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Grazing incidence to total internal reflection
fluorescence structured illumination microscopy
enabled by a prism telescope
HENNING ORTKRASS1*, GERD WIEBUSCH1, JASMIN SCHÜRSTEDT1,
KAROLINA SZAFRANSKA2, PETER MCCOURT2, AND THOMAS HUSER1
1 Biomolecular Photonics Research Group, Faculty of Physics, Bielefeld University, Bielefeld, Germany
2 Department of Medical Biology, UiT - The Arctic University of Norway, Tromsø, Norway
*hortkrass@physik.uni-bielefeld.de
Abstract: In super-resolution structured illumination microscopy (SR-SIM) the separation
between opposing laser spots in the back focal plane of the objective lens affects the pattern
periodicity, and, thus, the resulting spatial resolution. Here, we introduce a novel hexagonal
prism telescope which allows us to seamlessly change the separation between parallel laser
beams for 3 pairs of beams, simultaneously. Each end of the prism telescope is composed of 6
Littrow prisms, which are custom-ground so they can be grouped together in the form of a tight
hexagon. By changing the distance between the hexagons, the beam separation can be adjusted.
This allows us to easily control the position of opposing laser spots in the back focal plane and
seamlessly adjust the spatial frequency of the resulting interference pattern. This also enables
the seamless transition from 2D-SIM to total internal reflection fluorescence (TIRF) excitation
using objective lenses with high numerical aperture. In linear SR-SIM the highest spatial
resolution can be achieved for extreme TIRF angles. The prism telescope allows us to
investigate how the spatial resolution and contrast depend on the angle of incidence near, at,
and beyond the critical angle. We demonstrate this by imaging the cytoskeleton and plasma
membrane of liver sinusoidal endothelial cells, which have a characteristic morphology
consisting of thousands of small, transcellular pores that can only be observed by super-
resolution microscopy.
1. Introduction
Super-resolution structured illumination microscopy (SR-SIM) is a fluorescence-based
imaging method that, in its linear implementation, can overcome the diffraction limit by up to
a factor of two [1]. This is accomplished by exciting fluorescence in the sample via an
interference pattern with a high spatial frequency, which encodes sample information beyond
the diffraction limit into the passband of the modulation transfer function of the microscope
[2]. Even higher spatial resolution can be achieved by exploiting objective lenses with high
numerical aperture (NA) [3], and by further pushing the spatial frequency of the interference
pattern to higher values using counter-propagating evanescent waves generated by total internal
reflection fluorescence (TIRF) excitation [4–6]. Li et al. demonstrated that a spatial resolution
of down to 84 nm is possible by using a NA1.7 objective lens [3].
When fluorescence is excited by total internal reflection in super-resolution structured
illumination microscopy (TIRF-SIM), the interference pattern of standing evanescent waves is
created by focusing the interfering laser beams into the back focal plane (BFP) of a high NA
microscope objective lens at radii near the edge of the back aperture. In this case the beams
encounter the glass-sample interface at an angle beyond the angle of total internal reflection.
Near the critical angle, above which total internal reflection takes place, one can distinguish
three different special regions that describe how light behaves near the glass-sample interface.
For light rays impeding the glass-sample interface at angles near the critical angle, the
transmitted rays will adopt an angle very close to the glass-sample interface. This region is
called „grazing incidence“ and, when implemented in SR-SIM, it is called grazing incidence
structured illumination microscopy (GI-SIM) [7]. Rays that hit the air-sample interface at the
critical angle result in total internal reflection, hence, TIRF-SIM when implemented in SR-
SIM. Lastly, for rays that hit this interface at angles much larger than the critical angle we can
refer to as the „extreme TIRF“ region, resulting in extreme TIRF-SIM (eTIRF-SIM).
In current implementations of SR-SIM systems, the illumination pattern is often generated by
a spatial light modulator (SLM). These devices offer significant advantages in terms of
flexibility in generating the periodicity, angle and phase of the pattern and they can change
between patterns with very high speed [8]. Because of the finite pixel dimensions of the SLMs,
the patterns required for a specific type of illumination have to be carefully chosen for each
illumination wavelength. Finding the proper patterns to cover a wide range of SIM patterns that
generate illumination conditions for all wavelengths near the critical angle is difficult, if not
even impossible. And each modality will require the use of customized spatial filters that will
only let the first diffraction orders pass on to the microscope.
To overcome these issues, we have recently demonstrated a fiber-optics based SR-SIM system
[9,10]. Here, we show how a novel prism telescope allows us to seamlessly change the distance
between parallel laser beams focused to the BFP of a high NA objective lens. The radial
position of these laser foci in the BFP is directly related to the angle at which the beams will
intersect the glass-sample interface. By translating one part of the prism telescope, we can, thus,
seamlessly move from GI-SIM to TIRF-SIM to eTIRF-SIM. In this paper we describe the
construction and function of this prism telescope and we investigate how the spatial resolution
and the contrast changes as the illumination is moved from GI-SIM to eTIRF-SIM. We
demonstrate the versatility of this device by studying how the different conditions affect SR-
SIM images of liver sinusoidal endothelial cells, in particular their actin network and
transmembrane fenestrations, which are a characteristic morphological hallmark of these cells.
2. Results and discussion
2.1 Modified fiber-optic SIM microscope
All measurements were performed with a fiber-optics based SR-SIM microscope [10] that was
enhanced by a prism telescope as described in detail below. The interfering beams are launched
to free space from a hexagonal fiber array and focused into the BFP of the objective lens. A
custom-manufactured fiber collimator holds all fibers in a hexagonal pattern (angle between
pairs of beams: 60°), collimates the beams emitted from every fiber and refocuses the beams
individually through the prism telescope to the image plane of a 4
!
optical relay composed of
achromatic lenses (see Fig. 1). The beam foci are then projected by this optical relay into the
BFP of the objective lens. The objective lens is a 60x, 1.5NA (UPLAPO60XOHR, Olympus)
with a BFP diameter of approximately 9 mm. The fluorescence is epi-detected and imaged by
a Ploessl-type tube lens (Thorlabs Inc. ACT504-500-A, 2x) onto a scientific complementary
metal-oxide semiconductor camera (sCMOS, pco.edge 4.2).
2.2 Prism telescope
Between the hexagonal holder and the relay telescope we placed a custom-manufactured prism
telescope that allows us to continuously adjust the lateral beam position, which is otherwise
fixed by the hexagonal fiber holder, to a radius smaller than the radius of the BFP. This prism
telescope consists of identical pairs of 30°/60°/90° Littrow prisms (along the light propagation
axis, see Fig. 1d). At each end, the prisms are arranged in a hexagonal pattern (array) to allow
all beams emerging from the fiber collimator to be displaced evenly by the same amount and
Fig 1: 3D-rendering of a prism and the hexagonal prism telescope (a-d). a) Each prism is custom-ground
out of a commercial Littrow prism. b) The prisms are radially orientated in a hexagonal array and held in
a custom-manufactured aluminum frame. c) Side-view of the aluminum frame with prisms. d) Two
identical arrays are arranged on opposite ends along the optical axis to form a telescope, where the distance
between the prism holders can be varied by a piezo-driven translation stage (ELL17/M, Thorlabs Inc.). e)
Right hand side: this arrangement results in a variable, but symmetric beam displacement of the six off-
axis-beams. Left hand side: the beams propagating through the telescope are emitted by six single mode
fibers and refocused by a hexagonal fiber collimator array before passing through the prisms. Scale bar is
10 mm (a-e).
to redirect the beams parallel to the optical axis. As shown in Fig. 1a, each array consists of six
radially symmetrically arranged prisms. The side faces of the upper half of each prism were
custom-ground to a triangular shape to allow them to be placed in a tight hexagonal pattern (see
Fig. 1a). For each of the six off-axis beams, a first prism (at the front end of the telescope)
refracts the incoming beam towards the optical axis and a second prism (at the back end of the
telescope) refracts it back to paraxiality (see Fig. 1d). The lateral beam position is continuously
adjustable by changing the distance between the hexagonal prism arrays.
The two prism arrays are custom-ground out of uncoated Littrow dispersion prisms (Edmund
Optics Inc., #43-648) with a size of 12.7 x 12.7 x 22 mm³. They are cut from the sides by 30°
and the tip by 8° to allow them to be assembled very closely. The hole in the middle of the
prism array has a diameter of 3 mm and allows an on-axis beam to propagate unaffected. This
is useful for 3D-SIM or the alignment of consecutive optical elements. The minimum distance
between opposing beam pairs that can be achieved in the BFP is limited to 6.4 mm due to the
physical size and custom-manufacturing limitations of the prisms. The adjustable prism array
is mounted on stainless steel rods via ball guides and one of the prism holders is actuated by a
piezo driven translation stage (ELL17/M, Thorlabs Inc.). The motor and the mechanics allow
for a positioning resolution of the spot pattern of 30 µm in the BFP. It typically takes
approximately 100 ms to shift between beam displacements of less than 1 mm. See Fig. 1c for
a 3D CAD drawing of the fully assembled and motorized prism telescope.
Fig 2: Beam synthesis propagation and ray tracing simulation using the optics design software CodeV for
different optical configurations of the prism telescope. a) Without the prism telescope, the refocused beam
from the fiber is diffraction limited in the BFP. b) With an uncompensated prism telescope, significant
astigmatism occurs. c) By tilting the prisms by 8°, the astigmatism can be minimized. By axially shifting
the second prisms, the beam displacement can be seamlessly tuned. d) Image of the back focal plane of
the objective lens. This image is the superposition of three images with laser spot locations highlighted
by the colored ring diameters, which indicate the foci position for GI (red), TIRF (green) and the upper
TIRF limit (blue). The thin white cross and lines are generated by a Python script during the acquisition
and display of the camera frames and indicate the position of the optical axis (cross) and the lines along
which the spots should move in order to achieve the three SR-SIM angles. Scale bar is 100 µm (a-c) and
1 mm (d).
Since the prisms are passed by converging laser beams, astigmatism induced by the prisms has
to be taken into account. In the absence of the prism telescope, the beams are focused in the
excitation path of the SIM setup with a cone angle of 40 mrad (at
!
"!
intensity). This corresponds
to a focus spot diameter of 13 µm (see Fig. 2a). If the prisms are vertically oriented, such that
the beams enter the prism surface tilted by 30° in the Meridional plane, the focus diameter is
enlarged and deformed by astigmatism to a size of 40 µm (see Fig. 2b). To minimize the
astigmatism, the prisms are tilted by 8° in the Meridional plane. We confirmed that this angle
indeed best reduces astigmatism by simulating the effect with the optics design software CodeV
(Synopsis, Inc.) (see Figs. 2a-c). Due to this tilt, the beams enter the prisms through a 22° tilted
surface and exit them through a -8° tilted surface. The measured focus diameter is 15 µm (see
Fig. 2c and 2d). A diffraction limited focus diameter is necessary in order to obtain a
homogenous sinusoidal interference pattern in the sample plane. The low distortion of the
sinusoidal pattern ensures high modulation contrast over the entire field of view (FOV) of up
to 280 µm as well as highest SR-SIM image reconstruction quality without having to tile the
raw data.
The radial beam position depends linearly on the separation between the prisms. The beams are
tilted by 16.3° in the space between the prisms (see Fig. 1e). We confirmed that the paraxiality
of the beams as well as their azimuthal position after passing the prisms is maintained over the
full travel range of the prisms. This allows us to seamless tune the setup from generating a
coarse 2D-SIM pattern, through grazing incidence near the critical angle, and all the way to the
TIRF limit of the objective lens without reducing the quality of the sinusoidal interference
pattern. This is shown for three select spot patterns in the BFP in Fig. 2d. Here, the spot pattern
in the BFP for angles at the glass-sample interface of 63°, 72° and 81° were imaged and
overlaid. Colored circles (red: 63°, green: 72°, blue: 81°) indicate the rings in the BFP for the
corresponding angles. The thin white lines shown in the figure are generated by a Python script
during display of the BFP camera data and are used as guides to the eye during alignment. The
axial focus position varies for different beam distances in the BFP due to different optical path
lengths in the prism telescope. For beam distances corresponding to 2D-SIM and extreme
TIRF-SIM, the focus shifts axially by 0.5 mm in the BFP. This does, however, not distort the
excitation pattern significantly.
Since the angle of refraction in between the prisms slightly depends on the wavelength of the
excitation laser beams, the prism distance needs to be adjusted for each excitation wavelength
in order to maintain a certain radial beam position for the different wavelengths. For example,
for a wavelength of 647 nm the separation between the prism needs to be 1.3 mm larger than
for 488 nm to obtain the same separation between the beams.
2.3 Sample preparation
The first step of sample preparation is coating the #1.5 coverglass slides with fibronectin to
improve cell adhesion. To accomplish this, the 25 mm diameter cover glass slides are incubated
in fibronectin (0.2 mg ml-1 in Phosphate Buffered Saline (PBS)) for 45 minutes at room
temperature and are washed with PBS afterwards. The cryopreserved rat liver sinusoidal
endothelial cells (LSECs) are prepared as described previously [11]. The cells are thawed at
37°C until nearly all ice inside the vial is gone. The cells are gently pipetted to 24 ml of
prewarmed RPMI 1640 media. The cell suspension is centrifuged at 50g for 3 minutes to
remove any other remaining cell types after LSEC separation e.g. hepatocytes The supernatant
containing the LSECs is used for a second centrifugation step at 300g for 8 minutes. The cell
pellet is then resuspended in 10 ml fresh RPMI 1640 media. A volume of 1.5 ml of cell
suspension is added to each coated coverglass, resulting in a density of 100.000 cells per cm2.
After 1 hour in the incubator at 37°C and 5% CO2, the cells are washed with new media and
placed in the incubator for another 2 hours.
Subsequently, the LSECs are fixed with 4% paraformaldehyde in PBS for 10 minutes and
washed three times with PBS before staining. The membrane of the cells is stained with a 1:200
dilution of BioTracker 555 Orange Cytoplasmic Membrane Dye (SCT107, Sigma-Aldrich) in
PBS for 1 hour at room temperature. In addition, the actin cytoskeleton is stained with 1:40
Alexa Fluor 488 Phalloidin (A12379, ThermoFisher) in PBS for 2 hours at room temperature
without prior permeabilization. All SR-SIM experiments of the fixed and stained cells are
conducted in PBS.
2.4 Theoretical considerations about the resolution enhancement in TIRF-SIM
In SR-SIM the angle of incidence (AOI) at the glass (cover slip) - sample interface and the
resulting spatial frequency of the interference pattern is adjusted by shifting the radial position
of the beam foci in the back focal plane (BFP) of the objective lens. The radius of the beam
focus r in the BFP with regard to the Abbe sine condition is proportional to the sine of the angle
"
of the beam exiting the objective lens [12]:
# $ %&' "
.
The separation between two maxima of the interference pattern (pattern spacing) s in the sample
plane is given by:
( ) *
+,- %&' ".../
where
*
is the laser wavelength and NA is the numerical aperture of the objective lens. The
AOI
"
can therefore be calculated by the pattern frequency
0 ) !
#
obtained from the image:
" ) %&'$! *0
+,-
The critical angle
"%&'('%)*
for total internal reflection (TIR) is given by the ratio of the refractive
indices (RI) of the media comprising the interface. For cells, the refractive index of the plasma
membrane is between 1.46 and 1.54 [13], whereas the RI of the cytoplasm in living cells is
approximately 1.38 [14]. In fixed and mounted cells, the refractive index of the cytoplasm
changes due to the presence of the embedding medium to values between 1.33 and 1.38. Due
to the thickness of the cell’s plasma membrane, i.e. typically less than 13nm [15], the effect of
the membrane itself can be neglected [16]. In this case the TIR condition can be calculated by
the refractive index of the cover glass (
1!) 23425
) and the cytoplasm (
1+) 2366 7 2365
). The
critical angle is therefore:
"%&'('%)* )%&'$! 1+
1!
)823+9 7843:9
The maximum acceptance angle of the objective lens
",)-
is limited by the NA of the
objective lens (in our experiments a 60x Olympus UPLAPO60XOHR, NA= 1.50, is used):
",)- )%&'$! ."
/0
= 81.2°
The intensity of the evanescent field at the glass-cell interface for s-polarized beams also
varies depending on
"
and is [17]:
;1):<=#<> ?@% ">
2 A 1+
+
1!
+
where
=#
is the electric field amplitude of the s-polarized laser beam. The illumination intensity
thus decreases with increasing illumination angles as shown by the blue curve in Fig. 3a.
The theoretically achievable improvement in spatial resolution d by SR-SIM depends on the
Abbe resolution limit
B2"("%('3..
and the pattern spacing s:
B ) B2"("%('3.
(C 2
)
*", +,-
D
*"- +,- %&' "
D
C 2
)*", %&' "
*"-
C 2
For an excitation wavelength of
*"- ).
488 nm and a fluorescence emission wavelength
centered at
*", )
530 nm, the critical angle and the maximum acceptance angle of the objective
lens correspond to resolution improvements between d = 1.95 and d = 1.99, and up to d = 2.07.
The intensity of the evanescent field and the Abbe limit of the maximum spatial resolution that
can be obtained by linear SR-SIM are shown in Fig. 3a.
2.5 GI-SIM and TIRF-SIM imaging of biological samples
We next explored the ability of our prism telescope enhanced fiberSIM system to image
biological samples by GI-SIM and TIRF-SIM using different excitation pattern spacings.
Fluorescently stained actin filaments in fixed LSECs were imaged at 16 different angles near
the critical angle between the glass-sample interface ranging from 63.3° to 80.1°. Fig. 3b shows
a two-color SR-SIM image of LSECs, where actin filaments are displayed in blue and the
plasma membrane is overlayed in red. This image is an average of the 16 SR-SIM images that
were reconstructed for each color channel and, thus, contains information obtained from all
illumination angles. The image shows a doubly stained LSEC in full in the center of the image.
The membrane staining of the cells located on top and on the right side of the image is not
visible due to the contrast adjustments to emphasize the details visible in the central LSEC. The
difference in the intensity of the LSEC membrane staining using the BioTracker stain is not
fully understood. According to the manufacturer the dye is sensitive to the disruption of the cell
membrane which could have been affected by the individual cell viability prior to fixation,
fixation artefacts or the order of staining (the actin cytoskeleton was stained in the first step).
Fig. 3: a) The theoretical resolution limit for SR-SIM (using the Abbe condition) dependent on the
excitation angle (green curve); as well as the normalized TIRF and GI excitation intensity 50 nm above
the coverslip-sample interface (blue curve). b) TIRF-SIM image of rat liver sinusoidal endothelial cells
stained with phalloidin-AF 488 against actin and BioTracker 555 Orange against the plasma membrane (
Panel I). The cell imaged only partially on the right hand side did not take up any membrane stain. The
image is an average of 16 reconstructed images per channel, acquired with different GI and TIRF angles
between 63.3° and 80.1°. Panels II-IV show the actin cytoskeleton for GI excitation corresponding to an
angle of 64.5° (II) and TIRF excitation with angles of 68.4° (III), 75.2° (IV). Scale bar is 10 µm (Panel
I) and 1 µm (Panels II-IV).
The cell in the center exhibits several large gaps (several 100 nm to microns in diameter) in its
plasma membrane, but it also contains a significant number of small holes with diameters on
the order of 100 nm (some of which can be seen more clearly in the insets to Fig. 5). These
small holes are fenestrations, the main morphological hallmark of LSECs. As can be seen in
Fig. 3 and in more detail in Fig. 5, all of these transmembrane holes are surrounded by actin
fibers. Actin fibers also provide the main link of the circumference of the cells to the substrate.
In addition, several focal adhesions, where actin fibers inside the cell bundle up and connect
the cell to the substrate can also be identified. Panels II-IV in Fig. 3b show the actin
cytoskeleton for TIRF excitation corresponding to angles of 64.5° (GI-SIM, Panel II), 68.4°
(TIRF-SIM, Panel III) and 75.2° (eTIRF-SIM, Panel IV). The angles result in different
illumination pattern spacings as well as different decay lengths of the evanescent field in axial
direction, which explains why different parts of the actin cytoskeleton are visible in these
images, also noticeable in Fig. 5.
Next, we systematically evaluated the spatial resolution that can be obtained by the different
illumination angles for the actin channel (488 nm excitation wavelength). In order to obtain
quantitative information about the spatial resolution, two subsequently acquired SR-SIM
images taken at the same illumination angle were analyzed by Fourier ring correlation (FRC)
using an ImageJ plugin [18]. As can be seen from Fig. 4a, the spatial resolution improves from
100.8 nm for grazing incidence to 97.9 nm at the TIRF angle and reaches a plateau at 95.8 nm
for an eTIRF angle of 75°, corresponding to a resolution improvement of 2.07x over
conventional widefield fluorescence microscopy. For angles beyond this value, the signal-to-
noise ratio (SNR) of the images deteriorates due to the
E?@% FG+
dependence of the intensity of
Fig. 4: a) Spatial resolution obtained across the entire reconstructed image as measured by Fourier ring
correlation (FRC). The curve indicates that the lateral resolution continues to improve with increasing
illumination angles until the SNR limits the resolution, due to the simultaneously decreasing excitation
intensity. b) FRC curves for several select angles. The values at the 1/7 threshold are plotted in a). c)
Lineplots through several actin fibers lying in close proximity from images obtained at GI excitation angle
(63.3°, green), the TIRF angle (68.4°, orange) and an extreme TIRF angle (73.9°, blue).
the illumination, which causes the FRC-measured spatial resolution to increase, again. Fig. 4b
shows the FRC curves for several select angles. The experimentally determined spatial
resolution follows a similar curve as the theoretical prediction in Fig. 3a, and rapidly improves
with increasing angle. The resolution predicted by the Abbe criterion can, however, not be
reached experimentally. To determine if the resolution continues to improve beyond an angle
of approx. 72° would require to either adjust the excitation power or extend the acquisition time
for images taken at such extreme angles in order to improve their SNR.
We also evaluated qualitatively how the image contrast behaves for angles before and beyond
the critical angle. The result is shown in Fig. 4c in the form of cross sections through several
actin fibers that were taken from images consecutively obtained at a GI excitation angle (63.3°,
green), the TIRF angle (68.4°, orange) and an eTIRF angle (73.9°, blue). The cross sections are
taken at the same position in the reconstructed images and were normalized to the background.
As can be seen by this graph, while the overall signal to background level is similar for the
different angles, different actin fibers are emphasized at different angles and the overall signal-
to-background ratio continuously improves with increasing excitation angle.
To further demonstrate how spatial resolution and image contrast change with increasing
illumination angle, Fig. 5 shows select SR-SIM images of the rat LSECs shown in the overlay
in Fig. 3b for different GI and TIRF angles. The upper row shows the actin channel, while the
lower row shows the plasma membrane channel. The spatial resolution as determined by FRC
ranges from 103 nm - 98 nm for the actin channel and 115 nm - 111 nm for the membrane
channel. While contrast and spatial resolution do not visibly change between the different
plasma membrane images, a very different effect can be seen in the actin channel. Here,
depending on the illumination angle, different actin fibers begin to become visible, as is most
apparent by the change in brightness of the actin filaments in the outer circumference of the
cells. This can be best explained by changes in the penetration depth of the evanescent field
and this effect has previously been exploited in order to improve the axial resolution of TIRF-
SIM images, albeit so far only by varying the angle for TIRF illumination and not with the
corresponding SIM pattern [19].
4. Conclusion
We have introduced a novel prism-based telescope, which allows us to continuously vary the
excitation angle of SR-SIM illumination pattern. This is accomplished by translating the
separation between corresponding pairs of prisms along the optical axis. By using a piezo-
Fig. 5: TIRF-SIM image of the rat liver endothelial cells shown in Fig. 3 for different TIRF and GI angles.
The upper row shows the actin channel, while the lower row shows the plasma membrane channel. a) The
actin and membrane channels have a FRC resolution of 103 nm (actin) and 115 nm (membrane) for an
excitation angle of 64.5°, i.e. GI excitation. b) At the TIRF angle (68.4°) the FRC resolution is 98 nm
(actin) and 114nm (membrane). c) For the extreme TIRF angle of 75.2°, the FRC resolution is 99 nm
(actin) and 111nm (membrane). Scale bar is 10µm (1µm in section).
actuated translation stage, fine and continuous adjustments as well as rapid changes in the
separation of the resulting laser spots in the back focal plane of an objective lens can be
achieved. This enables us to continuously vary the SR-SIM excitation from objective-type
grazing incidence to TIRF-SIM excitation and allows us to reach extreme TIRF-SIM angles.
We have evaluated the spatial resolution and the changes in contrast provided by these different
illumination modalities and find that the spatial resolution continues to improve up to an
illumination angle of approx. 75°. Because of the simultaneously occurring decrease in the
illumination power at such extreme angles, the SNR for images obtained beyond an excitation
angle of 75° is so low, that the spatial resolution appears to decline, again. To compensate for
this effect, the excitation laser power or signal accumulation times would have to be adjusted,
which complicates the quantitative comparison of data acquired at these different angles.
Funding. Universitätsbibliothek Bielefeld (Open Access Publication Fund); European Regional Development Fund
(Fiber-SIM); European Union (101046928).
Acknowledgements. Funding for this project was provided by the European Regional Development Fund 2014 -
2020 programme "NRW-patent validation" under the project "Fiber-SIM". This project has also received funding from
the European Union’s European Innovation Council PATHFINDER Open Programme under grant agreement
No 101046928. We acknowledge support for publication costs by the Open Access Publication Fund of Bielefeld
University and the Deutsche Forschungsgemeinschaft (DFG)."
Disclosures. TH and GW submitted a patent application related to fiberSIM through their employer (Bielefeld
University). The other authors declare no conflicts of interest.
Data availability.
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the
authors upon reasonable request.
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