# Nanotomography of Cell Surfaces with Evanescent Fields

Authors: Michael Wagner; Petra Weber; Wolfgang S. L. Strauss; Henri-Pierre Lassalle; Herbert Schneckenburger
Journal: Advances in Optical Technologies
 (2008)

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## Abstract

The technique of variable-angle total internal reflection fluorescence microscopy (TIRFM) and its application to nanotomography of cell surfaces are described. Present applications include (1) 3D imaging of chromosomes in their metaphase to demonstrate axial resolution in the nanometre range, (2) measurements of cell-substrate topology, which upon cholesterol depletion shows some loosening of cell-substrate contacts, and (3) measurements of cell topology upon photodynamic therapy (PDT), which demonstrate cell swelling and maintenance of focal contacts. The potential of the method for in vitro diagnostics, but also some requirements and limitations are discussed.


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## Body

## 1. Introduction

Microscopy with subwavelength
resolution has been a challenge for many years. While lateral resolution has
been increased continuously to about 60 nm [1] in far-field microscopy and to
even less in optical near-field microscopy, axial resolution has remained in a
range above 100 nm. However, in close vicinity to a surface (e.g., cell-substrate
interface), further improvement in axial resolution may be achieved by total
internal reflection (TIR) techniques. Total internal reflection fluorescence
microscopy (TIRFM) has first been described in 1981 [2], and since that time
has been used increasingly for measuring cell-substrate contacts [3–5],
membrane or protein dynamics [6, 7], membrane proximal ion fluxes [8] as well as
endocytosis or exocytosis [9–11]. TIRFM is based on total internal
reflection of a light beam propagating through a medium of refractive index n1 (e.g., glass substrate) and meeting a second medium of refractive index n2<n1 (e.g., cell). Despite being totally reflected at all angles Θ≥Θc=arcsin(n2/n1),
the incident beam establishes an evanescent electromagnetic field Eev that
penetrates into the second medium and decays exponentially with the distance z from the interface.
According to the relation(1)d=(λ4π)(n12sin2Θ−n22)−1/2, penetration depths d between about
60 nm and more than 200 nm are attained for its intensity Iev∼Eev2,
depending on the wavelength λ and the angle of incidence Θ. Therefore, fluorescent dyes located within or close to the plasma
membrane can be examined almost selectively in living cells. In addition, upon
variation of Θ, the relation(2)IF(Θ)=AcT(Θ)te−Δ/d(Θ) between fluorescence intensity IF(Θ), penetration depth d(Θ), and cell-substrate distance Δ has been
established for fluorescent dyes (of constant quantum yield) distributed within
a thin layer of thickness t [4] with A corresponding to an experimental constant, c
to the local concentration of the fluorophore, and T(Θ)=4cos2Θ/[1−(n2/n1)2]
to the transmission factor of incident light through the cell-substrate
interface (on the condition that light is polarized perpendicular to the plane
of incidence). Similarly, a relation(3)IF=AcT(Θ)d(Θ)e−Δ/d(Θ) has been established for fluorescent dyes
distributed homogenously at distances larger than Δ [4],
again assuming a constant fluorescence quantum yield over the samples.On the basis of (2) fluorescence images of
a membrane marker recorded at different angles of incidence Θ can be
used to calculate the cell-substrate distance Δ with
nanometre precision, when ln[IF(Θ)/T(Θ)] is evaluated as a function of d−1(Θ). Similarly, fluorescence images of a
cytoplasm marker can be used for calculation of Δ, when ln[IF(Θ)/T(Θ)d(Θ)] is evaluated as a function of d−1(Θ) according to (3). In both cases, Δ results
from the slope of a straight line, which can be calculated for each pixel of a
fluorescence image.In the present paper, three examples of nanotomography
with variable-angle TIRFM are given.(1)
Chromosomes in metaphase stained with the
fluorescence marker DAPI are used to show the potential of 3D tomography.(2)
Human glioblastoma cells incubated with the
membrane marker laurdan are used to visualize changes of cell-substrate
topology upon depletion of cholesterol.(3)
Human glioblastoma cells loaded with
5-minolevulinic acid induced protoporphyrin IX are shown to change their
membrane topology upon photodynamic
therapy (PDT) [12].


## 2. Materials and Methods

All TIRFM measurements were carried out with
either chromosomes or cultivated cells. Chromosomes from Chinese hamster ovary
(CHO) cells stained with the DNA marker DAPI were stopped in the metaphase of
mitosis by colchicine and prepared on microscope object slides. Their typical
length was about 5μm and their diameter around 500 nm.U373MG human glioblastoma cells
obtained from the European Collection of Cell Cultures (ECACC no. 89081403)
were routinely grown in RPMI 1640 medium supplemented with 10% fetal calf
serum and antibiotics at 37°C and 5% CO2. After seeding of 150 cells/mm2, cells were
grown on microscope object slides for 48 hours prior to rinsing with
Earl's balanced salt solution (EBSS) and incubation for 1 hour with either
the fluorescent membrane marker laurdan (8 μM) or coincubation with laurdan
(8 μM) and methyl-β-cyclodextrin (MβCD; 1 mM or 4 mM) diluted in culture medium
without serum. Cholesterol depletion after application of MβCD is well documented in the literature [13],
and preliminary experiments proved that its intracellular amount is reduced by
about 30% (at 1 mM MβCD) or 50% (at 4 mM MβCD). After incubation for
60 minutes, cells were again rinsed with EBSS and examined in the fluorescence
microscope at room temperature.In a further experiment U373MG cells
cultivated under the same conditions were incubated for 6 hours at
pH = 7.2 with10−3 M 5-aminolevulinic acid (5-ALA; Medac Wedel, Germany), an intermediate in
porphyrin biosynthesis, and rinsed with EBSS prior to TIRFM measurements. Part
of the cells was transiently transfected with the plasmid encoding for the yellow
fluorescent protein (YFP)-focal
adhesion kinase (FAK) fusion protein [14] by lipofection (FuGene 6
Transfection Reagent, Roche Diagnostics GmbH, Mannheim, Germany) in order to
visualize focal adhesions. The YFP-FAK vector was kindly provided by A.R.
Horwitz, University of Virginia, USA.For TIR illumination of cultivated
cells a UV-laser diode (LDH 400 with driver PDL 800-B, Picoquant, Berlin,
Germany; wavelength: 391 nm, pulse energy: 12 pJ, repetition rate
40 MHz, average power: 0.5 mW) was adapted to a fluorescence
microscope (Axioplan 1, Carl Zeiss Jena, Germany) by single mode fibre optics
(Point Source, kineFlex-p-3-S-395, Southampton, UK). For measurements of DAPI
stained chromosomes, the 391 nm laser diode was replaced by a diode of the
same type, but emitting light at 375 nm. EYFP-FAK was illuminated by the 514 nm line of an
argon ion laser operated at 10 mW (Innova 90, Coherent, Santa Clara, USA).
TIR illumination was carried out with a custom made device [4] permitting to
vary the angle of incidence in steps of 0.5° with a resolution of 0.15°. Angles
were varied either between 66° and 72° (U373MG cells) or between 70° and 76°
(chromosomes). Assuming refractive indices n1=1.515
(glass substrate) and n2=1.37 (cytoplasm), penetration
depths of the evanescent electromagnetic field within cells varied between
about 160 nm and 70 nm according to (1). Fluorescence images were recorded with
an electron multiplying (EM-) CCD camera with Peltier cooling and a sensitivity
below 10−17 W/Pixel (DV887DC-BV, ANDOR Technology, Belfast, UK).
In one case, this camera was replaced by a Sony 3CCD Colour Camera (Model
MC-3254, AVT Horn, Aalen, Germany, in combination with the software
AxioVision, Carl Zeiss Jena, Germany).
A long pass filter for λ≥415 nm was used for detection of
DAPI stained chromosomes as well as for laurdan, whereas a long-pass filter for
λ≥590 nm was used for detection of
5-ALA induced protoporphyrin IX (PP IX), and a bandpass filter for 529 ± 20 nm (in combination with a notch
filter at 514 nm) was used for YFP-FAK. For experiments on photodynamic therapy sublethal
light doses of 4 J/cm2 at 633 nm
(resulting from a 25 mW helium-neon laser; Stabilite 124B, Spectra
Physics, Mountain View, USA) were applied under epi-illumination, and recording
of TIRFM images was repeated immediately afterwards.Cell-substrate distances were
calculated according to (2) or (3) with a custom made computer program from a
series of 8−12 TIRFM images each. For each image, the
background of the camera system was subtracted. Cell-substrate distances (or
chromosome-substrate distances) were evaluated for all pixels, where
fluorescence intensity exceeded this background level. The scheme and the
experimental setup of the TIR illumination device are depicted in Figure 1
together with a series of variable-angle TIRFM images of U373MG cells loaded
with the cytoplasm marker calcein.Illumination device (microscope condenser) for variable-angle TIRFM with an additional option for
transillumination and phase contrast microscopy (a) scheme, (b) experimental
setup, and (c) fluorescence images of the cytoplasm marker calcein in a U373MG
glioblastoma cell upon variable-angle illumination.
(a)(b)(c)


## 3. Results and Discussion

### 3.1. 3D Topology of Chromosomes

Fluorescence images of DAPI stained chromosomes
on a microscope slide are depicted in Figure2 upon epi-illumination
(conventional fluorescence microscopy) and TIR illumination at Θ=72.5°. Upon TIR illumination, a
pronounced increase of the signal-to-background ratio occurs. Figure 2 also
shows a topographic image calculated according to (3) from variable-angle
TIRFM images (since the fluorescent dye is distributed over the whole
chromosomes, the same fitting algorithm as for a cytoplasm marker is used).
According to this topographic image, the distance between the chromosomes and
the glass surface varies between 0 nm and 150 nm. Only when chromosomes are
overlapping (marked by an arrow) larger distances are observed. In Figure 2,
the axial resolution is far below 100 nm and typically reaches values around
10–20 nm.Fluorescence images of chromosomes from T47D breast cancer cells stained
with DAPI in metaphase. (a) Conventional fluorescence microscopy, (b) TIRFM atΘ=72.5°, and (c) tomographic imaging
of chromosome-substrate distances (fluorescence excited at 391 nm and
detected at λ≥415 nm; image size:
55 μm × 55 μm). The arrow marks overlapping
chromosomes.
(a)(b)(c)


### 3.2. Impact of Cholesterol on Cell-Substrate Topology

The fluorescence marker 6-dodecanoyl-2-dimethylamino-naphthalene (laurdan) is described in the literature as a polarity-sensitive probe, whose optical spectra
depend on membrane stiffness and fluidity [15, 16]. Its fluorescence spectrum
is composed by two emission bands with maxima around 440 nm and
490 nm, respectively. It is well documented that with decreasing membrane
stiffness the long-wave 490 nm band becomes more pronounced compared to the
short-wave 440 nm band, for example, upon temperature increase or
cholesterol depletion [17]. The effect of cholesterol depletion on laurdan
fluorescence in whole cells and plasma membranes is documented in Figure 3
upon (b) epi-illumination and (c) TIR illumination by real colour imaging. For a
comparison, (a) phase contrast images of these cells are added. Upon incubation
of the cells with laurdan (without any further treatment) the fluorescence
images appear dark blue, since they are dominated by the 440 nm band,
whereas upon coincubation with laurdan and MβCD (30% cholesterol
depletion) whole cells still appear dark blue, whereas the plasma membranes
appear blue-green due to an increasing contribution of the 490 nm band at
decreasing membrane stiffness.(a) Phase contrast, (b) whole
cell fluorescence, and (c) TIRFM images of U373MG glioblastoma cells upon
incubation with laurdan either without further treatment (upper series) or with
30% cholesterol depletion (by 1 mM MβCD; lower series). Fluorescence excited at 391 nm and
detected at λ≥415 nm; image size: 200 μm × 150 μm. Real colour images recorded
with a 3-chip CCD camera.
(a)(b)(c)The question now arises how
cell-substrate topology is affected by cholesterol depletion. This effect is well
illustrated in Figure4. First, upon incubation with laurdan but without any
further treatment, cell-substrate distances vary between about 20 nm and 120 nm.
Upon application of 1 mM MβCD (30% cholesterol depletion)
cell-substrate distances generally increase, and upon application of 4 mM MβCD
(50% cholesterol depletion), these distances are above 200 nm in the
central parts of the cells, whereas on the cell edges the smaller distances are
maintained. This proves that cell-substrate contacts are loosened in the
central parts of the cells, which finally may lead to dissolution of the cells.TIRFM images recorded atΘ=66° (left) and cell-substrate
topology (right) of U373MG glioblastoma cells upon incubation with laurdan
either (a) without further treatment or 30% cholesterol depletion (by 1 mM
MβCD; (b) or (c) 50% cholesterol depletion (by 4 mM MβCD). Fluorescence excited at 391 nm and
detected at λ≥415 nm; image size: 210 μm × 210 μm).
(a)(b)(c)


### 3.3. Cell-Substrate Topology upon Photodynamic Therapy

Adhesion of tumour cells upon
photodynamic therapy (PDT) [18, 19] is an important question with regard to a
possible formation of metastases. Therefore, variable-angle TIRFM appears very
promising for investigation of cell-substrate topology and focal adhesions.
Since 5-ALA induced protoporphyrin IX (PP IX) is partly located in the
plasma membrane, it can be used as a photosensitizer as well as a membrane
marker permitting to calculate cell-substrate distances according to (2). While
Figure 5(a) shows a TIRFM image (Θ=66°) of U373MG glioblastoma cells
incubated with 5-ALA, Figure 5(b) shows cell-membrane topology before, and Figure
5(c) after application of a nonlethal light dose of 4 J/cm2 (633 nm). While
cell-substrate distances between 100 nm and 150 nm are the most
frequent ones before PDT, these distances are decreasing after PDT, probably
due to an increase of the cell volume upon swelling. If, instead of the plasma
membrane, focal adhesions are visualized by YFP-FAK, it becomes evident from
Figure 6 that these focal adhesions are maintained upon PDT. While Figure 6(a)
shows a TIRFM image of focal adhesions, Figures 6(b) and 6(c) show their
topologies prior and after PDT, which obviously are almost equal. Results described
for PP IX and YFP-FAK have recently been verified with a larger number of
U373MG glioblastoma cells and HeLa cervix cancer cells [5], proving that
detachment of these cells from their substrate upon photodynamic therapy is
unlikely to occur.TIRF image of 5-ALA induced PP IX (A, Θ=66°) as well as (b) cell-substrate topology prior and (c) after
PDT (633 nm; 4 J/cm2). Fluorescence excited at 391 nm and
detected at λ≥590 nm; image size: 140 μm × 140 μm; cell-substrate distances
(colour scale) given in nanometres. Reproduced from [5] with
modifications.
(a)(b)(c)TIRF image of YFP-FAK (A, Θ=66°) as well as (b) topology of focal adhesions prior and (c)
after PDT with ALA-induced PP IX (633 nm; 4 J/cm2). Fluorescence excited at 514 nm and
detected at 520−550 nm; image size: 140 μm × 140 μm; cell-substrate distances
(colour scale) given in nanometres.
(a)(b)(c)


## 3.1. 3D Topology of Chromosomes

Fluorescence images of DAPI stained chromosomes
on a microscope slide are depicted in Figure2 upon epi-illumination
(conventional fluorescence microscopy) and TIR illumination at Θ=72.5°. Upon TIR illumination, a
pronounced increase of the signal-to-background ratio occurs. Figure 2 also
shows a topographic image calculated according to (3) from variable-angle
TIRFM images (since the fluorescent dye is distributed over the whole
chromosomes, the same fitting algorithm as for a cytoplasm marker is used).
According to this topographic image, the distance between the chromosomes and
the glass surface varies between 0 nm and 150 nm. Only when chromosomes are
overlapping (marked by an arrow) larger distances are observed. In Figure 2,
the axial resolution is far below 100 nm and typically reaches values around
10–20 nm.Fluorescence images of chromosomes from T47D breast cancer cells stained
with DAPI in metaphase. (a) Conventional fluorescence microscopy, (b) TIRFM atΘ=72.5°, and (c) tomographic imaging
of chromosome-substrate distances (fluorescence excited at 391 nm and
detected at λ≥415 nm; image size:
55 μm × 55 μm). The arrow marks overlapping
chromosomes.
(a)(b)(c)


## 3.2. Impact of Cholesterol on Cell-Substrate Topology

The fluorescence marker 6-dodecanoyl-2-dimethylamino-naphthalene (laurdan) is described in the literature as a polarity-sensitive probe, whose optical spectra
depend on membrane stiffness and fluidity [15, 16]. Its fluorescence spectrum
is composed by two emission bands with maxima around 440 nm and
490 nm, respectively. It is well documented that with decreasing membrane
stiffness the long-wave 490 nm band becomes more pronounced compared to the
short-wave 440 nm band, for example, upon temperature increase or
cholesterol depletion [17]. The effect of cholesterol depletion on laurdan
fluorescence in whole cells and plasma membranes is documented in Figure 3
upon (b) epi-illumination and (c) TIR illumination by real colour imaging. For a
comparison, (a) phase contrast images of these cells are added. Upon incubation
of the cells with laurdan (without any further treatment) the fluorescence
images appear dark blue, since they are dominated by the 440 nm band,
whereas upon coincubation with laurdan and MβCD (30% cholesterol
depletion) whole cells still appear dark blue, whereas the plasma membranes
appear blue-green due to an increasing contribution of the 490 nm band at
decreasing membrane stiffness.(a) Phase contrast, (b) whole
cell fluorescence, and (c) TIRFM images of U373MG glioblastoma cells upon
incubation with laurdan either without further treatment (upper series) or with
30% cholesterol depletion (by 1 mM MβCD; lower series). Fluorescence excited at 391 nm and
detected at λ≥415 nm; image size: 200 μm × 150 μm. Real colour images recorded
with a 3-chip CCD camera.
(a)(b)(c)The question now arises how
cell-substrate topology is affected by cholesterol depletion. This effect is well
illustrated in Figure4. First, upon incubation with laurdan but without any
further treatment, cell-substrate distances vary between about 20 nm and 120 nm.
Upon application of 1 mM MβCD (30% cholesterol depletion)
cell-substrate distances generally increase, and upon application of 4 mM MβCD
(50% cholesterol depletion), these distances are above 200 nm in the
central parts of the cells, whereas on the cell edges the smaller distances are
maintained. This proves that cell-substrate contacts are loosened in the
central parts of the cells, which finally may lead to dissolution of the cells.TIRFM images recorded atΘ=66° (left) and cell-substrate
topology (right) of U373MG glioblastoma cells upon incubation with laurdan
either (a) without further treatment or 30% cholesterol depletion (by 1 mM
MβCD; (b) or (c) 50% cholesterol depletion (by 4 mM MβCD). Fluorescence excited at 391 nm and
detected at λ≥415 nm; image size: 210 μm × 210 μm).
(a)(b)(c)


## 3.3. Cell-Substrate Topology upon Photodynamic Therapy

Adhesion of tumour cells upon
photodynamic therapy (PDT) [18, 19] is an important question with regard to a
possible formation of metastases. Therefore, variable-angle TIRFM appears very
promising for investigation of cell-substrate topology and focal adhesions.
Since 5-ALA induced protoporphyrin IX (PP IX) is partly located in the
plasma membrane, it can be used as a photosensitizer as well as a membrane
marker permitting to calculate cell-substrate distances according to (2). While
Figure 5(a) shows a TIRFM image (Θ=66°) of U373MG glioblastoma cells
incubated with 5-ALA, Figure 5(b) shows cell-membrane topology before, and Figure
5(c) after application of a nonlethal light dose of 4 J/cm2 (633 nm). While
cell-substrate distances between 100 nm and 150 nm are the most
frequent ones before PDT, these distances are decreasing after PDT, probably
due to an increase of the cell volume upon swelling. If, instead of the plasma
membrane, focal adhesions are visualized by YFP-FAK, it becomes evident from
Figure 6 that these focal adhesions are maintained upon PDT. While Figure 6(a)
shows a TIRFM image of focal adhesions, Figures 6(b) and 6(c) show their
topologies prior and after PDT, which obviously are almost equal. Results described
for PP IX and YFP-FAK have recently been verified with a larger number of
U373MG glioblastoma cells and HeLa cervix cancer cells [5], proving that
detachment of these cells from their substrate upon photodynamic therapy is
unlikely to occur.TIRF image of 5-ALA induced PP IX (A, Θ=66°) as well as (b) cell-substrate topology prior and (c) after
PDT (633 nm; 4 J/cm2). Fluorescence excited at 391 nm and
detected at λ≥590 nm; image size: 140 μm × 140 μm; cell-substrate distances
(colour scale) given in nanometres. Reproduced from [5] with
modifications.
(a)(b)(c)TIRF image of YFP-FAK (A, Θ=66°) as well as (b) topology of focal adhesions prior and (c)
after PDT with ALA-induced PP IX (633 nm; 4 J/cm2). Fluorescence excited at 514 nm and
detected at 520−550 nm; image size: 140 μm × 140 μm; cell-substrate distances
(colour scale) given in nanometres.
(a)(b)(c)


## 4. Conclusion

The potential of cell-substrate
tomography with nanometre resolution has been demonstrated for 3 examples.
Variable-angle TIRFM seems to be an ideal method, if a fluorescent dye is
distributed rather homogenously either within a thin layer (e.g., cell
membrane) or on top of a layer (e.g., within the cytoplasm or an isolated chromosome).
A user-friendly two-layer model was applied for all calculations reported
above, although up to 4 layers (glass substrate, buffer solution, cell
membrane, cytoplasm) may interfere. However, for thin cell membranes, and
cell-substrate distances which are smaller than the laser wavelength, the two-layer
model still holds with only few percent of error [20]. Similarly, the error
resulting from an inhomogeneous spot of incident light may become negligible,
if this spot is large compared to the object field [4]. Further requirements for
calculation of tomographic images are a stationary spot of illumination (upon
variation of the angle Θ), a constant fluorescence quantum yield over
the measured parts of the sample (as deduced in the case of laurdan from the
homogeneous distribution of fluorescence lifetimes) and a high signal-to-noise
ratio. Since TIRFM images are usually about 100 times weaker than conventional
fluorescence images, a high sensitivity of the detection system, for example, of
an EM-CCD camera, is needed. This kind of detection system also permits the use
of very low illumination doses (far below 1 J/cm2 for 10−12 TIRFM images), which may assure cell
survival in high-resolution microscopy and in vitro diagnostics.



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*Source: 254317-2008-06-25.xml*