photo-bleaching due to high laser paper • Specialized microscope set-up • Long acquisition times • Data fitting and interpretation is complex	• Diffusion of fluorescent lipid analogues • First detection of differences in lipid diffusion and lipid nanoclusters	(Eggeling et al., 2009) (Mueller et al., 2011)
Stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM)	Localization microscopy relies on the stochastic activation of conventional fluorophores (STORM) or photoswitchable fluorescent proteins (PALM). During one cycle, fluorescent molecules are photoswitched/activated, imaged and then photobleached, and the cycle repeated several thousand times.	• Localization of individual molecules with 10–25 nm precision in lateral direction and 60–100 nm in axial direction • Single molecule data	• Long acquisition time • Not suitable for all fluorophores • Photo-physical properties of fluorophores need to be understood for molecular counting • Complex analysis since each molecules needs to be fitted	• Molecular quantification of protein distribution and clustering	(Williamson et al., 2011) (Rossy et al., 2013) (Sengupta et al., 2011)

Figure 1. Different plasma membrane models. (A) Fluid mosaic model presents the plasma membrane as a two-dimensional liquid in which proteins (orange and blue shapes) are homogenously distributed in the lipid bilayers (dark blue). This model allows the possibility of rapid lateral diffusion of lipids and proteins within the membrane. (B) The lipid raft model proposes the existence of small regions (green shapes) enriched in cholesterol (red shapes) and sphingolipids. (C) The picket fence model suggests that transmembrane proteins act as “pickets” (orange pink and blue shapes) and the membrane skeleton as “fences” (dark orange net). The membrane skeleton confines the diffusion of membrane components due to collision. The membrane is depicted from cytosolic side. (D) The lipid shell model suggests that 25% of the cell membrane surface contains proteins (orange shapes) which are surrounded by lipid rings (purple and pink). (E) The models are exclusive and it is likely that different various membrane domains and compartments coexist in the plasma membrane.

Figure 2. Spectral and lifetime methods of analysis to detect lipid phases. (A) The plasma membrane is depicted with the membrane dye Laurdan (orange shapes) in a random distribution between liquid disordered phase (Ld, blue lipids) and liquid ordered phase (Lo, green lipids). (B) Schematic emission spectra of lipid-phase sensitive dye, Laurdan, with green and purple spectra corresponding to Lo and Ld phases, respectively. (C) Typical fluorescence lifetime decays correspond to liquid ordered (dotted line) and liquid disordered (solid) phases, respectively. (D) Transformation of a spectrum or decay into a phasor (vector) plot. Spots represent data for Lo (lower) and Ld (upper) phases.

Figure 3. Fluorescent approaches for detection of protein interactions and dynamics. (A) Fluorescence resonance energy transfer (FRET) with the most commonly used fluorescent protein pair, CFP-YFP. Middle scheme: FRET does not occur because of a large distance between the CFP and YFP proteins. Right scheme: FRET occurs when the distance (R₀) between donor and acceptor is below 10 nm. (B) Fluorescence correlation spectroscopy (FCS). Middle scheme: The cross correlation function between two proteins is constant because the proteins are not assembled into complexes or the distance between them is too large. Right scheme: the cross correlation function indicates the presence of a protein complex. (C) Fluorescence recovery after photobleaching (FRAP). Middle scheme: a region of interest is photobleached region (black square). Right scheme: recovery of fluorescence intensity.

Figure 4. Super-resolution methods. (A) Features in the plasma membrane such as lipid rafts are below (green PSF) the diffraction limit (black PSF). (B) Stimulated emission depletion (STED). Left panel: PSF of confocal beam. Right panel: PSF of STED beam where in a doughnut shaped STED beam is overlaid onto a confocal beam (green) which reduces the focal volume. (C) Combination of STED and FCS. The autocorrelation function is shifted towards shorter time when STED is applied to FCS, which allows a precise quantification of the diffusion coefficient. (D) Photo-activated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM). Single molecules are photo-activated so that there are a limited number of fluorescent events in each frame. The PSF of each fluorescent event is analysed to obtain the localizations of each molecule with nanometre precision. All localization are combined to produce a super-resolution image.

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