Mapping Membrane Topology Label Free and Corrected for

Changes in the Refractive Index of the Membrane on a Nanometer

Scale

Joachim Walter

, Natalia Schiefermeier

, Nina Hobi

3,4,5

, Benjamin Grau

, Christine E. Bandtlow

Lukas A. Huber

2,7

, Rainer Uhl

, Sebastian Munck

3,8

BioImaging Zentrum of the Ludwig-Maximilians, Universität München Martinsried, München, Germany

Divisions of Cell Biology and for Neurobiochemistry, Biocenter - Innsbruck Medical University, Innsbruck, Austria

Biocenter - Innsbruck Medical University, Innsbruck, Austria

Department of Physiology and Medical Physics, Division of Physiology, Innsbruck Medical University, Innsbruck, Austria

Institut for General Physiology, University Ulm, Ulm, Germany

Department of Developmental Neurobiology, Instituto Cajal, C.S.I.C., Madrid, Spain

Austrian drug screening institute, ADSI, Innsbruck, Austria

VIB Center for the Biology of Disease, KU Leuven, Leuven, Belgium

Keywords: Adhesion Sites, Membrane Dynamics, Interference Reflection Microscopy, Topography.

Abstract: The plasma membrane is the outer limit of the animal cell. As such, it is both a border separating inside

from outside and a signaling platform for interactions with the surroundings. Among these interactions are

extracellular matrix contacts and adhesion sites. The membrane and its contact sites together with the

underlying cytoskeleton undergo constant remodeling, which leads to changes of the cell shape. In addition

to spatial information micro-topographical maps provide, information about the z-dimension and describe

the position of the plasma membrane with respect to the distance to a given substrate. Here we address how

to measure height differences in the plasma membrane and how to create topographical maps of the plasma

membrane with nanometer resolution. We address the currently used methodologies along with their

advantages and drawbacks. Moreover, we delineate a label-free method to obtain topographic maps of the

plasma membrane that are corrected for differences in the refractive index of the membrane utilizing an

interferometric approach with multiple wavelengths and a normalization procedure to account for changes

in the refractive index in the membrane.

1 INTRODUCTION

The cell membrane separates the intracellular

content from the external environment of the cell. It

forms a hull over the cytoskeleton of the cell. The

shape of the cell can be compared to a tent plane

spanned over its poles, which leads to bending and

curvature of the membrane covering the

cytoskeleton. The cytoskeleton in turn anchors the

membrane to a substrate and is reported to be

involved in the compartmentalization of the

membrane through so called pickets and fences

(Ritchie et al., 2003). The adhesion points

themselves can act as signaling platforms (Cabodi et

al., 2010). Cell surface proteins transduce dynamic

extracellular signals to the cell interior (Spiller et al.,

2010) including interactions with the extracellular

matrix, modeling multiple cellular responses

including migration, differentiation and

proliferation.

Per se the plasma membrane is a self-organizing

organelle with strong interdependencies of its

constituents which are compartmentalized through a

special interaction and distribution pattern into so

called nano- and microdomains (Lingwood and

Simons, 2010).

In living cells, adhesion and migration are

fundamental cellular processes, which undergo

constant remodeling together with constant turnover

of the membrane itself. Adhesion and migration are

involved in all organizational levels of the organism,

from single cell translocation to tissue development

and movement of entire organisms. Consequently,

Walter J., Schiefermeier N., Hobi N., Grau B., Bandtlow C., Huber L., Uhl R. and Munck S..

Mapping Membrane Topology Label Free and Corrected for Changes in the Refractive Index of the Membrane on a Nanometer Scale.

DOI: 10.5220/0004752100310040

In Proceedings of the International Conference on Bioimaging (BIOIMAGING-2014), pages 31-40

ISBN: 978-989-758-014-7

 2014 SCITEPRESS (Science and Technology Publications, Lda.)

adhesion and migration are fundamental aspects of

development and appear early in the evolution of

higher organisms (Harwood and Coates, 2004).

Defects in adhesion have been implicated in several

diseases, for example in neuro-developmental

disorders (Olson and Walsh, 2002), and changes can

be associated with the carcinogenic transformation

of normal cells (Knudsen and Miranti, 2006).

Considering the interdependent role of the plasma

membrane, adhesion, cell surface movement,

diffusion of its constituents and the interactions with

the cytoskeleton have to be investigated as one

complex. How cells remodel their envelope during

early adhesion events and during spreading has been

highlighted before (Pierres et al., 2003, Ryzhkov et

al., 2010). Nanometer scale topographic maps of the

membrane can be used as a read-out for this

remodeling (Parthasarathy and Groves, 2004,

Sugiyama et al., 2012).

A topographic map is a representation that adds

the relief or height information to the classic x,y 2-D

information. Unfortunately the typical tool of the

cell biologist for retrieving 3-D information, a

confocal microscope does not have the resolution

that is required to accurately depict the typical

variations in height of cell membrane (Giebel et al.,

1999). Therefore, we want to explore a technique

that is not limited in depth, like for example TIRF

and that ideally is label free. While there is no

denying that efficient labeling techniques are

available, these techniques are offset by the time and

resources required to attach the probe to the

biomolecule of interest. The quality of the technique

is depending on the quality of the label and the

labeling process. In contrast, to this previously used

interferometric methods are label free. Here we

present an interferometric method that uses a multi

wavelength normalization procedure. By applying

this new method we are the first to compensate for

artifacts, which are introduced by differences in the

refractive index of the membrane, e.g. at sites of

adhesion between the membrane and the substrate

and thus get more reliable results for creating micro-

topographical maps of adhering cells.

2 DIFFERENT TECHNIQUES TO

CREATE TOPOGRAPHIC

MAPS

Several procedures can be applied to investigate the

membrano-cytoskeletonal complex reorganization

via topographic maps on a nanometer scale. Among

them are Atomic Force Microscopy (AFM), Total

Internal Reflection Fluorescence Microscopy

(TIRF); Surface Plasmon Resonance (SPR),

Fluorescence Interference Contrast (FLIC), Point

Spread Function (PSF) fitting, Foerster Resonance

Energy Transfer (FRET) and micro-interference

methods.

2.1 Atomic Force Microscopy

Atomic Force Microscopy (AFM) involves probing

a surface, e.g. a cell surface by scanning a sharp tip

over the cell. In AFM, the interaction between the

specimen and the tip is measured by the deflection

of the cantilever (the tip holder) (Zhang et al.,

2010). A topographic map of the cellular membrane

can be created using AFM (Duman et al., 2010). The

limitations of AFM include the time-resolution as a

consequence of the scanning process and the fact

that only the top membrane is accessible to the

scanning probe.

2.2 Total Internal Reflection

Fluorescence

In Total Internal Reflection Fluorescence (TIRF)

microscopy light is projected under an angle of total

internal reflection on an interface from higher to

lower refractive index. This process initiates an

evanescent wave that can be used for excitation of

fluorescence. The intensity of the fluorescent signal

scales exponentially with the distance from the

interface (Axelrod et al., 1984). This technique has

been used to investigate the adhesion contacts and

membrane dynamics (Reichert and Truskey, 1990,

Snijder-Van As et al., 2009). The artifacts that can

occur include interference fringes due to laser light

being scattered at non-uniformities in the light path

and anisotropic blur due to a mismatch in the

refractive index between the specimen and the

surrounding medium.

TIRF based topographic maps encounter the

problem decreasing fluorescence intensity with

increasing distance from the interface; therefore,

making measurements more difficult and more

inaccurate the further away the sample is from the

interface. The penetration depth depends on the

wavelength and the incident angle, which means that

measurements in practice are limited to ~200 nm

above the coverslip (Mattheyses and Axelrod, 2006)

which however was sufficient for dynamic cell

measurements (Ryzhkov et al., 2010).

BIOIMAGING2014-InternationalConferenceonBioimaging

2.3 Surface Plasmon Resonance

Surface Plasmon Resonance (SPR) measurements

are another possibility for creating topographic maps

and have been successfully used in the past to create

topographic maps (Giebel et al., 1999) . SPR are

electromagnetic waves, which are traveling parallel

to a metal or dielectric surface, if the frequency of

the photons matches the frequency of surface

electrons. SPRs are changed upon changes to the

dielectric surface like absorption or binding

processes. Since surface plasmons are used to

establish an evanescent wave, SPR shares some

similarity with TIRF microscopy.

2.4 Fluorescence Interference Contrast

Fluorescence Interference Contrast (FLIC) can be

used to create topographic displays with nanometer

resolution of the cellular envelope (Parthasarathy

and Groves, 2004). Since FLIC occurs only by the

interference of a light emitted by a fluorophore with

its reflection in vicinity of a reflecting surface

(Lambacher and Fromherz, 1996) it is not very

commonly used for biological measurements.

2.5 Point Spread Function Shape based

Methods

Another way to deduce height information on the

nanometer scale has been applied for single particle

tracking (Toprak et al., 2007). Here, the path of the

single molecule is corrected based on information

derived from the microscopic image itself and the

way the light is diffracted.

For a given spot, typically the shape of the Point

Spread Function (PSF) changes with height, which

means for an object with a known shape and a

corresponding 3-D PSF, that the z-position can be

measured.

Different attempts have been made so far to use

this paradigm. One way is to use a double plane

detection scheme, which allows imaging of different

optical planes at the same time allowing the 3-D

information of the PSF to be fitted. Another method

is to use a cylindrical lens as it changes the 3-D PSF

due to an introduced astigmatism and allows direct

conclusions to be made about the height (Kao and

Verkman, 1994).

Basically, if the path of a particle is read out in 3-

D, it can be corrected for movements in 3-D;

however, a topographic map would not necessarily

be created. This can be achieved using the 3-D

detection scheme for super-resolution microscopy

images as described by (Huang et al., 2008, Juette et

al., 2008). The quality of the measurement depends

on the quality of the PSF fit.

Concerning maps created by PSF based methods;

differences in the refractive index of the membrane

will as well influence the PSF. These distortions of

the PSF are difficult to compensate for. Moreover, if

a complete topographic map is measured on living

cells with a super-resolution method, the timing can

become problematic, as a multitude of images need

to be acquired for these methods (Schermelleh et al.,

2010). Another difficulty is that single molecule

super-resolution methods are pointillistic and depend

on the labeling density, which means they leave

gaps, requiring interpolation of the missing data

(Galbraith and Galbraith, 2011).

2.6 Fluorescent Resonant Energy

Transfer

FRET provides information about the interaction

between proteins at a resolution of a few

nanometers. As such, it is used for surface

interactions of proteins and was used to some extent

to create topographic maps (Parthasarathy and

Groves, 2004). However, due to the limited range of

the resonant energy transfer, the answer provided by

this technique was a contact or no contact answer

and not a differentiated read-out describing the

topography of the cell membrane over a longer

ranges.

2.7 Interferometric Methods

Interferometric methods represent another potential

strategy for the creation of topographic maps. Both

interference reflection microscopy (IRM) and

reflection interference contrast microscopy (RICM)

have been applied to investigate minute changes in

the height of membranes at nanometer resolution

(Weber, 2003). For example, these techniques were

used to investigate the dynamics of adhesion of

fibroblasts to several surfaces (e.g. see (Fang et al.,

2005)). However, repeatedly changing from dark to

bright due to the oscillatory nature of the

interference pattern over longer distances creates

some ambiguity due to lack of an absolute intrinsic

reference. This problem has also limited the

possibility for investigation of the z-direction of

objects with unknown shape. Nevertheless, recent

advantages in RICM have shown that absolute

distance measurements are possible in floating

vesicles with the application of more wavelengths

and quantitative image analysis (Schilling et al.,

MappingMembraneTopologyLabelFreeandCorrectedforChangesintheRefractiveIndexoftheMembraneona

NanometerScale

2004). Consequently dual wavelength or DW-

reflection interference microscopy is used recently

for adhesion, vesicle dynamics and further

developments of the technique (for examples see

(Contreras-Naranjo and Ugaz, 2013, Limozin and

Sengupta, 2009, Monzel et al., 2009, Mundinger et

al., 2012, Sugiyama et al., 2012)). One of the

advantages of the interference methods is that they

allow for label free investigation of the cell

membrane. However, earlier reports have shown that

interference microscopy and measurements of

adhesion force were not in agreement (Zheng et al.,

1994) and that the refractive index within the cell

membrane can vary, especially at adhesion sites

(Iwanaga et al., 2001). According to the Fresnel

equations (Hecht, 2002) changes in the refractive

index of the membrane lead to changes in

reflectivity of the membrane. Along the same lines,

it was shown that the reflectivity at adhesion sites

can be higher (Atilgan and Ovryn, 2011). Moreover,

we could not fit DW-RICM measurements of

cellular process to the theory. In order to overcome

these problems and to be able to create absolute,

correct and label free topographic maps that can

universally be applied to cells, DW-RICM

measurements need to be corrected for differences in

the refractive index.

Here we delineate a method to correct for

differences in the refractive index of the membrane

using a normalization procedure based on multiple

wavelengths. We provide a theoretical background

and show the proof of principle using a cellular

process.

Figure 1: (a) schematic of reflective interference imaging

of a glass bead. (b) interference fringes of a glass bead

imaged with the setup. Scale bar=5µm.

3 MATERIALS AND METHODS

The images were acquired using a Zeiss Axiovert

200M equipped with a 63x oil (NA 1.4), a 100x oil

(NA 1.45) objective lens and a RT monochromatic

camera (diagnostic instruments). The setup was

driven by the Metamorph software (Universal

Imaging). A 50/50 beam splitter was used for

reflected light illumination and to allow collection of

the reflected light. A HBO lamp was used as light

source using Köhler illumination and aperture and

field diaphragms relatively closed to restrict the light

exposure of the sample. Interference filters (center

wavelength as indicated) were used in the parallel

part of the illumination path to create

monochromatic light. A motorized filter wheel was

used to switch between the wavelengths.

The rat CNS neuroblastoma cell line B104

(Schubert et al., 1974) was grown at 8.5% CO

DMEM (Invitrogen) supplemented with 10% heat-

inactivated fetal calf serum and 1% antibiotics

(Sigma; 10,000 units penicillin/ml and 10 mg

streptomycin/ml). Cultures were passaged once a

week at approximately 70–80% confluence and the

growth medium was changed every two days. B104

cells were plated at a density of 3,000 cells/cm

growth-supplemented medium on glass bottom

tissue-culture dishes (Sarstedt, Newton, NC), which

coated with poly-D-lysine (Sigma) and laminin

(Sigma). A heated stage and a cover were used to

conserve optimal conditions for cell survival and

growth (Carl Zeiss, Jena).

Beads of approximately 2 mm diameter with

unmarked surfaces were created from glass droplets

with a Bunsen burner.

Images were analyzed using ImageJ (1.44

National Institutes of Health USA,

http://rsb.info.nih.gov/ij), with custom written

macros. Models were calculated using Mathcad (13,

Mathsoft Engineering & Education, Cambridge MA)

and Excel (Microsoft, Redmond).

For clarity, the background in the images with

the growth cones has been removed based on the

phase contrast image. Background in the acquired

images was measured with a region of interest in the

vicinity of the object. Images of Q’1, Q’2 and Q’3

were derived according to the theory section. For the

creation of the height maps, values were rounded

and compared to a theoretical look up table

generated in Excel. Correlation plots were created

using the cytofluorogram module in Jacob using Fiji

(Bolte and Cordelieres, 2006, Schindelin et al.,

2012).

BIOIMAGING2014-InternationalConferenceonBioimaging

4 RESULTS

4.1 Theory of Refractive Index-free

Interferometric Measurements

In interference microscopy, the sample is

illuminated in reflective mode. Part of the light is

reflected at the interface between the coverslip and

the medium with a lower refractive index (interface

1). At the interface between the medium and the cell

membrane, which has a higher refractive index,

again part of the light is reflected (interface 2).

These two reflected beams form the interference

pattern that is observed as a function of the distance

between the coverslip and the cell membrane. The

intensity distribution of the interference pattern for a

given wavelength was previously described

(Schilling et al., 2004):

12 12

4(,)

((,), ) 2 cos

nh x y

Ihxy I I II











 





(1)

with I(h(x,y,),λ) being the intensity on the

interferogram corresponding to a point whose lateral

position is defined by the coordinates (x,y) and the

height of the membrane above the coverslip is given

by h(x,y). I

and I

are the intensities of the beams

reflected from interface 1 and interface 2,

respectively.

δ is the difference of the phase shifts δ

1/2

both

beams undergo upon reflection. δ

1/2

is 0 if the beam

is reflected from an interface to a medium with

lower refractive index, such as interface 1. It is

π if

the beam is reflected from a medium with lower

refractive index such as interface 2 (Hecht, 2002).

Hence, in this application







(2)

The intensity distribution further depends on n being

defined as the refractive index of the medium and

λ,

the wavelength of the illumination light.

A glass bead was used as a test object as it has a

higher refractive index than water and a known

shape, permitting analysis of the interference pattern

under controlled conditions. As expected, the glass

bead shows the characteristic Newton fringes (see

Figure 1). When two wavelengths were applied, the

resulting pattern fits nicely to the relation given in

(1). When we consequently investigated cellular

processes we have observed that the phase shift and

the intensity do not match the relation given in (1).

This finding confirms previously published data,

indicating the variability of the reflection coefficient

especially at focal adhesion sites (Atilgan and

Ovryn, 2011, Iwanaga et al., 2001).

Consequently, our aim was to find a way to measure

the height of the membrane above the coverslip

without relying on the assumption of a constant

reflectivity of the plasma membrane.

In Equation (2), I

1/2

can be written as

2/12/1

RaI





(3)

a being the intensity of the incoming light and R

1/2

the reflectivity of the respective interface.

We used three wavelengths to correct for the

inhomogeneities in the reflectivity. For wavelength

,, inserting equations (2) and (3) into (1) yields

12 12

(, ) 2 cos

Ih a R R RR











  

















(4)

Similar equations hold for wavelengths λ

and λ

Scaling and subtracting the intensities for different

wavelengths cancels out the summand R

+ R

(, ) (, )

2cos cos

2coscos

Ih Ih

nh nh

aRR

nh nh

aRR









































 





















 









(5)

Finally, division by a similar expression with a third

wavelength eliminates the term

RR .



























































































nhnh

cos

),(),(

),(),((

(6)

As the denominator reaches zero,



contains

reoccurring discontinuities which can be avoided by

defining the denominator as

(, ) (, ) (, ) (, )

(, ) (, )

Ih Ih Ih Ih

Ih Ih





































































(7)

and defining new quotients as

MappingMembraneTopologyLabelFreeandCorrectedforChangesintheRefractiveIndexoftheMembraneona

NanometerScale

(( ( , ), ) ( ( , ), )

I hxy Ihxy













(8)

(( ( , ), ) ( ( , ), )

I hxy Ihxy













(9)

(, ) (, )

Ih Ih















(10)

The expressions





and





can be measured in

areas of the images, where only the interface

between the coverslip to the medium contributes to

the signal (cell-free areas) as from equation (3) it

follows



















(11)

An instructive display of the different wavelengths

(540 nm, 600 nm and 685 nm) illuminating the bead

(Figure 2 a-d), their relation to the height of the bead

and the relative intensity of the consequent quotients

Q’1, Q’2 and Q’3 in dependence of the measured

height of the glass bead can be seen in Figure 2 e-h.

The height of the lower hemisphere of the glass bead

can be calculated and is a function of the radius r

and distance x from the center where the glass bead

touches the coverslip (see (12)).

hxrr 

(12)

Overall, the intensity distributions follow the

theoretical descriptions. The different wavelengths

in Figure 2d show the described phase shift

(Schilling et al., 2004). For the RICM, the intensity

changes with height of the bead, which is in

agreement with previous findings (Contreras-

Naranjo and Ugaz, 2013). In contrast to DW-RICM

our approach normalizes for this effect.

4.2 Practical Test of Refractive Index

Free Interferomtric Measurements

Next the growth cone of a B104 cell, a widely used

neuronal model (Schubert et al., 1974), was

investigated as a more physiologically relevant

model (Figure 3). The sample was imaged with the

same three wavelengths as the glass bead described

above (Figure 3 a-c). The equations above were

applied to calculate Q’1, Q’2 and Q’3 (Figure 3 d-f).

Figure 2: Testing multi-wavelength interference

microscopy on glass beads. (a-c) interferometric images of

the glass bead using light of 540nm; 600nm and 685nm

wavelength. (d) is an intensity profile of the pictures (a),

(b) and (c) from the region indicated by the yellow line in

(a). The x axis represents the height if the glass bead based

on equation 12. The length of the region is about 30 µm.

Insert shows theoretical values (e-g) depicts the calculated

quotients Q’1-Q’3 based on (a-c). (h) the relative intensity

distribution of Q’

, Q’

and Q’

of (e-f) similar to (d).

Insert shows theoretical values. Scale bar = 5 µm.

To test DW-RIM under these conditions, a rectangle

of 50x50 pixels in the growth cone was selected and

is displayed in a correlative graph. It can be seen that

the points do not strongly follow a pattern and the

characteristic curl shaped relation of (Schilling et al.,

2004) does not become visible. Using the same

approach with our method and using the Q’1 and

Q’2 (Figure 3 h),) the expected oval shaped relation

of the two Q’s becomes recognizable. Next, we

explored calculating a 3-D topography of the

membrane. As the concatenation of

QQ 

can be

used for calculating the height in the range from 0 to

240 nm, which proved to be sufficient in this case, a

look-up table was generated and applied to the

values measured from the growth cone. Therefore,

an image with grey values representing the height in

nm can be deduced and is displayed as a surface plot

next to the phase contrast image of the growth cone

(Figure 3i,j). The result is in agreement with earlier

publications (Giebel et al., 1999). It was not possible

to map all pixels in the look-up table with the

measured values, which is probably due to noise in

the system; these values were consequently set to

zero. The overall noise was relatively low as can be

seen by the minimal variation in the relation of

BIOIMAGING2014-InternationalConferenceonBioimaging

Figure 3h, which depicts depicting the oval shaped

relation of Q’1 and Q’2 with little variation. To

demonstrate the dynamic behavior of the membrane

and the height changes, the same growth cone was

imaged s20s later (Figure 3 i,j). Figure 3 k,l shows

another example of a growth cone demonstrating

that the height distributions can vary in different

neuronal processes.

Taken together, we present a multi-wavelength

extension of RICM: MIRM. By applying different

wavelengths for illumination, we can reconstruct the

refractive index corrected 3-D shape of the cellular

membrane.

5 CONCLUSIONS

Here we have described different ways to measure

membrane topography presenting the advantages

and limitations of several methodologies. We

highlight the benefits of label free interferometric

measurements, which can measure over a longer

range then TIRF, SPR, FLIC and FRET

measurements. Different then AFM interferometric

methods can probe the cell membrane, where the

adhesion sites are located. For imaging without gaps

the PSF based techniques require dense labeling, but

need to be read out in a non-overlapping way. This

requires a single molecule approach, which entails

the acquisition of much more images as compared to

our method. Another advantage of interferometric

methods is that they can be designed to be

compatible with labeling techniques and thus allow

for a higher degree of multiplexing. So far the

labeling independent interferometric methods have

suffered from ambiguity due to changes in the

refractive index. Here we show that when corrected

for differences in refractive index, unambiguous

imaging of membrane topologies can be achieved on

a widefield system. In comparison to the earlier

described method of (Schilling et al., 2004), the

method we propose has the advantage that

differences in the refractive index of the membrane

can be cancelled out. This effect can be seen in the

comparison of Figure 3 g and h and in the fact that a

clear relationship only becomes visible when

differences in the refractive index are cancelled out,

emphasizing that the differences in the refractive

index are not only drastic but can also be corrected.

Furthermore, the framework presented here can

easily be expanded to longer distances by applying

more wavelengths and using different wavelength

triplets for correction.

We have noticed that the method is sensitive to

Figure 3: Testing multi-wavelength interference

microscopy for mapping height profiles in a refractive

index free manner on cellular processes. (a-c)

interferometric images of a neuronal growth cone using

light of 540nm; 600nm and 685nm wavelength. (d-f)

depicts the calculated quotients Q’1-Q’3 based on (a-c).

(g) correlation of the normalized intensities of (a) and (b)

of the region indicated by the blue rectangle in (a). Theory

in black. (h) correlation of Q’1 and Q’2 like in (g). Theory

in black. (i) phase contrast picture of a neuronal process.

(j) is a reconstruction of the membrane topology of the

cellular process in (i). (k) the same growth cone as in (i)

20 seconds later. (l) 3-D topographical map of the growth

cone in (k). (m) another example of a neuronal growth

cone in phase contrast. (n) 3-D topographical map of the

growth cone in (m).

For the reconstruction the relation between Q’

and Q’

with the wavelength triplet 540/600/685 nm has been

used.

Scale bars = 5 µm color/height range in (j,l and n) from

purple = zero to peaks in red = 200 nm.

background disturbances. Therefore, subtraction and

the relative normalization of the individual

wavelengths are critical for correct calculation.

Here the corrected membrane topography has

been reconstructed with an unprecedented ~30 nm

MappingMembraneTopologyLabelFreeandCorrectedforChangesintheRefractiveIndexoftheMembraneona

NanometerScale

resolution (Fig. 3). Theoretically, a much better

resolution in z-direction is possible than shown in

Figure 3. The limited resolution here is mainly due

to the use of the look up table. That relative

differences of ~5 nm can be practically be achieved

using interference measurements has been

demonstrated earlier (Llobet et al., 2003). The

achievable practical resolution depends on the

sensitivity of the detector and the wavelengths used.

Due to the nonlinearity in the obtained relation after

correction for the refractive index, the achievable

resolution cannot be equal at all distances. Like in

the PSF based methods, our implementation leaves

gaps; however these could be improved when the

measurements are fitted to the quotients instead of

using a look-up table.

It should to be emphasized that correction for

differences in height is needed for single particle

tracking. Simple arithmetic indicates that the error

calculated for a movement in the membrane is about

12%, e.g .if a height change of 150 nm is ignored

over a 300 nm distance. We believe an independent

measurement, undisturbed from refractive index

changes, is achievable with a label free technique

and is a good alternative to the currently used

methods for the correction (Kao and Verkman, 1994,

Toprak et al., 2007).

One of the advantages of the described method is

its speed. The three wavelengths for reflective

imaging can easily be acquired in the millisecond

range. The analysis can be implemented for almost

instantaneous results (see as well (Contreras-Naranjo

and Ugaz, 2013)). The fact that the method is label

free means that it is relatively cheap and is making it

attractive for large scale quantitative analysis and

screening tasks. One potential application we see is

screening for the transition between mesenchymal

and amoeboid modes of migration of tumor cell

upon drug treatment. This transition is relevant for

potential treatment as it has been put in the context

of tumor invasiveness and aggressiveness (Kosla et

al., 2013, Pankova et al., 2010). Another application

field is neuroscience as exemplified here. Neuronal

growth cones are the growing tips of neurons. These

exhibit fast remodeling of the cytoskeleton and the

membrane when growing towards their targets. The

dynamic of this remodeling is demonstrated by

Figure 3 i-l. Moreover, that fact that there are

different states of height maps is emphasized when

comparing Figure 3i-l with Figure 3 m and n.

Overall, we have only started to understand how

cells move and migrate; therefore it is important to

look at all processes and not only at the points of

contact. With topographical maps, the overall

remodeling of the cellular envelope with respect to

their movement and interaction with the substrate

can be observed. This aspect of the remodeling of

membrane has been neglected so far, potentially due

to the lack of good methods. However we believe it

poses an important aspect of the cell and the

migration process. Considering migration and

focusing on the adhesion sites would be like looking

solely at the feet when trying to figure out how a

human walks.

The analysis of the topography of the cell

membrane provides information on membrane

trafficking and the remodeling of the underlying

cytoskeleton as one complex that is remodeled

during cellular movement. We believe that the tool

presented here is a useful extension of the standard

repertoire to investigate adhesion and migration and

will contribute toward the illumination of these

processes in the future.

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