Imaging molecular order in cell membranes by polarization resolved fluorescence microscopy

Sophie Brasselet*, Patrick Ferrand, Alla Kress, Xiao Wang, Hubert Ranchon, Alicja Gasecka

Institut Fresnel, CNRS, Aix-Marseille Université, Ecole Centrale Marseille, Domaine Universitaire St Jérôme, 13013 Marseille, France

Abstract The use of light polarization properties in the analysis of fluorescence images has driven a large amount of research towards the measurement of orientational behavior of molecules in cells, in particular in their membranes. This field has been recently revisited to enlarge the possibilities of polarization resolved fluorescence microscopy. We show that this technique allows retreiving a wealth of information on the constraints that hinder rotational mobility of lipid probes and proteins in membranes, bringing thus new insights on inter-proteins and lipid-proteins interactions, on membrane morphology at the sub-diffraction length scale and on local membrane physical properties such as viscosity.

Keywords polarized fluorescence microscopy, fluorescence anisotropy, two-photon fluorescence, molecular order, cell membrane

Contents

1. Introduction 2. Defining molecular orientational order in cell membranes 3. Fluorescence and its relation to light polarization 4. Fluorescence anisotropy

4.1 Fluorescence anisotropy in isotropic media 4.2 Fluorescence anisotropy imaging in cell membranes and its limita-

tions 5. Polarization resolved fluorescence

5.1 Principle 5.2 Application to molecular order imaging in heterogeneous mem-

branes 5.3 Circumventing depolarization effects

6. Summary and outlook

2

1 Introduction

Molecular orientation is a parameter which is important to monitor in order to understand the interactions that drive the structure and morphology of biomolecu-lar assemblies and their consequences on related biological functions. This organi-zation, governed by molecular interactions such as those between proteins, be-tween lipids, or between proteins and lipids, plays an important role in biology. Proteins and lipids in cell membranes are for instance known to form structured assemblies (such as protein clusters or lipid domains) with collective molecular order participating in cell motility (Cheresh1999), vesicular trafficking (Anantha-ram2010) and signaling (Pentcheva2001,Fooksman2006,Benninger2009). Protein interactions in supramolecular complexes such as bio-filaments are also highly driven by orientational order (Borejdo1993,Brack2004,Vrabioiu2006). For in-stance, collagen and other protein filaments in the extra-cellular matrix undergo strong exchange forces with cells membranes proteins, which can be affected in the development of tumors. An orientational organization is quantified by the so-called "molecular order", which varies between complete disorder (isotropic me-dium) to complete order (such as in a crystalline medium).

Imaging such information quantitatively using optical microscopy remains however a challenge. Measuring molecular orientational diffusion in solutions has been performed since the 50’s using the well known fluorescence anisotropy technique (Weber1953), which consists in exciting the molecules by a linear polarization, and probing their fluorescence emission along two perpendicular analysis directions. This technique, based on the capacity of polarized light to photo-select only molecules which exhibit an orientation close to its polarization direction, gives access to molecular rotational diffusion time in solutions, local environment properties (temperature, viscosity) (Cantor1980), but also to phenomena related to depolarization mechanisms such as fluorescence resonant energy transfer (FRET) between neighbor molecules (Chan2011). Fluorescence anisotropy is however more delicate to implement in oriented media (for instance membranes or fibers) where the photo-selection also depends on the orientation of the sample itself. Historically, techniques dedicated to the study of the cell mem-brane have been therefore essentially concentrated on measuring the spatial locali-zation of molecules. Membranes are indeed highly heterogeneous and dynamics, constituted of different lipids types and molecules (such as cholesterol), as well as membrane proteins which are connected to the cytoskeleton in a complex way. Techniques such as Fluorescence Recovery After Photobleaching (FRAP) (Schlessinger1976), Fluorescence correlation spectroscopy (FCS) (Schwille1999) and single molecule tracking (SMT) (Schütz2000) have allowed a better under-standing of the mobility of proteins and lipid and their perturbation by specific conditions of the cells, bringing a considerable amount of information on the nano-scale organization of the cell membrane and its associated signaling dynam-

3

ics. Less attention has been directed towards protein and lipid orientations in cell membranes.

Applying fluorescence anisotropy to membrane imaging is in fact only success-ful under certain conditions, which limit the observation to specific parts of the membrane, since this technique only addresses two directions of detected polariza-tion. Nevertheless, it has permitted to bring a new insight into specific molecular mechanisms in cell membranes, such as proteins clustering or proteins conformational changes, which cannot be only addressed by pure lateral diffusion. Such studies, which can be performed either in the time domain (time resolved fluorescence anisoptropy) or averaged over time (steady state fluorescence anisotropy) bring new information on orientational behaviors which are complemetary to other fluorescence-related imaging techniques such as lifetime imaging (FLIM) (Oida1993), Fluorescence resonant energy transfer (FRET) imaging (Chan2011) of colocalization imaging (Manders1993). This polarization related information is also applicable to single molecule studies and super-resolution techniques (Gould2008).

This chapter describes the basic principles of fluorescence anisotropy imaging

and polarization resolved fluorescence imaging, in the context of steady state measurements, in either one-photon or two-photon excitation regimes. It illustrates the use of these techniques with recent experiments performed on lipids and pro-teins orientational behavior in cell membranes, focusing in particular on how this behavior is related to the cell lipid environment and to its mechanical properties, governed by the cytoskeleton. We introduce a more general polarization-resolved fluorescence imaging technique which can circumvent limitations of fluorescence anisotropy imaging, and allow a complete mapping of molecular orientation in cell membranes. This concept can be extended to a vast variety of mechanisms in cells and tissues.

2 Defining molecular orientational order in cell membranes

In ordered media, molecules undergo a certain degree of orientational mobility constraint, which depends on their interaction with their nearby environment. This information is formally contained in a normalized molecular angular distribution function f() which defines the range of orientations () explored by the molecules after time and space averaging over the integration time of the optical measurement and the focal volume of the objective (Fig. 1a). Polarization resolved fluorescence experiments aim at measuring accessible information on this distribution function, defined as the so-called “molecular order”. Molecular order, which can be used to report information on a local environment or on molecular interaction mechanisms, applies to a fluorescent “orientational probe” such as a lipid probe (for cell membrane architecture studies) or a protein label (for proteins

4

interactions studies). As in all fluorescence imaging experiments, the information retrieved is the direct signature of the behavior of this fluorescent probe, which can further be interpreted as an environment information. It is therefore important that this probe stays rigidly attached or linked to the bio-molecule that it brings information on. At last, molecular orientational information as measured by polarized fluorescence is performed in microscopy where spatial resolution is limited: therefore the measured molecular order is always an averaged information in time (over the integration time of the detector) and in space (over the whole molecular population within the excitation volume, defined by the focal spot of an objective).

In molecular media where one-dimensional molecular probes are constraint by

a known potential U() at thermal equilibrium T, the orientational distribution function of the probe f() follows the Boltzmann statistics:

,, exp

B

Uf

k T

(1)

where kB is the Boltzmann constant. In lipid membranes, the orientation of lipids or proteins is most often defined as

lying within a cone aperture (Fig. 1a) with an abrupt change of the probe potential at a defined aperture angle

(Fig. 1a) (Axelrod1979,Florine-Casteel1990). In such a model, the fluorophores lie statistically inside the cone aperture , meaning that they only explore a range of orientations within this cone. f() is therefore defined as:

2

1/ 2

4 sin ( / 4),

0

iff

otherwise

(2)

Other smoother functions have been defined to quantify molecular order in membranes, such as based on a Gaussian distribution (Benninger2005,Haluska2008) or more generally decomposed over spherical harmonics basis functions (Brasselet2011).

In what follows, we will focus on the cone model, which is straightforward to visualize. In a cell or lipid membrane, a lipid probe can lie either along the membrane perpendicular direction (when it is embedded in the memebrane leaflets) or along its contour, depending on the molecule structure and its local interaction with lipids (Fig. 1b). In both cases the cone distribution can be defined by two parameters: its aperture (, which is related to the "molecular order") and its average orientation in the sample plane.

5

Figure 1. Models for molecular order in membranes. (a) Cone aperture and molecular ori-entation. (b) Different distribution functions based on cones filled with lipid probes mole-cules perpendicular (left) or parallel (right) to the membrane. (c) Examples of protein GFP-based constructs with GFP linked in a rigid way to the protein body. Left : Doubly lipidated enhanced GFP construction of a G-protein complex (Lazar2011). Right: MHC Class I protein labeled with GFP rigidly attached (Marguet1999, Rocheleau2003, Kress2011). (d) Fluorescein arsenical hairpin binder (FlAsH) (Griffin1998,Spille2011). (e) Perturbations of the molecular orientational order in membranes (represented by a varia-tion of the distribution cone aperture): local environment properties (liquid order left, liq-uid disorder center) or local morphology (right).

In proteins, a fluorescent label is generally attached by using N- or C-terminal tagging by either a green fluorescent protein (GFP) mutant expression, or by a synthetized fluorophore. In the most general case this label is likely to rotate freely in the whole space, leaving no information on its orientation. To be able to use a protein label as a reporter of its orientation, this label needs to be at best rigidly linked to the protein body. Since a few years, a strong effort has been put on the engineering of such systems (Fig. 1c). A major histocompatibility complex class I (MHC I) construct has been obtained with a GFP probe located within the aminoacid sequence of the H2Ld protein, ensuring a limitation of its rotational freedom (Marguet1999,Rocheleau2003,Kress2011). A G-protein complex has also been labelled in its subunits using a doubly lipidated GFP construction (Lazar2011). Recently, a strategy has also been developped based on fluorophores which can attach to proteins in several sites positions: Fluorescein arsenical hairpin binder (FlAsH) contains for instance a biarsenical group group that can bind to a tetracysteine motif Cys-Cys-Pro-Gly-Cys-Cys in proteins (Griffin1998). This label has been used to study orientational dynamics of a G-protein-coupled receptor in cell membranes (Spille2011).

6

The information brought by the measurement of the molecular order can be

related to local environment properties or local molecular interactions in several ways (Fig. 1e). Among the most important mechanisms which govern molecular order in cell membranes, are:

- Local lipid phases of different viscosity/polarity/lipid content. In particular an ongoing research is dedicated to the imaging of lipid domains of different phases such as liquid order, liquid disorder or gel phases (Si-mons1997,Ikonen2001,Veatch2002,McConnell2003,Munro2003). These domains called rafts, are of sub-resolution size, highly dynamic, and are described as support platforms for proteins in different biological processes.

- Formation of proteins’ clusters in cell membranes and in the cytoplasm, which are often related to mechanisms in cell signaling, cell motility or cell trafficking (Pentcheva2001, Fooksman2006, Benninger2009).

- Local morphological changes in cell membranes due to either external interaction (with the extra-cellular matrix) or internal forces (due to actin, microtubule fibers and the cytoskeleton in general), which are related to biological processes such as cell migration or adhesion (Anantharam2010).

3 Fluorescence and its relation to light polarization

Fluorescence is the result of two successive processes, the absorption of an incident photon and the emission of a fluorescence photon of lower frequency. These processes are separated in time due to the delay necessary for the molecule to relax from its high energy excited state to a lower (fluorescent) excited state (Lakowicz1999). The steady state fluorescence efficiency is consequently proportional to the product of two probabilities: the absorption probability between the ground and the excited state, and the emission probability from the fluorescent state to the ground state. In the linear excitation regime (one-photon excitation: 1PE), the fluorescence intensity is proportional to:

1 1 .PEF PEabs emI P P (3)

where the proportionality coefficient contains collection efficiencies and normalization factors. The absorption probability 1PE

absP is proportional to

2

1PEabsabsP E

(4)

7

where E

is the polarization of the incident electromagnetic field, and

abs

the

absorption transition dipole of the molecule. The emission probability emP depends on the analysis directions of the fluores-

cence emission (denoted X and Y, which define the sample plane in microscopy):

2 2

, ,,em emem X em YP X P Y

(5)

where em

is the emission transition dipole of the molecule from its fluorescent

state down to the ground state. The proportionality coefficient contains here the fluorescence quantum yield of the molecule, and the characteristics of the radiated intensity.

Eq. (5) is written assuming a planar wave approximation for the emission, which is not applicable for a high numerical aperture objective collection. In the case of a high aperture collection, a mixture of radiations along the X,Y and Z di-rections occurs and Eq. (3) has to be replaced by (Axelrod1979):

2 2 2

, 1 2 3

2 2 2

, 2 1 3

em em emem X

em em emem Y

P X Y Z

P X Y Z

(6)

where 1,2,3 are polarization mixture coefficients which depend on the numeri-cal aperture of the objective with 1>2,3) (Axelrod1979).

In general

em

and abs

have different orientations, due to the fact that differ-

ent states are involved in the absorption/emission processes, involving therefore different molecular conformations. For the sake of simplicity, we will assume that these dipoles are pointing along a same direction of orientation (), which also defines the orientation of the molecular structure: ,em abs

. The fact

that both dipoles can have different angles can be addressed by using a more com-plete expression accounting for an additional angle.

Finally the one-photon fluorescence intensity, along the analyzis direction X

for a molecule oriented with an angle (), is written in the sample frame as:

21 , , ,PEFX XI E J

(7)

with 2 2 2

1 2 3, , , ,XJ X Y Z

.

8

Eq. (7) can be written similarly for 1 ,PEFYI . Since polarization analysis does

not depend on proportionality coefficients, they do not need to be specified here. This formalism can be extended to multiphoton fluorescence and in particular

two-photon excited fluorescence (2PEF) accounting for the two-photon excitation absorption probability 2PE

absP , which involves a higher order nonlinear excitation

(Brasselet2011). Since the emission process does not change:

42 , , ,PEFX XI E J

(8)

and similarly for 2 ,PEFYI .

Eq. (8) emphasizes the fact that the two-photon fluorescence intensity depends on the square of the incident intensity, whereas this dependence is linear in the case of one-photon fluorescence.

In both one- and two-photon excitation cases, one can see than when a linearly

polarized light excites a single molecule, the highest probability of absorption oc-curs when its transition dipole moment

abs

is oriented parallel to the incident po-

larization E

.This property, called angular photo-selection, is at the origin of the polarization-dependence of the fluorescence process. Denoting the angle be-tween the absorption/emission molecular transition dipole and the exciting polari-zation, the one-photon angular photo-selection is proportional to

1 2cosPEabsP whereas in the TPEF process this photo-selection is propor-

tional to 2 4cosPEabsP . This makes the two-photon photo-selection narrower

than for a one-photon excitation, which allows measuring molecular orientations with a finer precision.

Finally, in a collection of molecules one has to account for the fluorescence

emitted by an ensemble of these molecules contained in the focal volume of the objective, with an orientational constraint defined by the function ,f (Bras-

selet2011):

22

1

0 0

sin, , ,PEFX XI E J f d d

(9)

and similarly for 1PEFYI .

Eq. (9) permits to calculate the dependence of the collected fluorescence as a function of the incident polarization E

. This expression can therefore be used to

9

define fluorescence anisotropy (Section 4 of this chapter) or more refined polari-zation resolved signals (Section 5 of this chapter). It shows also that from a polar-ized fluorescence measurement, if E

is known one can derive information on the

orientational distribution ,f . In the case of a cone model, ,f is written

as in Eq. (2) and therefore depends only on the molecular order cone aperture and its orientation in the macroscopic sample frame. We will show in Section 5 that this information can be determined experimentally without ambiguity.

4 Fluorescence anisotropy

Fluorescence anisotropy has been widely developped since decades to study molecules rotational diffusion properties, media viscosity, proteins conformations and interactions. Here we develop the principles of fluorescence anisotropy imaging.

4.1 Fluorescence anisotropy in isotropic media

The idea to investigate a molecular structure by a polarization resolved fluorescence method was introduced by Perrin in 1926 (Perrin1926) and applied by Weber in 1953 to the structural study of the binding of small molecules to proteins (Weber1953). This method is based on the fact that even though in a solution the macroscopic medium is isotropic, the fluorescence emission is polarization-dependent since the microscopic structures investigated are anisotropic. In a traditional fluorescence anisotropy set-up (Fig. 2a), a solution is excited using a beam propagating along along the Z axis and polarized along X. The fluorescence is measured along the perpendicular propagation direction Y, and analyzed along the X and Z directions using a polarizer, the measured quantity being called //I and I respectively. A ratiometric analysis of these two

situations provides he so-called fluorescence anisotropy, independent on the intensity fluctuations and solely dependent on orientational properties:

//

// 2X Y

X Y Z

I II IA

I I I I I

(10)

The normalization denominator represents the total fluorescence intensity, summed over all possible analysis directions. The factor 2 originates from the fact that in a isotropic solution, Y ZI I I (Cantor1980).

10

Originally, the fluorescence anisotropy was used to measure rotational diffu-sion times of proteins in solutions. In an isotropic sample where molecules are fixed, then 2( , ) 1/ 2f in Eq. (9). Setting //E X

as the incident polarization

leads to A = 0.4 in a one-photon regime, and A = 0.57 in a two-photon excitation regime. These numbers are the maximum anisotropy values possibly reachable in an isotropic medium. In practice, the measured value can be lower than this limit for three main reasons: (i) the angle between

abs

and em

is not 0°, (ii) the mole-

cules undergo some rotational mobility, therefore having different orientations be-tween their absorption and emission events, (iii) the molecules transfer energy be-tween each other, therefore the absorption step takes place for one molecular orientation and the emission step for another, uncorrelated orientation. These three processes lead to depolarization, meaning that the absorption and emission events do not take place with identical orientations. Case (ii) has lead to numerous studies on proteins conformational changes and local viscosity monitoring (Fig. 2b).

Figure 2. Fluorescence anisotropy. (a) Traditional fluorescence anisotropy measurement in solution. (b) Typical anisotropy versus viscosity (, normalized by the fluorescence lifetime f) curve obtained from fluorophores Indo-1 and BCEF in solution of glycerol of increasing concentration. The values measured have been compared to anisotropy in MDCK cells (above), to retrieve cytoplasmic viscosity information (Dix1990). (c) Fluorescence anisotro-py imaging in microscopy.(d) Illustration of anisotropy changes in cells where protein (GPI-GFP) clusters form close to the membrane: the decrease of anisotropy is correlated with a depolarization due to energy transfer between proteins. Intensity (left), steady state anisotropy (middle), and cluster-size deduced from anisotropy analysis (right). The latter two images are binned to 80x80 pixels (Bader2009).

11

Fluorescence anisotropy has been more recently applied to microscopy imaging (Fig. 2c), where fluorescence excitation is performed in a wide field geometry us-ing an incident polarized excitation along a direction X in the sample plane (X,Y). IX and IY images are formed on a CCD camera along the X and Y analysis direc-tions, using a polarization splitter. This technique has been used to retrieve orien-tational diffusion rates in intracellular media (Dix1990, Foster2005).

Among the depolarization factors widely studied in fluorescence anisotropy

imaging is fluorescence resonance energy transfer between similar molecules (homo-FRET) (case (iii) mentioned above). Homo-FRET is the property of an ex-cited molecule to transfer its energy in a non-radiative way, through a dipole-dipole interaction, to a neighbor molecule which then emits fluorescence light (Clegg1996). Since the orientations of the two molecules are uncorrelated, the ab-sorption and emission steps are decoupled in the fluorescence process, which cre-ates depolarization and therefore a loss of orientational information (Chan2011). This process, which occurs at molecular inter-distances of a few nanometers (Pat-terson2000), has been used in cells to identify possible proteins clusters (Black-man1998,Varma1998,Gautier2001,Pentcheva2001,Bader2006) (Fig. 2d). Homo-FRET information can be derived in a quantitative way to measure clusters sizes, and is thus used to monitor oligomerization of protein constructs. This has been applied to glycosylphosphatidylinisotol (GPI)-anchored proteins labeled with GFP, revealing that GPI forms clusters close to cell membranes with an average size of more than two subunits (Fig. 2d) (Bader2006).

4.2 Fluorescence anisotropy imaging in cell membranes and its limitations

In microscopy imaging in oriented samples such as cell membranes, the

situation is different since the distribution ( , )f is no more isotropic but con-

fined in a cone of a priori unknown aperture and orientation. In this situation, the geometry of a photo-selection by a linear fixed polarization is no more adapted since the averaged orientation of the molecules in the sample is unknown. In order to avoid such photo-selection artifacts, the incident excitation is therefore often changed into a circular polarization (Fig. 3a). A new anisotropy factor is defined, which accounts for the symmetry of the set-up:

X Y

X Y

I IA

I I

(11)

12

with A measured for an incident circularly polarized light. The (X,Y) directions also define the sample plane (Fig. 3a). This new anisotropy factor is therefore essentially sensitive to the emission probability polarization dependence. One can deduce from Eq. (11) that A varies between -1 (for transition dipoles strictly oriented along the Y axis), and 1 (for transition dipoles strictly oriented along the X axis). In all intermediate situations, such as different orientation and different distribution functions, A will take intermediate values.

Figure 3b depicts the evolution of A for fluorescent molecules lying within a cone aperture , for different orientations of the cone in the (X,Y) plane (the cone is supposed to lie in the sample plane, therefore the membrane is imaged in its equatorial plane). This graph is generated using Eq. (9) in a two-photon excitatoin regime, assuming parallel absorption and emission dipole moments in the molecules. Similar curves are obtained for a one-photon excitation. One can immediately notice that in order to retrieve a molecular order information (a value) in a sample, the knowledge of the cone orientation in the sample plane () is required with high precision. Indeed determining a single experimental value A does not allow retrieving more than one parameter on the sample. In addition, this measurement is only applicable when the cone lies close to the X or Y axes, the case /4 leading to an indetermination (A=0) of the molecular order since both X and Y projections are equivalent. Retreiving from an anisotropy measurement is nevertheless feasible in a membrane of round shape, such as in a giant unilamellar vesicle (GUV) (Fig. 3c). All points of its equator can indeed be assigned with a position angle , which gives access to molecular order values even if the membrane is heterogeneous.

13

Figure 3. Fluorescence anisotropy confocal imaging in membranes. (a) Experimental scheme: the IX and IY analyzed intensities are recorded for an incident circular polarization (some schemes use a linear polarization). The sample is schematized as a spherical lipid membrane in which molecules are assembled within a cone aperture, normal to the mem-brane. All images (cell, GUVs) are performed in the equatorial plane of the membrane. (b) Theoretical dependence of the polarization factor A () as a function of the cone aperture of a filled cone distribution, for several tilt angles of the cone in the sample plane. (c) Anisotropy A measured for a Giant Unilamellar Vesicle made of DOPC (left) and a mix-ture of DOPC, Sphingomyelin and Cholesterol (1:1:1) which leads to heterogeneous lipid domains formation (right). The points of measurement M and N at the position = 90° are represented on the theoretical A () graph, leading to an estimation of the molecular order value at this position of the sample (Gasecka2009).

This method shows however its strongest limitations when applied to

membranes of arbitrary shapes where the local values are difficult to estimate visually from the fluorescence image. Applying fluorescence anisotropy imaging to the investigation of molecular order in cell membranes is finally only successful in cases restricted to simple distribution functions of cylindrical symmetry (such as a cone aperture), and necessitates an a priori knowledge of either the mean orientation of the molecular distribution or its shape (Dale1999,Gasecka2009,Kinosita1977,Florine-Casteel1990). Many fluorescence anisotropy imaging studies have been therefore performed in membranes of sphrerical shapes such as in swelling cells (Fig. 4a) or in particular regions of the membrane where the direction is identitied as being along X or Y (Fig. 4b,c, Fig. 5a). A large amount of investigations have been carried out in this field since the seminal work of Axelrod in 1979 (Axelrod1979), in which the orientation of long chain carbocyanine dyes was studied in spherical lipid membranes of red blood cells. All further studies in lipid membranes have been limited so far to simple spherical membranes geometries such as in artificial Giant Unilamellar Vesicles (GUVs) (Florine-Casteel1990), red blood cells (Blackman1996), swelling cells (Rocheleau2003), spherical cells (Benninger2005, Haluska2008) and spherical nuclear envelopes (Mattheyses2010). In these membrane studies, the measurement was performed on the perimeter of the spherical membrane and the in-plane average orientation of the molecular distribution function of the fluorescent probes in the lipid membrane was assumed to follow the direction normal to the membrane. Apart from membrane studies, fluorescence anisotropy imaging has been applied to the determination of the width of molecular angular distributions in biopolymers of cylindrical symmetry such as actin filaments (Borejdo1993), muscle fibers (Dale1999) and septin filaments (Vrabioiu2006). In these studies, the orientation of the fibers in the sample plane was visualized in the image and used as a known parameter to reduce the complexity of the studies.

Even though the limitations of fluorescence anisotropy imaging are quite

constraining, this technique is still able to probe interesting mechanisms in cell membranes such as drug treatments effects or mechanical perturbations. Changes

14

relative to lipid composition (varying the cholesterol content) have been observed on lipid probes which are seen to loose their orientational order when cholesterol is depleted from the membrane, indicating a relation between local rigidity and cholesterol content (Benninger2005). Cell membrane local morphology at a sub-resolution scale can also be probed using such technique. A recent observation has evidenced cell ruffling at the junction of two cells, demonstrated by a loss of anisotropy contrast of lipid probes in the junction (Benninger2009). We have recently demonstrated the modifications of the lipid probe’s molecular order when perturbing the cell membrane tension by a mechanical perturbation (by a hypotonic shock provoking cell swelling) or by drug treatments affecting the cytoskeleton (using latrunculin A, an actin polymerization inhibitor or cytochalasin D, an actin depolymerization inducer) (Kress2011). A higher orientational order is observed when the cell membrane is locally more tense due to the relaxed constraint from the cytoskeleton. This emphasizes the correlation between molecular order and local membrane morphology (Kress2011).

Of particular interest are recent studies on membrane proteins (Fig. 4). A large

amount of research is indeed dedicated to label proteins in a rigid way such as the attached fluorescent probe can report the orientation of the protein itself (Fig. 1c).

15

Figure 4. Fluorescence anisotropy imaging in cell membranes (see Fig. 1c for the scheme of the molecules involved). (a) MHC I rigidly labeled with intra-sequence GFP in cell blebs. The anisotropy contrast shown as a function of the contour angle around the round cell membrane is a signature of rigid labelling (Rocheleau2003). (b) 2-photon fluorescence ani-sotropy imaging of G-protein activation. The anisotropy is followed in time for a G subu-nit labeling using doubled lipidic attachment with yellow fluorescent protein (YFP), after addition of an agonist (norepinephrine) (Lazar2011) (Scale bar: 5 m). (c) Anisotropy im-ages of left image: the intracellular loops (IL3) of a G-protein coupled receptor (GPCR), labeled with FlasH connecting a bridge between its N- and C-termini, right image: the par-athyroid hormone receptor (PTHR) used as a nonrigid membrane FlAsH binding motif where the labeling takes place at the C-terminus (Scale bar: 10 m). Right: The anisotropy distribution taken in a particular region of the cell membrane shows a visible contrast (Spille2011).

A major histocompatibility complex class I (MHC I) construct rigidly labeled with GFP has been shown to exhibit strong anisotropy contrast in a cell bleb membrane, indicating a degree of orientation constraint as compared to other non rigid constructs where the GFP label is attached to the cytoplasmic tail of the protein (Rocheleau2003) (Fig. 4a). Recent constructs applied to protein receptors have also allowed measuring, using two-photon fluorescence anisotropy, changes of proteins conformation upon G-protein activation. In particular interactions involving the different subunits of heterotrimeric G-proteins could be visualized in the presence of a suitable G-protein-coupled receptor (GCPR), responding to the presence of a receptor agonist by a conformation change, probed by a modification of anistropy (Fig. 4b) (Lazar2011). The decrease of anisotropy in the superior region of the cell membrane is indeed a signature of a change of relative orientation angle compared to the membrane, indicating a folding of the labeled subunit towards the membrane direction. Labelling engineering using fluorophores attached at two sites of a protein has also permitted to probe orientational order in membrane receptors (Fig. 4c) (Spille2011).

The relation between orientational properties of membrane proteins and the

cell membrane mechanical properties has been further explored by applying treatments affecting the cytoskeleton (Fig. 5) (Kress2011). Contrary to what was observed for lipid probes, we recently observed that membrane proteins MHC I rigidly labeled with GFP in COS-7 cells undergo a loss of molecular order when the cytoskeleton is disrupted by a hypotonic shock (Fig. 5b) or drug treatments (Latrunculin A, cytochalasin D). Similar effects could be observed when perturbing the microtubule network by a colcemid treament. This shows that the orientational constraint of membrane proteins is highly governed by their mechanical or chemical link to nearby cytoskeleton molecules, as previously emphasized in other studies based for instance on lateral diffusion observations (Edidin1991;Edidin1994, Bunnell2001, Barda-Saad2005,Treanor2010). A new construct of similar MHC I rigidly labeled but with a truncated cytoplasmic chain has shown similar loss of order, confirming the role of the attachement of the protein to the cytoskeleton in its orientational behavior (Fig. 5c). The MHC I

16

orientational behavior was also probed in the endomembrane, showing higher disorder due to the more complex endomembrane composition and morphology as compared to the plasma membrane, and to the less mature stage of the protein in this location before its migration to the outer region of the cell (Fig. 5). Interestingly, the behavior of MHC I in the endomembrane can be also be affected by cytoskeleton drug treatments, revealing mechanical relations between the plasma membrane and the interior of the cell (Schauer2010).

Note that in these studies, it is important to ensure that homo-FRET does not take place, since it would lead to depolarization and therefore to an underestima-tion of the anisotropy values. This characterization can be done using photo-bleaching studies, measuring possible changes of the anisotropy while molecules are progressively removed in the samples by photo-bleaching (therefore lowering the probability of homo-FRET) (Kress2011).

Figure 5. Fluorescence anisotropy imaging in MHC I proteins rigidly labeled with GFP. (a) Left: Fluorescence image. Middle: Map of anisotropy and selected region of interest for structural studies. Right: A() graph used to retrieve a quantitative number of the molecu-lar order . Several points of measurements are shown. (b,c) Changes in molecular order in the plasma membrane and the endomembrane for (b) MHC I protein rigidly labeled with GFP perturbed by a hypotonic shock (Kress2011), (c) truncated construct where the cytoplasmic tail has been removed.

At last, note that one photon versus two-photon fluorescence leads to a slightly

different behavior in the anisotropy versus molecular order dependence. As men-

17

tioned above, the photo-selection is indeed finer when using a two-photon excita-tion process, which leads to a more refined dependence. Two-photon fluorescence imaging is however subject to other drawbacks such as a higher probability to de-teriorate the cell upon intense pulsed illumination, which is necessary to overcome the lower absorption probability cross sections of fluorophores. Among the exam-ples found in the literature, both techniques have been used to profit from their re-spective advantages.

5 Polarization resolved fluorescence

In general, membranes are highly heterogeneous and exhibit shapes and features that cannot be fully explored using fluorescence anisotropy, due to the limitations mentioned above. In order to be able to map molecular order in the whole membrane contour, more refined techniques need to be implemented.

5.1 Principle

Polarization resolved fluorescence has been developed in order to circumvent limitations of fluorescence anisotropy imaging. In this technique, the incident polarization is no more purely linear along one direction or circular, but is rotated between 0° and 180° (relative to the X sample horizontal direction), with angle steps small enough to retrieve a complete information on molecular order (typically 20° or less).

Polarization resolved fluorescence techniques (Fig. 6a) have been developed in both one-photon (Kress2011, Kress2012) and two-photon excitation regimes (LeFloch2003,Brasselet2004, Gasecka2009). An incident laser excitation (either continuous for one-photon or pulsed such as from a tunable Ti:Sapphire laser, which delivers 150 fs pulses at a repetition rate 80 MHz, for two-photon) is used in a confocal mode. The laser beam is reflected by an appropriate dichroic mirror and focused on the sample by a microscope objective which Numerical Aperture (NA) can vary between 0.6 to 1.2. The use of high apertures presents the advantage of a higher optical resolution (typically 300nm) and a more efficient signal collection in the backward epi geometry, however it exhibits more polarization distortion and mixing for the collected signal, which should be accounted for in the signal analysis. In a epi geometry set-up, the backward emitted signal is collected by the same objective and directed to a polarization beamsplitter that separates the beam towards two detectors (avalanche photodiodes or photomultipliers). Images are performed by scanning either the sample on a piezoelectric stage, or the beam position by galvanometric mirror piezoelectric scanning. In the first case, the sample is imaged and then a precise

18

location is chosen for the measurement of the polarimetric response. The second case is more advantageous in terms of decrease of the acquisition time (typically a rate of 1 image per second is reached), and consists in recording one image per incident polarization state. In order to vary the incident linear polarization, a half waveplate, mounted on a step rotation motor, is placed before the dichroic mirror. An increase of the polarization switching speed can be obtained by the use of fast linear polarization controller, such as a Pockels cell placed before a quarter waveplate. For each value of the polarization angle (relative to X) from 0° to 180° (the second half of the measurement being redundant), the emitted signal is recorded on the two perpendicular directions X and Y (Fig. 6a). This set-up can be extended to forward detection schemes, the forward emitted signals being generally detected through a low aperture objective (NA ~ 0.5).

Figure 6. Polarization resolved Fluorescence. (a) Set-up. (b) Example of different fluores-cence polarimetric responses depending on the sample explored, from a solution to oriented molecules in a cone aperture. From top to bottom: molecular dipoles represented as ar-rows, distribution seen in the plane of the sample, polarimetric responses in a one-photon regime IX() and IY () represented as polar plots, total polarimetric sum response Ix() + Iy(). (c) Illustration in a COS-7 cell membrane labeled with di-8-ANEPPQ. Left: fluores-cence image, middle: anisotropy image, right : polarimetric responses measured in the two points A and B visible on the anisotropy image. The distribution parameters and are deduced from a fit of the polarimetric responses.

19

The resulting data are no more one anisotropy value as described above, but rather a so-called polarimetric response in both X and Y analysis directions, which will be denoted IX() and IY(). Examples of polarimetric responses are given in Fig. 6b as polar plots with respect to . In isotropic media, the polarimetric responses can take different shapes depending on the degree of depolarization in the sample: for fast rotating molecules (or complete depolarization cases), both IX() and IY() resemble a circle. When molecules are fixed in an isotropic distribution, IX() and IY() are no more identical, even though the total polarization response IX()+IY() is a circle since the distribution is still isotropic. This is an indication that an analysis of the signal in both X and Y directions can bring considerable information on the depolarization mechanisms taking place in the sample. In oriented samples, the polarimetric response takes various two-lobes shapes which aperture and orientation are respectively related to the molecular order , and its average orientation in the sample plane. From a fit of such polarimetric responses, it is therefore possible to retrieve a direct and unambiguous determination of both parameters (, . An illustration of this is shown in Fig. 6c: independently on the point taken around a labelled cell membrane, the simultaneous fit of polarimetric responses IX() and IY() leads to the determination of both parameters of the distribution function modelled as a cone. This situation therefore completes advantageously the anisotropy image which is not exploitable in all points of the image. Here, the information can be retreived over the whole contour of the cell equatorial plane.

The fit of polarimetric curves relies on the model developed in Eq. (9), using

for E

a function of (ideally, for a perfectly controlled linear rotating polariza-

tion: cos ; sinX YE E ), supposing abs

and em

parallel, and tak-

ing for ( , )f the cone distribution described above with two unknown parame-

ters (, However one has to take care of different deviations from this perfect model, in order to account for instrumental and sample artifacts:

- In a polarization resolved fluorescence set-up, controlling and correcting

for polarization distortions is determining. Indeed the polarization state of the exciting field has to be perfectly known. Calibration experiments are therefore necessary in order to introduce possible ellipticity and dichroism factors, introduced principally by the dichroic mirror which reflects the in-cident laser excitation (Schön2008).

- The high numerical aperture focusing induces a non homogeneous polari-

zation state within the focal excitation volume. In particular, small contri-butions polarized in the axial Z direction appear on the edge of the focal spot (Richard1959). Numerical analyses have shown that these distortions are not determining when molecules lie mostly in the sample plane (Schön2010).

20

- The assumption of parallel absorption and emission dipole moment does

not apply to all fluorescent molecules. The angle between the two dipoles should be known beforehand and introduced in the model. It has been shown however that below 20°, this angle has no detrimental effect on the deformation of polarimetric responses (Gasecka2009). In many probes of molecular orientation, this angle is found to lie in this range (Muller1996, Moyano2006).

- The assumption that the cone aperture lies in the sample plane relies on the

fact that measurements are performed in the equator of the membrane. If the measurement is performed out of this plane, a known tilt angle has to be introduced in the model (angle represented in Fig. 3a). Nevertheless studies have shown that the in-plane approximation leads to only a few de-grees of error on for angles below 45° (Gasecka2009, Kress2012).

- At last, this technique is sensitive to depolarization mechanisms, in par-

ticular homo-FRET between nearby molecules. When such mechanisms occur, the polarimetric response from a cone aperture loses its characteris-tics and tends to equalize the analyzed IX() and IY() responses (Gas-ecka2009). Similarly to anisotropy measurements, the absence of homo-FRET needs to be ascertained to avoid any bias on the retrieved molecular order angles. In Section 5.3, we describe a way to circumvent such bias.

5.2 Application to molecular order imaging in heterogeneous membranes

The main advantage of polarization resolved fluorescence is to provide a spatial information of molecular order independently on the membrane shape or hetero-geneity. This technique has been applied to the investigation of the orientational organization in coexisting liquid phase with short-range liquid order (Lo) and liq-uid disordered (Ld) fluid domains of micrometric sizes in artificial membranes (Giant Unilamellar Vesicles (GUV)), and in cell membranes of non-spherical shapes.

Giant Unilamellar Vesicles (GUV) made of lipid mixtures are considered as

model systems for the investigation of lipid interactions (Die-trich2001,Samsonov2001,Scherfeld2003,Baumgart2003). They can exhibit coex-isting domains with different fluidity, elasticity and polarity properties. This phase segregation into gel, liquid ordered and disordered environments, is closely related to fundamental cell processes, in particular in cell signaling where the existence of lipid-specific functional "rafts" platforms is still a debate (Si-

21

mons1997,Ikonen2001,Veatch2002,McConnell2003,Munro2003). So far, imaging lipid domains has relied on the use of dedicated fluorescent probes specifically partitioning in regions of known lipid composition or local polarity (Baga-tolli1999,Kim2007,Baumgart2007). Such probes are also expected to undergo specific orientational order detectable by fluorescence anisotropy (Baga-tolli1999,Gidwani2001,Kim2007). Recent studies have been dedicated to molecu-lar order monitoring in such phases (Gasecka2009,Farkas2010).

Figure 7 shows typical two-photon fluorescence (2PEF) polarimetric responses of fluorescent molecules di-8-ANEPPQ in GUVs formed from a ternary mixture of the lipids sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol (Dietrich2001). In such mixtures, Lo domains are known to be es-sentially constituted by enriched sphingomyelin and cholesterol regions, whereas DOPC is mainly present in disordered liquid regions (Ld). Although the Lo or Ld environments cannot be identified in a pure fluorescence image, the 2PEF polari-metric analysis permits to directly create an image of the spatial distribution of molecular order (Fig. 7a). Typical cone aperture values range between 30° < < 80° (Lo phases) and 90° < < 170° (Ld phases) depending on the probe molecule (Fig. 7b). In the Ld phase, where the lipid acyl chains are highly disordered, the aperture angle is therefore significantly increased. 2PEF polarimetry shows overall that molecular order information is dependent on the fluorescent probe structure, primarily because it is driven by lipid-fluorophore interactions which are influ-enced by the molecular head position. In particular takes larger values when the molecule inclusion localization takes place in the periphery part of the membrane (Chong1993,Kim2007,Haluska2008).

Similar studies have been performed on cells. One- and two-photon polarimetry

measurements performed on di-8-ANEPPQ probes in COS-7 cells (Fig. 7c) show that the molecules mean orientation roughly lies along the membrane normal di-rection. In many points the membrane is of complex shape and its global orienta-tion is difficult to visualize. The simultaneous fitting on both and parameters makes it possible to avoid speculating on the local membrane contour as usually done with fluorescence anisotropy. The measured cone aperture angle on COS-7 cell membranes shows that the di-8-ANEPPQ probes behave in a slightly more disordered way than in DOPC GUV membranes. This is most probably due to a more perturbed membrane spatial morphology, originating from folding at spatial scales much below the diffraction limit (from 20nm to 100nm) (Cheresh1999,VanRheenen2002,Adler2010,Anantharam2010).

These studies show that membrane morphology features below the 300 nm op-tical resolution limit can be probed at any position of a membrane using polariza-tion resolved fluorescence: any folding of the membrane at nanometer scales (due to ruffling, vesiculation…) (Sund1999, Benninger2009) will indeed lead to a loss of the measured molecular order.

22

Figure 7. (a,b) Polarization resolved two-photon fluorescence in artificial lipid membranes labeled with di-8-ANEPPQ: Giant unilamellar Vesicles (GUV) made of Ld and Lo phases from a DOPC:Sphingomyelin:Cholesterol (1:1:1) mixture. (a) Left: fluorescence image. Right: retrieved color-coded molecular order image showing the angle value obtained from a fit of polarimetric responses in several points of the GUV. (b) Typical polarimetric responses, represented in polar plots, for three points of measurements shown in (a). The fit (continuous line) of the 2PEF polarimetric data (markers) is performed using a filled cone model of orientation in the sample plane and aperture . Two populations could be found, characteristics of ordered and disordered phases (Gasecka2009). (c) Polarization re-solved 2PEF in a COS-7 cell labeled with di-8-ANEPPQ, which exhibit higher molecular disorder representative of local membrane morphology features (represented schematically from (VanRheenen2002)).

5.3 Circumventing depolarization effects

In order to avoid the sensitivity of polarization resolved fluorescence to depolarizatoin mechanisms as mentioned above, we developed recently a technique, derived from polarization resolved fluorescence but purely governed by photo-selection (Kress2012). In this method, the incident polarization is varied but the emission is not analyzed along a given direction, therefore the sum I()=IX()+IY() is detected. This way, the possible decorrelation between

23

absorption and emission events is not visible in the data, which are only sensitive to the photo-selection taking place in the sample. This technique may appear less precise for the estimation of molecular order parameters, since only one polarization response is recorded instead of two, howerer this information is mathematically sufficient to retrieve the paramerters required for the quantification of molecular order.

The data analysis is based the Fourier analysis of the polarimetric response I() (Fig 8a). Given the square modulus factor in Eq. (9), this response can be fully described using the finite Fourier series:

0 2 2cos 2 sin 2I A A B

(12)

Using the initial derivation of Eq. (9) for a cone aperture model, it can be shown that the normalized A2/A0 and B2/B0 coefficients are directly related to the molecular distribution parameters and , with no ambiguity of determination. This allows to build up a 2D look-up table, that gives for a wide set of (A2/A0,B2/A0) couples, the values of and . In particular the sinusoidal depend-ence of Eq. (12) has an amplitude related to the molecular order (), and a phase related to the orientation of the molecular distribution in the sample plane () (Fig. 8b). Since the coefficients A2/A0 and B2/B0, as Fourier coefficients, can be com-puted from the experimental data at every pixel of the image (Fig 8), and can be retrieved directly using the look-up table. Note that the obtained values are sys-tematically validated by performing a normalized 2 test, that quantifies the agreement between experimental data and the expected theoretical polarimetric re-sponse. In practice, a 2 value larger than 5 is a good indicator of measurements that were affected by photobeaching, sample drift, etc. Such measurements are systematically discarded (Kress2012). As compared to the previous data fitting process, this Fourier analysis approach has permitted to considerably speed up the data processing, allowing a complete polarimetric analysis over a whole cell con-tour in a few seconds.

24

Figure 8. Fourier decomposition of the polarization dependent fluorescence signal and ap-plication to molecular order imaging. (a) A COS-7 cell doped with di-8-ANEPPQ is imaged by one-photon confocal fluorescence. A stack of images is recorded with one image per in-cident polarization angle. Fourier coefficients are directly deduced from the polarimetric response I() of each pixel, leading to the determination of both parameters and . (b) Il-lustration of polarimetric response I() obtained from an ordered region in the cell mem-brane (left), and disordered region (right). The amplitude of the curves is related to the mo-lecular order parameter, while its phase is related to the orientation .

This technique has been applied to the analysis of molecular order in cell and

articial membranes labeled with di-8-ANEPPQ, using one-photon confocal polar-imetry analysis (Fig. 9) (Kress2012). Strong heterogeneities could be visualized in cells, contrary to GUV, with values confirming the ranges previously measured on a complete polarization dependence fit with two-photon fluorescence. Several conclusions were drawn from these studies:

- The GUV molecular order in a homogeneous lipid composition is highly homogeneous, with molecular order distributions widths close to the noise limit (Fig. 9a).

- In single cells, histograms of the values are much larger than GUVs, signature of a high degree of heterogeneity (Fig. 9b). The histogram over one population of 10 cells is typically of same shape as for one single cell.

- In cells, local parts of high disorder can be visualized, signature of the fact that the observed heterogeneity is spatially distributed along the cell con-tour (Fig. 9c).

25

Figure 9. Imaging molecular order by one-photon confocal polarization resolved fluores-cence around the cell contour. (a) DOPC GUV labeled with di-8-ANEPPQ. From left to right: fluorescence image superimposed with its local values; angle map; 2 mean square error map (values above 5 correspond to discarded pixels). Histogram of the val-ues obtained over the whole contour. A comparison between an untreated cell and cells treated by a hypotonic shock is shown. (c) Zoom on a cell membrane showing macroscopic and sub-micrometric size ruffling sites with high orientational disorder.

As supported by previous measurements in fluorescence anisotropy, the high

heterogeneity visualized in cells is most probably due to membrane local changes of morphology. In particular membrane treatments provoking a perturbation of the actin cytoskeleton network (hypotonic shock, Latrunculin A, cytochalasin D) in-duced a gain in molecular order around the whole cell contour (Fig. 9b). This con-firms that the molecular order indicator is a sensitive reporter of membrane lo-cal morphology, in particular due to its nanometric-size folds which are not visible in a standard fluorescence image. Polarization resolved fluorescence with un-polarized detection, with the additional capacity of being depolarization-insensitive, therefore allows a map of membrane features which are not accessible by fluorescence anisotropy. This technique might be able to reveal new infor-mation at particular points of a cell membrane or in proteins assemblies undergo-ing physical or chemical perturbations.

26

6 Summary, Conclusions, Outlook

The polarization dependence of fluorescence signals in microscopy carries rich information on the orientational behavior of molecular probes. During the last decades, considerable progress has been done in terms of instrumentation, polarized fluorescence image analysis and more importantly, in the development of fluorescent probe attachement strategies which now permit to reveal the orientation behavior of proteins and lipids in cell membranes with high precision.

Polarization signatures being a reporter of local environment rigidity and morphology, this technique is therefore likely to evolve towards the determination of important complementary information to already existing fluorescence-related techniques. Polarization resolution is indeed now introduced in a large scope of techniques such as super-resolution fluorescence imaging, single molecule spectroscopy, as well as in a wide variety of contrasts such as in nonlinear optics in tissues (second harmonic and third harmonic generation, coherent anti Stokes Raman scattering).

Acknowledgements

The authors thank H. Rigneault, P. Réfrégier, J. Duboisset (Institut Fresnel, Marseille, France), as well as D. Marguet, H.T. He, T. Trombik (Centre d’Immunologie de Marseille Luminy, Marseille, France), for helpful discussions and advices. The Institut Fresnel work mentioned in this chapter was supported by CNRS, Agence Nationale de la Recherche, the region Provence Alpes Côte d’Azur.

References

Adler J., Shevchuk A. I. et al (2010) Plasma membrane topography and inter-pretation of single-particle tracks. Nature Methods. 7:170–171

Anantharam A., Onoa B. et al (2010) Localized topological changes of the plasma mem-brane upon exocytosis visualized by polarized TIRFM. J. Cell Biol. 188:415–428

Axelrod D. (1979) Carbocyanine dye orientation in red cell membrane studied by mi-croscopic fluorescence polarization. Biophys. J. 26: 557–573

Bader A.N., Hofman E.G., Voortman J., van Bergen en Henegouwen P.M.P., Gerritsen H.C. (2009) Homo-FRET Imaging Enables Quantification of Protein Cluster Sizes with Subcellular Resolution, Biophys. J.97:2613-2622

Bagatolli, L. A., Gratton E. (1999) Two-photon fluorescence microscopy observation of shape changes at the phase transition in phospholipid giant unilamellar vesicles. Bio-phys. J. 77:2090–2101

27

Barda-Saad M., Braiman A. et al (2005) Dynamic molecular interactions linking the T cell antigen receptor to the actin cytoskeleton. Nat. Immunol. 6:80–89

Baumgart T., Hess S. T., Webb W. W. (2003) Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature. 425:821–824

Baumgart, T., Hunt G., Farkas E. R., Webb W. W., Feigenson G. W. (2007) Fluorescence probe partitioning between Lo/Ld phases in lipid membranes. Biochim. Biophys. Acta. 1768:2182–2194

Benninger R. K. P., Vanherberghen B., Onfelt B. (2009) Live cell linear dichroism im-aging reveals extensive membrane ruffling within the docking structure of natural killer cell immune synapses. Biophys. J. 96:L13–L15

Benninger, R. K. P., Onfelt, B. et al (2005) Fluorescence imaging of two-photon linear di-chroism: cholesterol depletion disrupts molecular orientation in cell membranes. Bio-phys. J. 88:609–622.

Blackman S. M., Cobb C. E. et al (1996) The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization micros-copy. Biophys. J. 71:194–208

Blackman, S. M., Piston D.W., Beth. A. H. (1998) Oligomeric state of human eryth-rocyte band 3 measured by fluorescence resonance energy homotransfer. Biophys. J. 75:1117–1130

Borejdo, J., Burlacu S. (1993) Measuring orientation of actin filaments within a cell: orien-tation of actin in intestinal microvilli. Biophys. J. 65:300–309

Brack A. S., Brandmeier B. D. et al (2004) Bifunctional rhodamine probes of Myosin regu-latory light chain orientation in relaxed skeletal muscle fibers. Biophys. J. 86:2329–2341

Brasselet S. (2011) Polarization resolved nonlinear microscopy: application to structural molecular and biological imaging. Advances in Optics and Photonics 3:205–271

Brasselet, S., LeFloc’h V., Treussart F., Roch J., Zyss J. (2004) In situ diagnostics of the crystalline nature of single organic nanocrystals by nonlinear microscopy. Phys. Rev. Lett. 92:207401

Bunnell S. C., Kapoor V. et al (2001) Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT. Immunity. 14:315–329

Cantor C. R., Schimmel P.R. (1980) Biophysical Chemistry, Part 2: Techniques for the Study of Biological Structure and Function. W. H. Freeman, San Francisco

Chan F.T.S., Kaminski C.F., Kaminski Schierle G.S. (2011) HomoFRET Fluorescence An-isotropy Imaging as a Tool to Study Molecular Self-Assembly in Live Cells. Chem Phys Chem. 12:500-509

Cheresh D., Leng J., Klemke R. (1999) Regulation of cell contraction and membrane ruf-fling by distinct signals in migratory cells. J. Cell Biol. 146:1107–1116

Chong, P. L. G., Wong P. T. T. (1993) Interactions of Laurdan with phosphatidylcholine liposomes: a high pressure FTIR study. Biochim. Biophys. Acta. 1149:260–266

Clegg, R. M. (1996) Fluorescence imaging spectroscopy and microscopy: fluorescence res-onance energy transfer. In Chemical Analysis 137, 13th Ed. Wiley, New York

Dale R. E., Hopkins S. C. et al (1999) Model-independent analysis of the orientation of flu-orescent probes with restricted mobility in muscle fibers. Biophys. J. 76:1606–1618

Dietrich C., Bagatolli L. A., Volovyk Z. N., Thompson N. L., Levi M. (2001) Lipid rafts reconstituted in model membranes. Biophys. J. 80:1417–1428

Dix J. A., Verkman A. S. (1990) Mapping of fluorescence anisotropy in living cells by ratio imaging. Biophys. J. 57:231–240

Edidin M., Kuo S. C., Sheetz M. P. (1991) Lateral movements of membrane glyco-proteins restricted by dynamic cytoplasmic barriers. Science 254:1379–1382

28

Edidin M., Zuniga M. C., Sheetz M. P. (1994) Truncation mutants define and locate cyto-plasmic barriers to lateral mobility of membrane glycoproteins. Proc. Natl. Acad. Sci. USA. 91:3378–3382

Farkas E.R., Webb W.W. (2010) Multiphoton Polarization Imaging of Steady-State Molec-ular Order in Ternary Lipid Vesicles for the Purpose of Lipid Phase Assignment. J. Phys. Chem. B 114:15512–15522

Florine-Casteel K. (1990) Phospholipid order in gel- and fluid-phase cell-size liposomes measured by digitized video fluorescence polarization microscopy. Biophys. J. 57:1199–1215

Fooksman D. R., Grönvall G. K. et al (2006) Clustering class I MHC modulates sensitivity of T cell recognition. J. Immunol. 176: 6673–6680

Foster T.H., Pearson B.D., Mitra S., Bigelow C.E. (2005) Fluorescence anisotropy imaging reveals localization of meso-tetrahydroxyphenyl chlorin in the nuclear envelope. Photo-chem. Photobiol. 81:1544-1547

Gasecka, A., Han T.-J. et al (2009) Quantitative imaging of molecular order in lipid mem-branes using two-photon fluorescence polarimetry. Biophys. J. 97:2854–2862

Gautier I., Tramier M. et al (2001) Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins. Biophys. J. 80:3000–3008

Gidwani, A., Holowka D., Baird B. (2001) Fluorescence anisotropy measurements of lipid order in plasma membranes and lipid rafts from RBL-2H3 mast cells. Biochemistry. 40:12422–12429

Gould T.J., Gunewardene M.S., Gudheti M.V., Verkhusha V.V., Yin S.R., Gosse J.A., Hess S.T. (2008) Nanoscale imaging of molecular positions and anisotropies, Nat Methods. 5:1027–1030

Griffin B. A., Adams S. R., Tsien R. Y. (1998) Specific covalent labeling of recombi-nant protein molecules inside live cells. Science 281:269–272.

Haluska, C. K., Schröder A. P. et al (2008) Combining fluorescence life-time and polariza-tion microscopy to discriminate phase separated domains in giant unilamellar vesicles. Biophys. J. 95: 5737–5747.

Ikonen E. (2001) Roles of lipid rafts in membrane transport. Curr. Opin. Cell Biol. 13:470–477

Kim H. M., Choo H.-J., Jung S.-Y., Ko Y.-G., Park W.-H. (2007) A two-photon fluorescent probe for lipid raft imaging: C-Laurdan. ChemBioChem. 8:553–559

Kinosita K., Kawato S., Ikegami A. (1977) A theory of fluorescence polarization decay in membranes. Biophys. J. 20:289–305

Kress A., Ferrand P., Rigneault H., Trombik T., He H.T., Marguet D., Brasselet S. (2011) Probing MHC Class I Protein and Lipid Order in Cell Membranes by Fluorescence Po-larisation-resolved Imaging. Biophys. J. 101:468–476

Kress A., Wang X., Ranchon H., Ferrand P., Brasselet S. (2012) Revealing molecular ori-entation and order in cell membranes of arbitrary shape, submitted

Lakowicz J. R. (1999) Principles of Fluorescence Spectroscopy, Springer Lazar J., Bondar A., Timr S., Firestein S.J. (2011) Two-photon polarization microscopy re-

veals protein structure and function. Nature Methods 8:684–690 LeFloc’h V. (2003) Monitoring of orientation in molecular ensembles by polarization sensi-

tive nonlinear microscopy. J. Phys. Chem. B. 107:12403–12410 Manders E. E. M., Verbeek, F. J., Aten, J. A. (1993). Measurement of co-localisation of ob-

jects in dual-colour confocal images. Journal of Microscopy 169:375-382 Marguet D., Spiliotis E. T. et al (1999) Lateral diffusion of GFP-tagged H2Ld molecules

and of GFP-TAP1 reports on the assembly and retention of these molecules in the endo-plasmic reticulum. Immunity. 11:231–240

29

Mattheyses A. L., Kampmann M. et al (2010) Fluorescence anisotropy re-veals order and disorder of protein domains in the nuclear pore complex. Biophys. J. 99:1706–1717

McConnell H. M., Vrljic M. (2003) Liquid-liquid immiscibility in membranes. Annu. Rev. Biophys. Biomol. Struct. 32:469–492. 24

Moyano, F., Biasutti M. A., Silber J.J., Correa N. M. (2006) New insights on the behavior of Prodan in homogeneous media and in large unilamellar vesicles. J. Phys. Chem. B. 110:11838–11846

Muller J. M., Harryvan D. H., Verhagen J. C. D., E. E. v. F, and van Ginkel G. (1996) The orientation of the transition dipole moments of TMADPH embedded in a poly(vinyl al-cohol) film. Chem. Phys. 211:413–420

Munro S. (2003) Lipid rafts: elusive or illusive? Cell. 115:377–388. 26 Oida T., Sako Y., Kusumi A. (1993) Fluorescence lifetime imaging microscopy (flim-

scopy). Methodology development and application to studies of endosome fusion in sin-gle cells. Biophys. J. 64:676-685

Patterson G. H., Piston D. W. Barisas B. G. (2000) Förster Distances between Green Fluo-rescent Protein Pairs Analytical Biochemistry 284:438-440

Pentcheva T., Edidin M. (2001) Clustering of peptide-loaded MHC class I molecules for endoplasmic reticulum export imaged by fluorescence resonance energy transfer. J. Im-munol. 166:6625–6632

Perrin F. (1926) La fluorescence des solutions. Polarisation. Vie moyenne des molécules dans l'état excité.J. de Phys. 7:390-401

Richards B., Wolf E. (1959) Electromagnetic Diffraction in Optical Systems. II. Structure of the Image Field in an Aplanatic System. Proc. R. Soc. London Ser. A. 253:358-379

Rocheleau J. V., Edidin M., Piston D. W. (2003) Intrasequence GFP in class I MHC mole-cules, a rigid probe for fluorescence anisotropy measurements of the membrane envi-ronment. Biophys. J. 84: 4078–4086

Samsonov A. V., Mihalyov I., Cohen F. S. (2001) Characterization of cholesterol-sphingomyelin domains and their dynamics in bilayer membranes. Biophys. J. 81:1486–1500

Schauer K., Duong T. et al (2010) Probabilistic density maps to study global endomem-brane organization. Nat. Methods 7:560–566

Scherfeld D., Kahya N., Schwille P. (2003) Lipid dynamics and domain formation in model membranes composed of ternary mixtures of unsaturated and saturated phosphatidylcho-lines and cholesterol. Biophys. J. 85:3758–3768

Schlessinger J., Koppel D. E. et al (1976) Lateral transport on cell membranes: mobility of concanavalin A receptors on myoblasts. Proc. Natl. Acad. Sci. USA. 73:2409–2413.

Schön P., Behrndt M., Ait-Belkacem D., Rigneault H., Brasselet S. (2010) Polarization and Phase Pulse Shaping applied to Structural Contrast in Nonlinear Microscopy Imaging. Phys. Rev. A 81:013809

Schön, P., Munhoz F. et al (2008) Polarization distortion effects in polarimetric two-photon microscopy. Opt. Express. 16:20891–20901

Schütz, G. J., Kada G. et al (2000). Properties of lipid microdomains in a muscle cell mem-brane visualized by single molecule microscopy. EMBO J. 19:892–901.

Schwille, P., Haupts U. et al (1999) Molecular dynamics in living cells observed by fluo-rescence correlation spectroscopy with one- and two-photon excitation. Biophys. J. 77:2251–2265.

Simons K., Ikonen E. (1997) Functional rafts in cell membranes. Nature. 387:569–572 Spille J.H., Zürn A., Hoffmann C., Lohse M.J., Harms G.S. (2011) Rotational Diffusion of

the α2a Adrenergic Receptor Revealed by FlAsH Labeling in Living Cells Biophys. J. 100:1139-1148

30

Sund S. E., Swanson J. A., Axelrod D. (1999) Cell membrane orientation visualized by po-larized total internal reflection fluorescence. Biophys. J. 77:2266–2283

Treanor B., Depoil D. et al (2010) The membrane skeleton controls diffusion dynamics and signaling through the B cell receptor. Immunity. 32:187–199

van Rheenen J., Jalink K. (2002) Agonist-induced PIP(2) hydrolysis inhibits cortical actin dynamics: regulation at a global but not at a micrometer scale. Mol. Biol. Cell. 13:3257–3267

Varma R., Mayor S. (1998) GPI-anchored proteins are organized in submicron domains at the cell surface' Nature 394:798-801

Veatch S. L., Keller S. L. (2002) Organization in lipid membranes containing cho-lesterol. Phys. Rev. Lett. 89:268101

Vrabioiu A. M., Mitchison T. J. (2006) Structural insights into yeast septin organiza-tion from polarized fluorescence microscopy. Nature. 443:466–469

Weber G. (1953) Rotational Brownian motion and polarization of the fluorescence of solu-tions. Adv. Protein Chem 8:415-459