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Fiber Optics
Broadband Polarization-maintaining
­Fiber Optics
RGBV beam combiner enables TIRF microscopy of motor proteins
Anja Knigge, Ulrich Oechnser, Bernhard Brenner, and Tim Scholz
Modern fluorescence microscopy allows the fundamental building blocks
of all life to be studied at a level of detail and accuracy that would have been
inconceivable just a few years ago. One
example is the movement of intracellular motor proteins responsible for
muscle contraction or for the transport of biological cargo along microtubules within the cell. To study these
systems using fluorescence microscopy, laser light over a wide range of
wavelengths is required that has to be
coupled precisely into the microscope.
With free-beam optics, this can only
be done to a limited extent and at considerable cost.
The effective solution is to couple the
laser beam sources of the desired wavelengths into a single polarization-maintaining (PM) singlemode fiber using
special apochromatic optics and dichroic multiplexing (Fig. 1) for the wavelength range 400 – 660 nm.
Using fiber optics to deliver the laser
light has several advantages. The light
sources and the measurement system
are allowed to be physically separated,
so that they are mechanically and thermally decoupled, preventing any mutual
disturbance. The light guidance itself is
totally enclosed resulting in a lower laser safety class, is insensitive to vibrations and exhibits long-term stability
in terms of temperature fluctuations.
In many cases, a measurement setup already equipped with laser beam sources
can simply be upgraded to fiber optics,
bringing a significant increase in stability and convenience without the need
for costly and tedious adjustment of
mechanically unstable, free-beam components.
Fig. 1 An RGBV beam combiner combines the radiation of four different lasers sources
(405 nm, 488 nm, 514 nm and 635 nm) into one single polarization-maintaning fiber. The
resulting light is then used to investigate the movement of e.g. motor proteins using TIRF
microsocopy.
TIRF microscopy of individual
motor proteins
In conventional studies using large ensembles of molecules, many crucial
functional parameters are not accessible
or could be masked by ensemble averaging. Thus, to characterize motor protein
function or pathological malfunction
properly it is important to study motor
proteins at the single molecule level.
This is done in the group of Prof. Brenner at the Hannover Medical School
(MHH) using total internal reflection
fluorescence (TIRF) microscopy (also
known as evanescent field microscopy).
The technique allows the simultaneous
detection of motor proteins like fluorophore-labelled kinesin, dynein or myosin along with fluorescently labelled
ATP molecules or molecules associated
with the cytoskeleton, such as the Alzheimer-related protein Tau at the single
molecule level (Fig. 1). Fluorophores are
molecules that emit light in response to
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
a defined excitation. The TIRF microscopy utilizes the optical principle of total
reflection which is described in detail in
the box (p. 20).
RGBV fiber optic components for
the TIRF analysis
For multicomponent experiments characteristic excitation wavelengths are
needed for different individual fluorophores, so an ideal light source consists
of several narrow-band excitation wavelengths that can be switched individually.
This can be realized by using different
laser diode beam sources, each coupled
into an individual PM fiber using laser
beam couplers. If a low noise laser source
is needed for the experiment, then fibercoupled laser sources of the type 51nano
are used. These are special low noise
laser sources (< 0.1 % RMS, < 1 MHz)
with a reduced coherence length and a
low speckle contrast. Laser source qualOptik&Photonik
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Dichroic
beam
combiner
TIRF Microscope
RGBV
Beam Combiner Unit
Interface to application:
ity (especially high pointing stability and
good polarization characteristics) as well
as the use of high quality polarizationmaintaining fibers are essential (here
with a polarization extinction ratio of
more than 32 dB measured at 405 nm),
as the efficiency of the following components are polarization sensitive.
OUT
PMC
20
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51Nano
e.g. 488 nm
51Nano
e.g. 635 nm
RGBV
60SMS
RGBV
60SMS
60SMS
PMC
Laser
beam coupler
51Nano
60SMS
e.g. 514 nm
PMC
51Nano
e.g. 405 nm
Fig. 2 Optical Scheme of the RGBV laser beam combiner using dichroic multiplexing
and laser beam couplers 60SMS. The combined laser light is then coupled into a broadband PM fiber using a laser beam coupler equipped with an apochromatic lens (RGBV
60SMS).
RGBV beam combiner unit
The transmission of the dichroic mirrors (here configured as long-pass filters) determines which wavelengths
can be combined. Fig. 2 shows an optical scheme of the dichroic multiplexing
using dichroic mirrors which have been
adjusted to the nominal wavelengths
(here e.g. 405 nm, 488 nm, 514 nm and
635 nm). The first dichroic reflects the
green light and allows the red light to
pass with high transmission. The following two dichroics reflect the blue
TIRF Microscopy
TIRF microscopy (optical scheme see
Fig. 3) utilizes the optical principle of total
reflection. An aqueous solution containing
the fluorescently labeled sample proteins
is applied to a cover glass of a reaction
chamber and mounted onto an objective
lens. In an objective-type TIRF microscope,
the light of suitable excitation lasers (1) is
focused off-axis into the back focal plane
of the objective lens such that it is sufficiently inclined to the glass-water interface
to become totally reflected. To achieve an
inclination angle shallow enough to cause
total reflection, an objective lens with a
sufficiently large numerical aperture (NA >
1.33) has to be used. Total internal reflection at the glass-water interface generates
an evanescent field, i.e., an electromagnetic
field of exponentially decaying intensity in
the aqueous solution adjacent to the glasswater interface. The exponentially decaying
evanescent field penetrates the sample solution to only approximately 100 – 200 nm.
Excitation of fluorophores attached to the
molecules of interest is thus limited to a very
thin layer at the glass-water interface. This
60SMS
RGBV PMC
Broadband polarization-maintaining fibers with end caps
For laser sources with larger powers
and especially for smaller wavelengths,
the PM fibers are equipped with end
caps. End caps lead to an increase in the
mode field diameter at the fiber endface which reduces the power density at
the fiber-air interface significantly. This
prevents damage to the fiber from e.g.
scorching or photocontamination.
The different input wavelengths introduced through the PM fibers are
combined using an RGBV Beam Combiner (Fig. 2), which means that they are
first collimated using standard laser
beam couplers (60SMS), combined using dichroic mirrors and the resultant
beam is finally coupled into one collective single broadband polarizationmaintaining fiber, designed for wavelengths 400 – 660 nm.
PMC
Aqueous solution
cover slip
Immersion oil
1
2
Back focal plane
Excitation
Light trap
Fig. 3 Optical Scheme of the excitation path
in a TIRF microscope
limitation results in a dramatic reduction of
diffuse background fluorescence as fluorophores deeper in solution do not get excited
and therefore do not emit light. The dramatically reduced fluorescence background then
facilitates the detection of individual fluorophores (2) which would otherwise disappear
in conventional fluorescence microscopy.
and violet light respectively, while the
combined wavelengths are transmitted
with high efficiency. A precise adjustment of the dichroic mirrors is required
for the particular spectral range and
wavelength configuration to be combined. The polarization dependency
determines that the polarization of
the input laser beams must be clearly
defined, which makes the use of PM
fibers essential for a stable beam combination.
The combined laser light is then coupled into the PM fiber using a laser beam
coupler equipped with an apochromatic
lens. This lens was specially designed to
minimize the chromatic focal shift for
the wavelength range 400 – 660 nm as
well as for spherical aberration, so that
a highly efficient fiber-coupling can
be achieved over the total wavelength
range. The resulting combined laser
light is then coupled into, for example,
the fluorescence microscope.
The combiner unit is an example of
a so called fiber port cluster. These are
compact opto-mechanical units that
split the radiation from one or more polarization-maintaining (PM) fibers into
one or multiple output polarizationmaintaining fiber cables with high efficiency and variable splitting ratio. The
beam delivery system consists of compact, modular opto-mechanic units.
The modularity ensures that almost any
desired system can be assembled and
provides stable and compact, as well as
sealed, beam delivery units.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Fiber Optics
optics is required, especially in the ultraviolet range.
Tests have shown that the standard
laser beam couplers (made from nickel
silver) have a power stability during
temperature cycling (15 – 35 °C) with
a typical maximum deviation of only
3 %, in a test setup with a focal length of
4.5 mm at 405 nm [1].
Standard fiber port clusters made up
with modular aluminum cluster units
and standard nickel silver laser beam
couplers, have already been used e.g.
in zero-G experiments and are already
very stable and resilient against outside
influences.
The RGB beam combiner unit or
any fiber port cluster can be further enhanced by using titanium components.
Titanium has the highest strengthto-weight ratio of any metal and a low
coefficient of expansion. It is corrosion
resistant and also amagnetic, so that titanium components can be used in environments that require very defined
magnetic fields.
Stable fiber coupling at short
wavelengths
In the RGBV Laser Beam Combiner,
the light is collimated and coupled into
the final fiber using standard laser beam
couplers, The laser beam coupler is a
fundamental component of a fiber port
cluster or in this case the beam combiner
unit. The stability and efficiency of the
total beam delivery is directly attributable to the stability of the laser beam
coupler.
When coupling back into the polarization-maintaining fibers, the
laser beam couplers produce a diffraction-limited spot that matches the mode
field diameter and the numerical aperture of the fiber. It is only when this
condition is met that fiber coupling with
high coupling efficiencies of up to 80 %
are achieved. Typical mode field diameters range from 3 µm (405 nm, NA 0.12)
to 5 µm (780 nm, NA 0.12). The required
pointing stability of the laser beam coupler can be visualized with an example:
For a focal length of 5 mm, an angular
misalignment of the coupler of a mere
0.2 mrad (0.01°) would result in a lateral
displacement between the laser spot and
the mode field of the fiber of 1 µm. A displacement only of 0.4 µm at λ = 400 nm
and NA 0.12 is enough to decrease coupling efficiency by as much as 10 %. Thus,
for high coupling efficiencies and longterm stability, a sub-micron precision
and pointing stability of the coupling
Functional analysis of motor proteins
Using the RGBV setup in combination
with TIRF microscopy, individual motor proteins can be characterized. For
localization of individual molecules,
including their movement over time
(Fig. 4), fluorophores are attached to
the proteins. The emitted fluorescence
a
b
signal, which is shifted towards longer
wavelengths, is collected by the same objective lens used to generate the evanescent field (see box). For an experimental
arrangement comprised of several different molecules of interest, each component has to be labelled with a distinct
fluorophore (Fig. 4a). Therefore, several
individually switchable, characteristic
excitation wavelengths of tunable intensities are needed to excite different
individual fluorophores in a defined
sequence, one at a time.. This challenge
of exciting and detecting individual
fluorescently labelled molecules leads to
the problem of photo-bleaching, since
bleaching of a fluorophore causes an immediate loss of signal. Photo-bleaching
can be reduced by a biochemical enzymatic approach to deplete reactive oxygen from the sample solution. To further
reduce bleaching pulsed excitation can
be used. Pulsed excitation has been reported to reduce photo-bleaching as
well as to increase fluorophore survival
and fluorescence yield [2]. Direct modulation of the laser diode sources or using
switchable elements such as AOTFs or
AOMs allows generation of fast excitation pulses of different wavelengths,
which permits intermitted relaxation
of fluorophores from vulnerable excited
states to their ground states.
The TIRFM technique offers an experimental way to directly visualize individual kinesin molecules moving along
their microtubule tracks [3, 4]. Current
d
c
f
e
E
Fig. 4 Experimental arrangements and results of single-molecule TIRF microscopy experiments on motor protein functions.
a) Scheme of a kinesin motor molecule (blue) moving on an immobilized microtubule (red-blue) in the presence of Tau molecules (green).
b) Kymograph (time-vs-position plot) of kinesin molecules (blue) moving on an immobilized microtubule (red) in the presence of diffusing Tau molecules (green). c) Kymograph of diffusing Tau molecules (green). d) Scheme of a single-molecule ATPase measurement using
immobilized myosin (grey) and Cy3-labelled ATP molecules (green). e) TIRFM image of fluorescently labelled ATP molecules bound to
surface immobilized myosin molecules (grey and white spots). f) Time course of Cy3-ATP fluorescence (bright white spot in e)) at one
individual myosin molecule. (Data in e, f: H. E. Huhnt, MHH)
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Company
Schäfter+Kirchhoff
Hamburg, Germany
Schäfter+Kirchhoff has accumulated substantial
experience in the development of optomechanical and optoelectronic systems for use in
research, aviation and in space, as well as for
demanding medical and industrial applications.
Schäfter+Kirchhoff designs and manufactures
their own CCD line scan camera systems, laser
sources, beam-shaping optics and fiber-optic
components, including laser beam couplers,
fiber collimators and fiber port clusters for customers worldwide.
www.sukhamburg.com
models of Alzheimer disease regard increased levels of the microtubule-associated protein Tau as possible obstacles for
microtubule-dependent motor proteins,
such as kinesin and dynein, thus impairing long-haul axonal transport in neurons. To test this hypothesis, it was necessary to detect both motor proteins and
Tau molecules on microtubules simultaneously. Fig. 4b shows a time sequence of
TIRF images in a so-called kymograph –
a time-vs.-position plot. In a kymograph,
stationary molecules are represented by
vertical lines, while moving molecules
deviate to the left or right. Following the
experimental scheme shown in Fig. 4a,
green fluorescent protein (GFP) labelled
kinesin molecules (shown in blue, excitation at 488 nm) moved from left to
right along a Cy5-labelled microtubule
(shown in red, excitation at 633nm) in
the presence of the tetramethylrhodamine- (TMR-) labelled microtubule-
associated protein Tau (shown in green,
excitation at 514 nm). Over a period of
several seconds, directed movement of
multiple individual kinesin molecules
could be observed, whereas the green Tau
molecules did not behave as proposed by
current models and did not remain stationary bound (Fig. 4b). Tau protein dilution to the low picomolar range and the
tracking of individual Tau molecules by
TIRF microscopy led to the discovery of
Tau diffusion along microtubules [5, 6].
To study not only the mechanical action
of motor proteins but the biochemical
enzymatic ATPase activity at the single
molecule level one can make use of fluorescently labelled ATP molecules. Due
to the dramatically reduced background
fluorescence in the TIRFM arrangement
it is possible to detect individual fluorophores attached to ATP molecules. Such
labelled ATP molecules only become visible as fluorescence signals when they get
bound by surface-immobilized myosin
molecules (Fig. 4d, e). Following the time
course of ATP fluorescence at different
myosin molecules (Fig. 4f) allows studying characteristic differences among or
changes in the kinetic behaviour of individual myosin molecules even in mixed
populations of pathologically altered
and wild-type myosin or mixtures of different motor isoforms [7, 8].
Summary
The coupling of laser light into PM fibers
provides an opportunity for the efficient
use of laser applications in both research
and industry. After beam combination,
the resultant single beam can be used
as a light source for fluorescence microscopy, by creating a defined interface
between the light source and the microscope. Exceptionally sensitive measurement systems (e.g., when observing individual motor proteins) benefit from
the physical separation of beam combiner and measurement system (e.g.,
fluorescent microscope), while also
avoiding costly, time-consuming and
tedious beam adjustment. Optical and
mechanical components that meet the
highest technical standards enable the
creation of modular and compact fiber
optic systems with remarkable longterm stability.
DOI: 10.1002/opph.201500041
[1] Physics Best, April 2013, Wiley-VCH
[2] R. T. Borlinghaus: MRT letter: high speed
scanning has the potential to increase fluorescence yield and to reduce photobleaching, Microsc. Res. Tech. 69 (2006) 689-692
[3] A. Seitz et al.: Single-molecule investigation
of the interference between kinesin, tau and
MAP2c, Embo J. 21 (2002) 4896-4905
[4] A. Rump et al.: Myosin-1C associates with
microtubules and stabilizes the mitotic
spindle during cell division, J. Cell. Sci. 124
(2011) 2521-2528
[5] M. H. Hinrichs et al.: Tau protein diffuses
along the microtubule lattice, J. Biol. Chem.
287 (2012) 38559-38568
[6] T. Scholz et al.: Transport and diffusion of
Tau protein in neurons, Cell Mol. Life Sci.
71 (2014) 3139-3150.
[7] M. Amrute-Nayak et al.: Inorganic phosphate binds to the empty nucleotide binding pocket of conventional myosin II, J.
Biol. Chem. 283 (2008) 3773-3781
[8] M. Amrute-Nayak et al.: ATP turnover by
individual myosin molecules hints at two
conformers of the myosin active site, Proc.
Natl. Acad. Sci. USA, 111 (2014) 25362541.
Authors
Anja Knigge
studied
Physics at the
University of
Würzburg with
a focus on the
description of
ultrashort laser
pulses and
quantum control. She joined Schäfter+Kirchhoff
in 2011 and now works in optics
development.
Ulrich
Oechsner
studied
Physics before
completing
his docto­
ral thesis at
the Uni­versity
of Hamburg.
After research
in the fields of electrophysiology
and physiological optics, he joined
Schäfter+Kirchhoff in 2000. He
became managing director in 2015.
Bernhard
Brenner is the
head of the
Department
of Molecular
and Cell Phy­
siology at
the Hannover
Medical School
(MHH). He and
his group work on the function of
muscles and motor proteins of different kinds.
Tim Scholz
studied Bio­
chemistry at
the University
of Hannover.
Since his doctoral thesis at
the Hannover
Medical School
(MHH) he
works on the functional characterization of motor proteins at the
single molecule level.
Anja Knigge, Dr. Ulrich Oechsner, Schäfter + Kirchhoff GmbH, Kieler Str. 212, 22525 Hamburg, Germany, Tel.: +49 40 85 39 97-0, Fax: +49 40 85 39 97-79, [email protected],
www.SuKHamburg.com; Prof. Dr. Bernhard Brenner, Dr. Tim Scholz: Hannover Medical School (MHH), Department of Molecular- und Cellphysiology, Carl-Neuberg-Str. 1, 30625 Hannover,
[email protected], [email protected], Tel. +49 511 532 6396
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