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MesoTIRF: a Total Internal Reflection Fluorescence illuminator for axial super-resolution membrane imaging at the mesoscale
MesoTIRF: a Total Internal Reflection Fluorescence illuminator for axial
super-resolution membrane imaging at the mesoscale
S. Foylan,1W. B. Amos,2J. Dempster,3L. Kölln,2, 4, 5 C. G. Hansen,4, 5 M. Shaw,6, 7 and G. McConnell1
1)University of Strathclyde, Department of Physics, Glasgow G4 0NG, UK
2)University of Strathclyde, Department of Physics, Glasgow, G4 0NG, UK
3)Strathclyde Institute for Pharmacy & Biomedical Sciences, University of Strathclyde, Glasgow, G4 0RE,
UK
4)University of Edinburgh, Centre for Inflammation Research, Edinburgh, EH16 4TJ, UK
5)University of Edinburgh, Institute for Regeneration and Repair, Edinburgh, EH16 4UU,
UK
6)Department of Chemical and Biological Sciences, National Physical Laboratory, Teddington, TW11 0LW,
UK
7)Department of Computer Science, University College London, London, WC1E 6BT, UK
(*Electronic mail: shannan.foylan@strath.ac.uk)
(Dated: 19 August 2022)
Total Internal Reflection Fluorescence (TIRF) illumination bypasses the axial diffraction limit of light by using an
evanescent field to excite fluorophores close to a sample substrate. TIRF illumination significantly improves image
contrast, allowing researchers to study membrane structure and dynamics with localized reductions in photobleaching.
However, a significant limitation of most TIRF microscopes is the relatively small field of view (FOV). TIRF objectives
require a high numerical aperture (NA) to generate the evanescent wave. Such lenses invariably have a high mag-
nification and result in a ∼50 µm diameter imaging field, requiring many subsequent images for accurate statistical
analysis. Waveguide and prism-based TIRF systems are, in principle, compatible with lower magnification lenses to
widen the FOV but these have a correspondingly low NA and lateral resolution. To overcome these limitations, we
present a prism-based TIRF illuminator for the Mesolens - a specialist objective lens with the unusual combination of
low magnification and high NA. This new imaging mode - MesoTIRF - enables TIRF imaging across a 4.4 mm x 3.0
mm FOV. We demonstrate evanescent wave illumination of cell specimens, and show the multi-wavelength capability
of the modality across more than 700 cells in a single image. MesoTIRF images have up to a 6-fold improvement
in signal-to-background ratio compared to widefield epi-fluorescence illumination, and we illustrate the benefit of this
improved contrast for the detection and quantification of focal adhesions in fixed cells. Fluorescence intensities and
resolvable structural detail do not vary considerably in homogeneity across the MesoTIRF FOV.
I. INTRODUCTION
Total Internal Reflection Fluorescence (TIRF) microscopy
is an established imaging technique in cell biology1. It
relies on delivering excitation light such that it is incident
on a refractive index boundary at the microscope specimen
plane at a super-critical angle. The subsequent Total Internal
Reflection (TIR) results in a rapidly decaying evanescent
field which penetrates to a depth on the order of several
hundred nanometres. Such illumination allows structure
below the axial diffraction limit to be visualized with high
contrast, while minimizing photobleaching of the specimen
by reducing the illumination volume. TIRF microscopy has
been used extensively in cell biology to image cell contacts1,
study cell adhesion2,3,4, ion channels5, endocytosis6and the
self-assembly of filamentous proteins7. Generating TIR at
the specimen plane can be achieved using a TIRF objec-
tive, a waveguide8, or a prism9. To support super-critical
illumination, TIRF objectives have a numerical aperture
(NA) between 1.45-1.5. However, due to their limited size,
these objective lenses have an associated high magnification,
typically 60x or 100x. As such, the field of view (FOV)
is restricted to around 50 µm x 50 µm and the microscope
can only capture a few cells in a single image10. Stitching
and tiling methods can be used to image larger specimen
areas, but these methods are time-consuming and routinely
introduce artefacts into the resultant image. Waveguide-based
TIRF obviates the need for a high NA objective lens allowing
images of up to 0.5 mm in diameter containing tens of cells
using custom-designed chips10,11 . Prism-based TIRF uses
off-the-shelf components and is therefore lower cost and
easier to implement than other methods, and, like waveguide
TIRF, is compatible with any objective lens. This potentially
allows for larger FOV imaging than possible with TIRF ob-
jectives. However, for both waveguide and prism-based TIRF,
the detection objective lens remains a fundamental limitation
when considering FOV. Low magnification objective lenses
that support wide FOV imaging typically have a low NA,
which in turn leads to low resolution images.
Here, we report MesoTIRF - prism-based TIRF microscopy
using the Mesolens for imaging over a large FOV with high
lateral and axial resolution. The Mesolens12 combines low
magnification with a high numerical aperture (4x/0.47 NA),
and our dual-wavelength MesoTIRF illuminator generates an
evanescent wave to exploit its full FOV (4.4 mm x 3.0 mm).
We present details of the optical setup and show the utility
of MesoTIRF for high-contrast, high-resolution imaging of
fluorescently-labeled proteins in fixed cells.
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MesoTIRF: a Total Internal Reflection Fluorescence illuminator for axial super-resolution membrane imaging at the mesoscale 2
II. METHODS
A schematic diagram of the optical set-up is shown in
Figure 1. The illumination laser source was a tunable
wavelength (Chameleon Ultra II, Coherent) Titanium Sap-
phire laser pumping an optical parametric oscillator (OPO)
(Compact OPO-Vis, Coherent). The second harmonic of
the signal wavelength output of the OPO was used as the
laser source, with 500 nm and 585 nm selected for dual-
wavelength TIRF imaging. This choice of wavelengths
was informed by the specification of the custom 100 mm
diameter Pinkel-type13 chromatic reflector and barrier filters
used for Mesolens imaging which allows fluorophore excita-
tion/emission combinations of 505 nm ± 25 nm/542.5 ± 7.5 nm
and 575.5 ± 22.5 nm/677.5 ± 72.5 nm. For comparison of the
performance of the MesoTIRF illuminator with widefield
epi-fluorescence illumination (WF epi) we used 504 nm
(bandwidth = 19.4 nm) and 584 nm (bandwidth = 27 nm)
light emitting diodes for wide-field illumination (pE-4000,
CoolLED).
The optical power of the OPO output beams at the
specimen plane were adjusted using a combination of a
polarizing beamsplitter cube and a variable neutral density
filter wheel. Next, the beam was expanded by a Keplerian
telescope consisting of 50 mm and 100 mm focal length
plano-convex lenses (anti-reflection coated for 350- 700 nm).
A first surface reflector in a kinematic mount was used to
adjust the angle of incidence of the beam to 86° at the top
surface of a 45° borosilicate glass dove prism which served
as the MesoTIRF prism. The theoretical evanescent field
depth for a wavelength of 504 nm in a borosilicate prism
(n=1.51) at this incidence angle is 56 nm 14. The 25 mm thick
prism has a top surface of 20 mm by 70 mm, and was placed
on top of a computer-controlled specimen stage (ProScan III,
Prior Scientific) for accurate positioning of the prism in three
dimensions. To capture the large, high-resolution images pro-
duced by MesoTIRF we used a chip-shifting camera sensor
(VNP-29MC; Vieworks) which recorded images by shifting a
29-megapixel CCD chip in a 3 x 3 array15. Reconstruction of
each image (260 Megapixels, 506 MB) took approximately 5
s on a typical computer workstation.
To confirm evanescent illumination, we prepared a speci-
men of murine fibroblast cells (3T3-L1) labeled with both a
fluorescent nuclear marker (SYTO Green) and an antibody
labeling against paxillin, a focal adhesion component16. We
hypothesized that the fluorescent emission from the nuclear
marker would be visible in WF epi but with MesoTIRF the
cell nucleus would be too far above the basal membrane to be
excited by the evanescent wave. To image the SYTO Green
stain, the camera exposure time and the camera gain were set
to 2 s and 1 X, respectively for both WF epi and MesoTIRF.
In WF epi, the 504 nm LED power was adjusted to 25 mW to
excite fluorescence from the stained nuclei without saturation.
For MesoTIRF, the maximum available laser power at the
specimen plane of 3.48 mW was used at a wavelength of
500 nm. To image the antibody against paxillin that was
conjugated to Alexa Fluor Plus 594, the camera exposure
time and camera gain were set to 2 s and 70 X, respectively
for both WF epi and MesoTIRF. The optical powers of the
584 nm LED (22 mW) and the 585 nm laser (3.48 mW) were
adjusted to produce images of the fluorescently labeled pax-
illin with a similar fluorescence signal intensity. To estimate
the number of cells, the ‘Surfaces’ model in Imaris (Imaris
9.8, Oxford Instruments) was used for object detection of
SYTO Green labeled nuclei in the WF epi image.
A comparison of signal-to-background ratio (SBR) in
WF epi and MesoTIRF from this dual labeled sample was
obtained by taking line intensity profiles in ImageJ17 through
focal adhesions in the images from each modality. Following
transfer of this data to Python, the peaks of each focal
adhesion where detected using the find_peaks() function in
the SciPy18 library and scaled against the minimum signal
intensity.
To evaluate the capability of MesoTIRF for dual-
wavelength imaging including an assessment of the uni-
formity of illumination, a fixed HeLa cell specimen was
prepared, using an anti-paxillin antibody that was conjugated
to Alexa Fluor Plus 594 and fluorescein phalloidin which
stains F-actin. For each sequential image, an exposure of 2 s
and a camera gain of 30 X was used.
To evaluate the improvement in SBR in MesoTIRF
compared with WF epi, the same specimen was imaged but
only the anti-paxillin conjugated to Alexa Fluor Plus 594
was excited. To measure the SBR Trainable Weka19 was
used to identify and segment objects from both WF epi and
MesoTIRF images. A selection mask was extracted from this
segmentation, which enabled us to derive the mean detected
signal in a cell from the raw images using ImageJ17. The
background signal was calculated for each image by selecting
three background regions of interest, calculating the mean
intensity, and averaging these measurements. This workflow
was carried out for six ROIs separated by at least 0.5 mm
across the full FOV to evaluate variation in SBR, and the
uniformity of illumination.
To demonstrate MesoTIRF in another cell type, the human
mesothelial cell line MeT-5A was fixed and labeled with an-
tibodies against paxillin and tubulin, which were conjugated
to Alexa Fluor 488 and Alexa Fluor Plus 594 respectively.
These (adhesion and cytoskeletal) proteins are within the
reach of an evanescent field. This data set is included as
Supplementary Information.
Cell culture, fluorescent labeling and specimen preparation
methods are included as Supplementary Information.
III. RESULTS
Figure 2 shows a comparison of WF epi with MesoTIRF
images of dual-labeled fixed 3T3-L1 cells prepared with
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MesoTIRF: a Total Internal Reflection Fluorescence illuminator for axial super-resolution membrane imaging at the mesoscale 3
Figure 1: Schematic of MesoTIRF: PL: Titanium Sapphire pump laser (Ultra II, Coherent), IR M1: Infrared mirror (BB1-E03,
Thorlabs), OPO: Optical Parametric Oscillator (Chameleon OPO-Vis, Coherent), PBS: polarising beam splitter
(CCMS-PBS201/M, Thorlabs), BD: beam dump, M1-6: visible broadband dielectric mirrors (Thorlabs, BB1-E01), FW: filter
wheel with 5 neutral density filters (Thorlabs, FW1A), L1: 50 mm planoconvex lens (LA1131-A-ML, Thorlabs), L2: 100 mm
planoconvex lens (LA1509-A-ML, Thorlabs), M3-6 mounted in right angled cage mounts (KCB1C/M, Thorlabs), P:45°
borosilicate glass prism (Mesolens Ltd.), S: sample, Im: immersion fluid (distilled water), Meso: Mesolens objective element
(12), D: dichroic filter & E: emission filter(custom from Chroma), C: chip-shifting camera sensor (VNP-29MC; Vieworks),
LED: 504 nm and 584 nm LEDs from LED module (pE-4000, CoolLED)
fluorescent staining for nuclei (green) and paxillin (magenta).
The full FOV WF epi image is shown in 2A, with a region
of interest (ROI) indicated by a yellow box that is digitally
zoomed in 2B. Figure 2C shows the same area of the spec-
imen imaged using dual-wavelength MesoTIRF, with the
same ROI expanded in 2D.
Using WF epi, the cell nuclei are clearly visible in 2B.
These nuclei disappear, as expected, when imaged with
MesoTIRF as shown in 2D, thus confirming that the evanes-
cent wave in MesoTIRF is restricted to a shallow depth
close to the coverslip and does not penetrate sufficiently
deep into the sample to excite fluorescence from the labeled
nuclei. Non-specific binding or binding of the anti-paxillin
antibody to cytosolic protein is apparent when imaged with
WF epi, but this fluorescence signal also disappears when
using MesoTIRF illumination. This is further evidenced by
the intensity profiles through two neighboring nuclei (Figure
2E) and through 3 neighboring focal adhesions (Figure
2F) for both the WF epi image (dark blue) and MesoTIRF
(cyan). The difference in background is clearly evident,
with MesoTIRF yielding a 4.2-fold reduction in background
through the neighboring focal adhesions over WF epi, with
a less noisy baseline than that of the WF epi image. While
the focal adhesions are still visible in WF epi, the contrast
enhancement afforded by MesoTIRF allows for the elongated
features to be easily distinguished from background. An
SBR improvement of 4.84 X/3.9 X/3.87 X was observed in
focal adhesions 1, 2 and 3 respectively when switching from
imaging with WF epi to MesoTIRF.
Furthermore, there is negligible nuclear signal in the
MesoTIRF image (Figure 2E) because, as discussed pre-
viously, the 86° incident beam (resulting in a calculated
evanescent field depth of 56nm) does not penetrate the cell
specimen deep enough to excite the nuclear stain. Using
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MesoTIRF: a Total Internal Reflection Fluorescence illuminator for axial super-resolution membrane imaging at the mesoscale 4
Figure 2: Comparison imaging of WF epi and MesoTIRF: fixed 3T3-L1 cells labelled with SYTOGreen stain visualizing
nuclei (shown in green) and with an anti-paxillin antibody conjugated to Alexa Fluor Plus 594 (shown in magenta). A: WF epi
image with 504 nm and 584 nm LEDs, B: ROI digital zoom of (A) with line ROIs in yellow and orange to study nuclei and
paxillin respectively, C: MesoTIRF image obtained with 500 nm and 585 nm OPO SHG illumination, D: ROI digital zoom of
(C), E: Yellow line profile intensity plot of neighboring nuclei in WF epi (dark blue) and MesoTIRF (cyan). F: Orange line
profile intensity for three neighboring focal adhesions in WF epi (dark blue) and MesoTIRF (cyan). Fluorescently labelled
nuclei are visible in WF epi data but disappear when imaged with MesoTIRF. A considerable reduction image background
signal is also observed in the MesoTIRF images.
the ‘Surfaces’ feature in Imaris on the nuclear channel, 743
cells were counted in this single image. For the purposes of
presentation, each image presented in Figure 2 has undergone
the ‘Enhance Local Contrast (CLAHE)’ function in ImageJ17
but all analysis has been performed on raw image data.
The application of MesoTIRF for imaging of dual-labeled
specimens is shown in Figure 3. Figure 3 shows a 4.4 mm
x 3.0 mm FOV dual-colour MesoTIRF image with focal
adhesions in magenta and F-actin in cyan. Yellow boxes
show digitally zoomed images of six separate ROIs separated
by a minimum distance of 0.5 mm. In all images, focal
adhesions and the F-actin network adjacent to the basal cell
membranes are clearly visible. We note there is a less than
a 50% decrease in fluorescence signal from the centre to the
edge of the imaged field, which we attribute to the Gaussian
intensity profile of the illumination yielded from the optics
chosen in Figure 1.
Supplementary Figure 1 shows a further example of two-
color MesoTIRF imaging with a third cell type, MeT-5A, la-
beled for paxillin and tubulin. We see the same improve-
ment in image quality and contrast in this data set as demon-
strated by MesoTIRF presented here, illustrating the use of
the modality for a variety of cellular imaging.
IV. DISCUSSION
We have demonstrated MesoTIRF imaging of a range
of different biological samples. Coupling a custom prism
TIRF illuminator with the Mesolens12 provides an unprece-
dented combination of a large FOV with sub-micron spatial
resolution in three-dimensions. The optical throughput of
the Mesolens is over 20 times greater than a commercial
objective lens with a low magnification12. This presents
an advantage for MesoTIRF imaging, which enables lower
optical power specimen illumination with corresponding
reductions in photobleaching and phototoxicity. MesoTIRF
utilized the comparatively cheap prism illumination method,
allowing for ease when changing evanescent field depth by
varying the incidence angle of the incident beam.
In our confirmation of TIRF illumination using the spec-
imen which was dual-labeled with a nuclear stain and an
antibody against the focal adhesion protein paxillin we note
that the position of the nuclei is dependent on where in their
life-cycle the 3T3-L1 cells were at the point of formaldehyde
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MesoTIRF: a Total Internal Reflection Fluorescence illuminator for axial super-resolution membrane imaging at the mesoscale 5
Figure 3: Uniformity of MesoTIRF: fixed HeLa cells labelled with an anti-paxillin antibody conjugated to Alexa Fluor Plus
594 (magenta) and Fluorescein Phallodin which stains the actin cytoskeleton (cyan). A full FOV MesoTIRF image is shown in
the centre, with six ROIs indicated by yellow boxes. These show digital zoomed areas from the original dataset, and confirm a
small variation in fluorescence intensity and little difference in resolvable detail across the multi-millimeter FOV.
fixation20. However we expect that the nuclear envelopes
of each imaged cell is distal from the basal cell membrane,
and therefore outside the reach of an evanescent field from
MesoTIRF20.
The ability of the MesoTIRF modality to capture fine
details and the contrast improvement over WF epi was
examined using a fixed mammalian cell line labelled for the
focal adhesion component paxillin16. Using the intensity
signals through neighbouring focal adhesions, an average
4.2-fold improvement in SBR was measured in MesoTIRF
over WF epi, with the improvement in contrast allowing for
many more focal adhesions to be resolved with this novel
modality (Figure 2).
The drop off in intensity from the centre to the edge of
the MesoTIRF FOV is to be expected for an evanescent field
generated using a Gaussian beam. This can be corrected for
with flat field correction, a commonplace post-processing
technique for many imaging modalities. However, as evident
from the chosen ROIs in Figure 3, the level of structural detail
resolvable even in these dimmer peripheral areas remains of
the quality expected of TIRF.
Excitation wavelengths for MesoTIRF are presently limited
to the two discussed here by the large diameter Pinkel-type
custom filters used for fluorescence detection. Additional
custom filters would allow this to be extended for further
wavelengths.
Mesolens data are rich in information21 but we recognise
that an imaging rate of 0.2 Hz for MesoTIRF is insufficient
for several applications in vitro, such as cell signalling
studies as reported by Crites et al22. However, with recent
innovations in camera technologies, notably the development
of cameras using large, high resolution 250 Mpixel sensors
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MesoTIRF: a Total Internal Reflection Fluorescence illuminator for axial super-resolution membrane imaging at the mesoscale 6
such as the Canon 2U250MRXSAA CMOS sensor, tenfold
higher imaging speeds (2.4 fps) can be achieved by avoiding
the need for chip shifting. In combination with environmental
control, this will offer opportunities to study faster dynamic
processes, for example, the action of fast-acting antimicrobial
peptides23 or imaging of calcium transients in the plasma
membrane24.
MesoTIRF may have applications in high-content
screening10 or wound healing models25, where large cell
populations must be imaged to obtain statistically significant
results. However, at present MesoTIRF is only compatible
with imaging at room temperature as there is no environmen-
tal imaging chamber that is compatible with the Mesolens.
We are presently considering chamber designs that would
be suitable for long-term imaging applications including
MesoTIRF.
A present limitation of MesoTIRF is the numerical aperture
of the Mesolens: at 0.47, this is much lower than a typical
TIRF lens and hence the lateral resolution is around three-
fold poorer than a commercial objective TIRF microscope.
However, with the principle of MesoTIRF now proven, an ob-
vious next step is to add structured illumination to produce
MesoTIRF-SIM26. Achieving SIM on the Mesolens is not
a trivial task and would require either further optics in the
MesoTIRF path to impose variable modulation patterns on
the incident excitation beam or utilizing computational meth-
ods such as blind-SIM27 algorithms. This would facilitate ap-
plications in single molecule localization microscopy in cell
specimens approximately two orders of magnitude larger than
current technology can image. At present we are again limited
by the chip-shifting camera technology, but we are carefully
following developments in this field.
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