Download Total internal reflection fluorescence spectroscopy and microscopy

Variable-angle total internal reflection fluorescence microscopy (VATIRFM): realization and application of a compact illumination device
Karl Stock1,2, Reinhard Sailer1, Wolfgang S.L. Strauss1, Marco Lyttek2, Rudolf Steiner1 and
Herbert Schneckenburger1,2,*
1
Institut für Lasertechnologien in der Medizin und Messtechnik an der Universität Ulm,
Helmholtzstr. 12, 89081 Ulm, Germany;
2
Fachhochschule Aalen, Institut für Angewandte Forschung, 73428 Aalen, Germany
*Correspondence to: Dr. Herbert Schneckenburger
Institut für Angewandte Forschung
Fachhochschule Aalen
73428 Aalen, Germany
Tel. +49-7361-568229
FAX: +49-7361-568225
E-mail: [email protected]
Running Titel: VA-TIRFM
Keywords: fluorescence microscopy, evanescent waves, TIR illumination, cell-substrate
topology, calcein, laurdan
2
Summary
A novel compact illumination device in variable-angle total internal reflection fluorescence
microscopy (VA-TIRFM) is described. This device replaces the standard condensor of an
upright microscope. Light from different laser sources is delivered via monomode fiber and
focused onto identical parts of a sample under variable angles of total internal reflection.
Thus, fluorophores in close proximity to a cell-substrate interface are excited by an evanescent
wave with variable penetration depth, and localized with high (nanometer) axial resolution. In
addition to quantitative measurements in solution, fluorescence markers of the cytoplasm and
the plasma membrane, i.e. calcein and laurdan, were examined using cultivated endothelial
cells. Distances between the glass substrate and the plasma membrane were determined using
the mathematical algorithm of a four layer model as well as a Gaussian shaped intensity
profile of the illumination spot on the samples. Distances between 0 nm and 30 nm in focal
contacts and between 100 nm and 250 nm in other parts of the cell were thus determined. In
addition to measurements of cell-substrate topology, the illumination device appears
appropriate for numerous applications, where high axial resolution is required, e.g.
experiments on endocytosis or exocytosis, as well as measurements of ion concentrations
proximal to the plasma membrane. The compact illumination device is also suitable to
combine
TIRFM
with further innovative techniques, e.g. time-resolved fluorescence
spectroscopy, fluorescence lifetime imaging (FLIM) or fluorescence resonance energy transfer
(FRET).
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1. Introduction
High lateral and axial resolution are challenges in modern optical microscopy, e.g.
fluorescence microscopy. Both resolutions are generally restricted by the wavelength  of
optical radiation as well as by the numerical aperture A of the microscope objective lens. For
light emitting samples the lateral resolution is commonly given by x = 0.61  / A, which
corresponds to the radius of a diffraction limited spot. Assuming a fluorescence wavelength
 = 550 nm and a high numerical aperture A = 1.30, the lateral resolution is about 260 nm. A
measure for axial resolution is given by the depth of focus z = n  / A² with n being the
refraction index of the immersion medium between the sample and the microscope objective
lens (e.g. oil or water). Assuming n = 1.50 and A = 1.30, a value z  500 nm is attained for
 = 550 nm. Object diameters, however, are often larger, and images from the focal plane are
superimposed by out-of-focus images from adjacent parts of the sample. In the past years this
disadvantage has been overcome by confocal laser scanning microscopy with the object being
located precisely in the focus of an incident laser beam. A pinhole within the image plane
permits selective detection from this focal plane. Microscopic images are obtained by
horizontal scanning of the laser beam; 3-dimensional information is achieved by moving the
sample in vertical direction.
More recently, in addition to confocal laser scanning microscopy, two-photon or multiphoton
microscopy has been established, where only those molecules of a sample are excited which
are precisely in the focus of one or several laser beams, as first described by Denk et al.
(1990) and later summarized by König (2000). Out-of focus molecules are not excited and
therefore not detected fluorometrically. Using a so-called 4Pi geometry with two opposing
lenses of high numerical aperture together with deconvolution techniques, an axial resolution
around 150 nm has been attained (Schrader & Hell, 1996). However, this value is still large
compared with the diameters of cell membranes and several other intracellular structures. In
addition, when using tunable picosecond or femtosecond laser systems, multiphoton
techniques are becoming very complex and expensive. This also holds for the so-called
stimulated emission depletion microscopy, where a first laser pulse is used to excite a
diffraction limited spot of the sample, and a second pulse to deactivate the molecules at the
edge of the sample, such that fluorescence arises only from its central part with a diameter
down to 33 nm (Dyba & Hell, 2002).
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Whereas confocal and multiphoton microscopy were developed during the past two decades,
the first applications of Total internal reflection fluorescence microscopy (TIRFM) have been
reported more than ten years earlier (Axelrod, 1981). Although its applicability is limited to
cell compartments in close proximity to a cell-substrate interface, i.e. plasma membranes and
adjacent cellular sites, TIRFM remains the method with the highest axial resolution. TIRFM
techniques use an evanescent electromagnetic field for the excitation of fluorophores. This
field arises upon total reflection of the excitation light on the cell-substrate interface,
penetrates into the cell and decays exponentially with perpendicular distance z from the
interface, as depicted in Figure 1. Penetration depth depends on the wavelength and the angle
of incidence of the excitation light as well as on the refraction indices of the optical media.
Typically, penetration depths can be varied between 50 nm and more than 200 nm (Burmeister
et al., 1994). Thus, fluorophores located within or close to the plasma membrane are excited
selectively. So far, total internal reflection fluorescence microscopy (TIRFM) has been applied
for (1) studies of the topography of cell-substrate contacts (Axelrod, 1981; Truskey et al.,
1992; Hornung et al., 1996), (2) measuring dynamics (Sund & Axelrod, 2000) or selfassociation (Thompson et al., 1997) of proteins at membranes, (3) detection of membraneproximal ion fluxes (Omann & Axelrod, 1996) and (4) imaging of endocytosis or exocytosis
(Betz et al., 1996, Oheim et al., 1998). Previous work of the authors using TIR illumination
was dedicated to the investigation of photosensitizers (compounds with tumour-localizing
properties) in close proximity to the plasma membrane (Strauss et al., 1998, Sailer et al.,
2000).
In general, two different technical solutions for TIR illumination are etablished. In the first
case, the cell substrate is optically coupled to a glass or quartz prism of cubic, hemicylindrical or hemi-spherical shape. Only when using a hemi-cylindrical or a hemi-spherical
prism (Oheim et al., 1999) or a multiple laser scanning system (Ölveczky et al., 1997), the
position of the illuminating light spot on the sample is maintained when the angle of incidence
is varied (VA-TIRFM). Experimental equipments for VA-TIRFM, however, have so far been
rather complex. The second technical solution is based on extreme dark field illumination by
an objective lens of high numerical aperture (prismless TIRFM; Stout & Axelrod, 1989). This
technique has been used e.g. for single molecule detection within thin cell layers (Sako et al.,
2000; Iino & Kusumi, 2001). However, even for A = 1.45, the aperture angle (72°) exceeds
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the critical angle for total internal reflection only by about 7°. Therefore, prismless VATIRFM is difficult to perform.
In the present paper, a compact illumination device for VA-TIRFM is described, where the
sample is optically coupled to a hemi-cylindrical glass prism, and where the incident light
(delivered from a monomode fiber) is deflected onto the sample under variable angles by an
adjustable mirror. Using a concave mirror as a focusing unit and folding the light beam twice,
the whole illumination device becomes very small and can replace the standard microscope
condensor.
2. Theory
When a light beam propagating through a medium of refractive index n1 meets an interface to
a second medium of refractive index n2, total internal reflection occurs at all angles of
incidence , which are greater than the critical angle C = arcsin (n2/n1). Despite being totally
reflected the incident light beam evokes an evanescent electromagnetic field that penetrates
into the second medium and decays exponentially with perpendicular distance z from the
interface according to I(z) = I0 e-z/d(). I(z) corresponds to the intensity of the electromagnetic
field and d() to the penetration depth at wavelength , as given by
d() = (/4) (n1² sin² - n2²)-1/2
(1).
As reported by Gingell et al. (1987) as well as Reichert and Truskey (1990), the intensity of
the evanescent electromagnetic field at z = 0 (I0), corresponds to the intensity of the incident
light Ie multiplied with the transmission factor T() and the ratio n2/n1. If the electric field
vector of the incident light beam is polarized perpendicular to the plane of incidence, this
transmission factor is given by
T() = 4 cos²  / [1 – (n2/n1)2]
(2).
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For calculating the fluorescence intensity in TIRFM measurements, light absorption dI/dz
within thin layers dz has to be considered. With () being the molar extinction coefficient
and c(z) the concentration of absorbing molecules, absorption is calculated according to
dI/dz = () ln10 c(z) I(z) = () ln10 c(z) Ie (n2/n1) T() e-z/d()
(3).
Fluorescence is obtained from Equation 3 by multiplication with the fluorescence quantum
yield  and the detection angle , as well as by integration over the layers where fluorophores
are located. If emission is assumed to be isotropic, the result for fluorescence intensity is
IF () = () ln10  (/4) Ie (n2/n1) T()  c(z) e-z/d() dz
(4),
or
IF () = A T() c(z) e-z/d() dz
(5),
if all factors which are independent from the angle of incidence  and the coordinate z are
included within the experimental constant A.
According to Equation 5, the fluorescence intensity IF () can be calculated for

a continuum, where fluorophores are distributed homogeneously (at a constant
concentration c) above the interface. In this case, the integral has to be calculated from
z = 0 to z = , thus giving IF = A c T() d();

a homogeneous distribution of fluorophores (c = const.) for z > , e.g. within the
cytoplasm of cells having a distance  from the interface. In this case, the integral has to
be calculated from z =  to z = , thus giving IF = A c T() d() e-/d();

a distribution of fluorophores within a thin layer of thickness t at a distance  from the
interface, e.g. within cell membranes. In this case, the integral has to be calculated from
z =  - t/2 to z =  + t/2, thus giving IF = A c T() t e-/d(), if the concentration c is
considered to be constant within the layer, and if t is small as compared with .
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For a well defined location of fluorophores (e.g. within the cytoplasm or the plasma
membrane), the distance  as well as the concentration c can basically be determined by VATIRFM using one of the equations described above. However, the following limitations have
to be considered:
(1) Optical dipole radiation in the near field of a dielectric interface is anisotropic, and the
collection efficiency by a microscope lens depends on the polarization and the distance z of
the dipoles from the interface as well as on the numerical aperture of the objective lens
(Burghardt & Thompson, 1984; Hellen & Axelrod, 1987). For objective lenses with apertures
A < 0.90, angles of incidence  between 64° and 80° and a polarization perpendicular to the
plane of incidence, deviations of collection efficiency were within + 2 % and could be
therefore neglected (Stock, 2001).
(2) When using a Gaussian shaped laser beam, the intensity I0 of the evanescent wave has an
elliptic profile according to
I0 (r,,) = exp [-2 (r/r0)]²
(6),
where the beam radius r0 depends on the angle of incidence  and the azimuth angle 
according to r0,y = r0 and r0,x = r0 / cos  for the axis of the ellipse. For an arbitrary angle , r0
can be calculated according to
r0 (,) = r0,y / [1 - (r0,x² - r0,y2) cos² / r0,x²]
0.5
(7)
and used for correction of the fluorescence intensity in TIRFM experiments.
(3) For TIRFM experiments on cell surfaces, any model using two refractive indices n1 and n2
has to be regarded as an approximation, since in reality four layers interfere: the glass
substrate (ns = 1.52), the aqueous extracellular medium (ne = 1.33), the plasma membrane
(nm = 1.45) and the cytoplasm (nc = 1.38). This four layer system has been described
theoretically by Gingell et al. (1987), but no analytic solution for the distance  = f(IF()) is
availableHowever, in the present paper IF() was measured and the distance  between the
plasma membrane and the glass substrate was calculated by non-linear regression. As shown
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by Reichert & Truskey (1990), the comparatively thin plasma membrane can be neglected for
calculation of IF() with an error of less than 2.5 %. In the remaining three-phase model,
effective values of the critical angle c, transmission factor T() and penetration depth d()
can be used. In case of a fluorescent membrane marker, and if the layer of the extracellular
medium is small compared with the wavelength of the incident light, the effective values c
and T() can be approximated by the values obtained from the two-phase model using the
refractive indices of the substrate and the cytoplasm. In contrast, the effective value of the
penetration depth d() can be approximated using the two-phase model with the refractive
indices of the substrate and the extracellular medium. For a fluorescent cytoplasm marker the
two-layer-model with refractive indices of the substrate and the cytoplasm has previously been
used (Ölveczky et al., 1997).
3. Materials and methods
3.1 Cells and solutions
For calibration and quantitative measurements, solutions of the fluorophore calcein dissolved
in distilled water were used at concentrations between 1 µM and 100 µM. Small droplets of
calcein solution were given onto microscope object slides which were optically coupled to the
glass prism (used for TIR illumination) by immersion oil and which had almost the same
refractive index as the prism. Prior to the experiments object slides were cleaned carefully
using a detergent and acetic acid, treated by ultrasonic waves and dried at 180°C.
BKEz-7 endothelial cells from the calf aorta (Halle et al., 1984) were routinely cultivated on
sterile object slides using Eagle’s Minimum Essential Medium (MEM Eagle) supplemented
with 10 % fetal calf serum, 2 mM glutamine and antibiotics at 37°C and 5 % CO2. 150 cells
per mm² were seeded 48 h prior to microscopic measurements. For staining the cytoplasm,
cells were incubated with calcein acetoxymethylester (calcein-AM; 5 µM, 40 min). After
10 min. reincubation in calcein-free medium, fluorescence measurements of calcein were
performed using the 488 nm excitation of an argon ion laser (Innova 90, Coherent, Santa
Clara, USA). For membrane staining, cells were incubated with 6-dodecanoyl-2dimethylamino-naphthalene (laurdan; 4 µM, 40 min). The blue-green fluorescence (with
9
maxima around 440 nm and 490 nm, as reported by Parasassi et al., 1990) was evaluated
using the 364 nm excitation of the same argon ion laser.
3.2 Compact device for VA-TIRFM
In preliminary TIRFM experiments (Strauss et al., 1998, Sailer et al., 2000) a cube-shaped
prism was used for evanescent field illumination. However, this setup had the inherent
disadvantage that the exciting laser beam was displaced on the sample surface when the angle
of incidence  was varied. To overcome this problem, the cubic prism was replaced by a
prism which together with the microscope object slide (thickness: 1 mm) formed a hemicylinder (radius: 10 mm) with the sample (illuminated cells) being located in its center. As
shown in Figure 2, the light spot on the adjustable mirror was imaged on the sample using a
concave focusing mirror (focal length f = 50 mm) and two deflection mirrors, such that the
illuminating beam was folded twice. The adjustable mirror was illuminated by a parallel
collimated beam. When this mirror was rotated, a well defined part of the sample could be
illuminated under variable angles  ranging from 60° to 80°. For  > C, the penetration
depth of the evanescent wave could thus be varied. To rotate the mirror, a lever was moved
sinusoidally by rotation of an oblique cylinder mounted on the axis of a step motor. This step
motor was controlled by a personal computer with a resolution of 0.15° per step. For the total
imaging system (including the concave mirror and the prism) an amplification factor of 0.6
was selected, such that the illuminating light spot corresponded to an ellipse with a minor axis
of 400 µm and a major axis of more than 1 mm (dependent on ). The reflected beam was
absorbed by a light trap.
Excitation light was usually coupled to the device using a monomode quartz fiber in
combination with a collimating lens at its exit in order to obtain a parallel beam of 0.7 mm
diameter. Fiber tip and lens were fixed within a cylindrical unit, which could be attached to
the device and adjusted in x,y direction by two pairs of screws. Fibers and their collimating
units could be easily changed when different light sources were required. So far, the argon ion
laser mentioned above was used with different monomode fibers for visible (458 nm-514 nm)
and ultraviolet (364 nm) excitation light. Polarization of the incident light could be varied by
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rotation of the (polarization maintaining) fiber. So far, an electric field vector oriented
perpendicular to the plane of incidence has been applied in TIR experiments.
For calibration of the angle of incidence , the hemi-cylindrical prism was optically coupled
to a rectangular prism. In this case, the laser beam was no longer totally reflected and could be
imaged on a screen to determine  and its divergence  by triangulation.  was found to
be + 0.25°. The critical angle C for total internal reflection of a 488 nm laser beam on a
glass / water surface was used for calibration of the setup prior to each measurement. Using
the refractive indices of 1.522 and 1.337 for BK7 glass and water at 488 nm, respectively, a
value C = 61.435° was calculated. The corresponding critical angle at which total internal
reflection was observed in the experiment, was 61.435° + 0.25° = 61.685°.
The VA-TIRFM device was attached to an upright microscope (Axioplan1, Carl Zeiss Jena,
Germany) replacing the standard condensor.
3.3 Fluorescence detection
Fluorescence excited by TIR illumination was detected using a 40 /0.75 objective lens and
appropriate long pass filters (calcein:  > 515 nm; laurdan:  > 410 nm). For additional
experiments under epi-illumination a mercury high pressure lamp (HBO 50) was used in
combination with an excitation filter (calcein: 450-490 nm; laurdan: 365 + 5 nm), a dichroic
beam splitter (calcein: 510 nm; laurdan: 395 nm), and a long pass filter (s. above).
Fluorescence images were recorded by a digital slow scan CCD camera with thermoelectrical
cooling and a 16 bit A/D converter (TE / CCD-512EFT, Princeton Instruments, München,
Germany). For each image, background fluorescence was determined by measurements in 25
spots outside the cells and fitting of a two-dimensional Gaussian function according to the
Equations 6 and 7. Smoothing of low intensity images (laurdan) resulted from the operation
I(x,y) = 0.5 [IM (x,y) + 1/8  IN ]
(8),
where IM (x,y) corresponds to the measured fluorescence intensity of the pixel (x,y), and IN to
the intensities of the 8 adjacent pixels. For detection of fluorescence intensities without spatial
11
resolution (calcein solutions) the CCD camera was replaced by a polychromator in
combination with an image intensifier and a diode array (IMD 4562, Hamamatsu Photonics,
Ichino-Cho, Japan). This device, which also permits the detection of fluorescence spectra, is
described in detail elsewhere (Schneckenburger et al., 1998). The background of experiments
with calcein solutions was determined using object slides with a droplet of water.
3.4 Mathematical modeling
Cell-substrate topologies were determined for BKEz-7 endothelial cells, which were loaded
either with the cytoplasmic marker calcein or with the membrane marker laurdan. In the case
of calcein, a homogeneous distribution in the cytoplasmic portion close to the plasma
membrane was supposed. For laurdan, a homogeneous distribution within the plasma
membrane was assumed. Distances  between the cell surface and the glass substrate were
determined from VA-TIRFM experiments using the four layer model (Gingell et al., 1987)
and a non-linear regression based on the Levenberg-Marquardt algorithm (Press et al., 1989).
In addition, computer simulations on the angular dependence of fluorescence intensity were
performed for variable distances  between the substrate and the plasma membrane using the
four layer model as well as the three layer model (omitting nm of the plasma membrane) and
the two layer model (omitting nm of the plasma membrane and ne of the extracellular
medium). Refractive indices ns = 1.522, ne = 1.337, nm = 1.45 and nc = 1.37 were used for the
cytoplasmic marker calcein (excitation wavelength: 488 nm), whereas the values ns = 1.535,
ne = 1.347, nm = 1.45 and nc = 1.38 were used for the membrane marker laurdan (excitation
wavelength: 364 nm). Again, the non-linear regression algorithm was used for the four layer
and the three layer model, whereas an analytical solution according to Equation 5 was
obtained for the two layer model. In addition, fluorescence intensities obtained from a
Gaussian shaped profile of the laser source (with an 1/e² radius of 175 µm) were compared
with the fluorescence intensities which would result from homogenous illumination.
Deviations were determined for distances of 50 µm, 100 µm and 150 µm from the center of
the illuminating ellipse (along its major axis).
4. Results
4.1 Solutions
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Measurements of aqueous calcein solutions were performed at concentrations ranging from
1 µM to 100 µM. When using the excitation wavelength of 488 nm, a broad emission band
with a maximum around 510 -515 nm was observed. The experimental values of the
fluorescence intensity IF (measured in the maximum) as a function of the angle of incidence
, are depicted in Figure 3 for calcein concentrations up to 25 µM and 62.5° <  < 80°. The
experimental fits according to the two layer model with a continuous distribution of
fluorophores are also shown (Fig. 3). The fitting curves are in a good agreement with the
experimental values.
The shape of the emission spectra did not change with calcein
concentration, and fluorescence intensities were proportional to concentrations of up to
100 µM for all angles of incidence with a regression coefficient R² > 0.996. This linearity is
exemplified in the inlay of Figure 3 for  = 65.3° and 1 µM < c < 25 µM.
4.2 Cell-substrate topology
The fluorescence of BKEz-7 endothelial cells incubated with calcein-AM is depicted in
Figure 4. Cells illuminated under total internal reflection (TIR) at  = 68° or 72° (using
488 nm laser excitation) are shown in the upper part of the figure, whereas cells exposed to
epi-illumination (by the Hg lamp at 450-490 nm) are shown in its lower part (left). During
epi-illumination, diffuse fluorescence arose from the whole cells with brightest signals around
the cell nucleus (thickest part of the cells). In contrast, fluorescent spots or fluorescent stripes
were mainly detected during TIR illumination. With increasing angle of incidence, decreased
fluorescence intensity and reduced extension of these fluorescent structures was observed. The
distances , as calculated from the four layer model (using the Gaussian shaped profile of
illumination), are depicted in the lower part (right) of Figure 4. They varied between 0 and
30 nm (bright fluorescent spots), indicating the presence of very tight cell-substrate contacts,
which probably represent focal contacts. Typical lateral distances between different focal
contacts were found to be 4-6 µm. Close adhesion was also found at the cell borders. Cellsubstrate distances for the other parts of the cells varied between 100 nm and more than
300 nm. A profile of cell-substrate distances along the line “A” is depicted in Figure 6 (upper
part).
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The fluorescence of BKEz-7 endothelial cells incubated with the membrane marker laurdan is
depicted in Figure. 5. Cells illuminated under TIR at  = 68° or 73° (using 364 nm laser
excitation) are again shown in the upper part, whereas cells exposed to epi-illumination (by
the Hg lamp at 365 nm) are shown in the lower part (left) of the figure. During epiillumination fluorescence arose from the whole cells indicating some accumulation of laurdan
also in intracellular membranes. Bright fluorescent areas in close proximity to the non- or only
weakly fluorescing cell nucleus may originate from laurdan in the endoplasmatic reticulum or
the Golgi apparatus. In contrast, fluorescence images obtained during TIR-illumination were
dominated by fluorescent spots and stripes originating from the plasma membrane. The
distances  between the plasma membrane and the glass substrate were calculated according
to the four layer model (with the fluorophore being located within one thin layer) and depicted
in the lower part of Figure 5 (right). Similar results as for the cytoplasmic marker calcein were
obtained:  was 0-30 nm within the tight cell-substrate contacts (probably focal contacts) and
varied between 100 nm and more than 300 nm in other parts of the cells. A profile of cellsubstrate distances along the line “B” is depicted in Figure 6 (lower part).
4.3 Computer simulations
Computer simulations were performed to calculate fluorescence intensities as a function of the
angle of incidence  and the distance  between the glass substrate and the plasma
membrane. If the fluorescence intensities calculated for the four layer model were used for
computer fits according to the three layer model or to the two layer model, some deviations of
 were obtained. At cell-substrate distances  < 200 nm and angles of incidence
66° <  < 73°, deviations of  were within + 6 nm when using the three layer model and
between - 15 nm and + 7 nm when using the two layer model. Deviations of  which would
result, if a homogenous light spot were used instead of a Gaussian shaped illumination profile
were within + 3 nm at a distance from the beam axis x = 50 µm, within + 8 nm at
x = 100 µm and within + 25 nm at x = 150 µm. In the presented experiments the measured
field was limited to 192 µm  192 µm, such that the maximum distance from the beam axis
was 96 µm.
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5. Discussion
A compact illumination device for total internal reflection fluorescence microscopy was
developed, which permits a variation of the angle of the incident light in steps of about 0.15°,
and therefore an axial resolution in the nanometer range. Due to its miniaturization this device
is so far unique and can be attached to an upright microscope replacing the standard
condensor. It has been adapted to a Zeiss Axioplan microscope, and with some minor
mechanical modifications it can be combined with other types of microscopes. However,
when TIRFM is combined with transillumination microscopy (e.g. for adjusting the samples),
the quality of the transmitted beam is rather poor, since part of this beam is shaded off and
Köhler’s illumination is becoming impossible. To overcome this problem, the hemicylindrical prism depicted in Figure 2 is presently replaced by a hemi-spherical prism which
together with a further collimating lens permits Köhler’s illumination. By moving the
collimating optics and the deflection mirrors aside, high contrast transillumination (and phase
contrast) microscopy can be combined with TIRFM in future experiments.
The present illumination device can be combined with any type of microscope objective lens.
In addition to its high angular resolution, this is a further advantage over prismless (objective
type) TIRFM, which is limited to high aperture and high magification lenses and therefore to
the detection of small object fields (typically below 100 µm diameter). In comparison with a
setup for VA-TIRFM using several scanning mirrors (Ölveczky et al., 1997), the presented
illumination device requires only one scanning mirror, which enables a fairly compact design.
In addition, by using a mirror-based optical system, chromatic aberration is excluded, and
different excitation wavelengths can be used. Disadvantages of further VA-TIRFM setups
using an acousto-optic modulator and telecentric lense optics with wavelength dependent
deflection angles and varying image planes (Oheim et al., 1999; Rohrbach, 2000) are also
avoided. Furthermore, fiber-based laser light delivery allows a simple change between
different laser systems even in different locations, thus minimizing electromagnetic
disturbancies and improving laser safety.
For variable angles of incidence, i.e. for variable penetration depths of the evanescent wave,
fluorophores can be determined quantitatively by TIR microscopy, as shown in Figure 3. In
15
addition, the angular dependence of the TIRFM signal due to fluorescent markers of either the
cytoplasm or the plasma membrane can be used for mapping the cell-substrate topology, as
depicted in the Figures 4 and 5. Comparable cell substrate distances were measured after
staining the cytoplasm (calcein) or the plasma membrane (laurdan). Cell substrate distances
around 10 nm are typical for focal contacts (Izzard & Lochner, 1976; Ölveczky et al., 1997).
However, major parts of the cell membrane show distances between 100 nm more than
300 nm from the substrate. In the case of laurdan, blurring could not be avoided due to low
intensity images. A four layer model with distinct refraction indices for the glass substrate, the
extracellular medium, the plasma membrane and the cytoplasm is most suitable to determine
the distances between the glass substrate and the plasma membrane using a non-linear
regression algorithm. When using a three layer model by neglecting the refractive index of the
plasma membrane solely the formulas are simplified, but nonlinear regression is still
necessary for calculation of the cell-substrate distances (Reichert and Truskey, 1990). An
analytical solution for a membrane or a cytoplasm marker is deduced from Equation 5 when
using a simplified two layer model. This model may be justified under certain experimental
conditions ( < 200 nm;  < 73°), if a high accuracy of cell-substrate distances  is not
needed. Comparative simulations for a fluorescent cytoplasm marker using the four-layermodel and the two layer model (refractive indices of the substrate and the cytoplasm) show
deviations below  15 nm for distances  < 400 nm (Ölveczky et al., 1997). Also, deviations
of , which would result, if a homogenous light spot were used instead of a Gaussian shaped
illumination profile can be kept small, if the evaluated object field is small in comparison with
the beam diameter. In our case of a 192 µm  192 µm object field, these deviations would be
less than  8 nm, i.e. for an average distance  = 100-150 nm less than 5-10 %. It should be
emphasized that to our knowledge the four layer model as well as the Gaussian shaped profile
of the illuminating laser beam were used for the first time to evaluate two-dimensional
TIRFM images.
So far, light scattering was not considered in our theoretical model. Scattered light may arise
from focal contacts where the membrane curvature is rather high, penetrate deeply into the
sample and induce some background of the fluorescence intensity IF () measured at variable
angle. However, when fitting the experimental curves IF (), this background was negligible,
indicating that the impact of scattering was rather small.
16
Further microscopic techniques allowing to examine cell-substrate contacts and cell-substrate
distances of living cells include interference reflection microscopy (IRM) (Izzard & Lochner,
1976; Gingell & Todd, 1979), surface plasmon resonance microscopy (Giebel et al., 1999)
and fluorescence interference contrast microscopy (FLIC, Iwanaga et al., 2001). However,
these methods exhibit some inherent difficulties or are only applicable to cells grown on
special substrates or substrate coatings.
In comparison with existing TIRFM equipments, the presented compact illumination device
enables the variation of the angle of incidence  more easily, more precisely and over a larger
range. This is useful for measurements of cell-substrate topology, e.g. in experiments on cell
growth on various substrates, in measurements on biocompatibility or in pharmacology (with
pharmaceutics affecting membrane properties). In addition, experiments on endocytosis or
exocytosis, as well as measurements of ion fluxes through membranes can be performed with
high axial resolution. TIRFM can also be combined with further innovative methods, e.g.
time-resolved fluorescence spectroscopy (Schneckenburger et al., 1998; Sailer et al., 2001),
fluorescence lifetime imaging (FLIM; Szmacinski et al., 1994; Sanders et al., 1995; Bastiaens
& Squire, 1999; Murata et al., 2000) or fluorescence resonance energy transfer (FRET;
Mahajan et al., 1998; Schneckenburger et al., 2000; Harpur et al., 2001). These measurements
may be performed even on a single molecule level when thin layers are illuminated in TIRFM
experiments.
ACKNOWLEDGMENTS
The authors thank Prof. K.-F. Klein, Fachhochschule Gießen-Friedberg, for providing a uvtransmitting monomode fiber and C. Hintze, Fachhochschule Aalen, for technical assistance.
The project was supported by the Bundesministerium für Bildung und Forschung (BMBF),
grants no. 1706698 and 13N7514.
17
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Figure Captions
Fig.1. Total internal reflection fluorescence excitation using an evanescent electromagnetic
field. The refractive indices of the substrate (ns), extracellular medium (ne) and
cytoplasm (nc) as well as the average distance  between the reflecting surface and the
plasma membrane are indicated.
Fig. 2. Compact illumination device for variable-angle total internal reflection fluorescence
microscopy (VA-TIRFM) in original scale.
Fig. 3. Angular dependence of the fluorescence intensity of aqueous calcein solutions at
concentrations of 1, 5, 10, and 25 µM in TIRFM experiments (after correction for
variations in the spot size of illumination). Inlay: Fluorescence intensity of calcein as a
function of its concentration measured at  = 65.3°.
Fig. 4. Fluorescence images of BKEz-7 endothelial cells after incubation with the cytoplasm
marker calcein-AM (5 µM; 40 min.) using TIR illumination at 488 nm (upper part) or
epi-illumination at 450-490 nm (lower part, left). Cell-substrate topology as deduced
from VA-TIRFM measurements using the four layer model (ns = 1.522, ne = 1.337,
nm = 1.45, nc = 1.37) are depicted in the lower part (right). “A” indicates the region of
the line scan depicted in Figure 6.
Fig. 5. Fluorescence images of BKEz-7 endothelial cells after incubation with the membrane
marker laurdan (4 µM; 40 min.) using TIR illumination at 364 nm (upper part) or
epi-illumination at 365 nm (lower part, left). Cell-substrate topology as deduced from
VA-TIRFM measurements using the four layer model model (ns = 1.535, ne = 1.347,
nm = 1.45, nc = 1.38) are depicted in the lower part (right). “B” indicates the region of
the line scan depicted in Figure 6.
Fig. 6. Line scans of cell substrate distances of BKEz-7 endothelial cells after incubation with
the cytoplasm marker calcein-AM (A) or the membrane marker laurdan (B).
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6