A guide to structured
illumination TIRF microscopy at high speed with multiple colors
AUTHORS:
Young, Laurence J
Department of Chemical
Engineering and Biotechnology
University of Cambridge
Cambridge, United Kingdom
Ströhl, Florian
Department of Chemical
Engineering and Biotechnology
University of Cambridge
Cambridge, United Kingdom
Kaminski, Clemens F
Department of Chemical
Engineering and Biotechnology
University of Cambridge
Cambridge, United Kingdom
CORRESPONDING AUTHOR:
Young, Laurence J
KEYWORDS:
Optical super-resolution,
structured illumination microscopy, fluorescence, high speed imaging, TIRF,
bioimaging
SHORT ABSTRACT:
This article provides an in depth
guide for the assembly and operation of a structured illumination microscope
operating with total internal reflection fluorescence illumination (TIRF-SIM)
to image dynamic biological processes with optical super-resolution in multiple
colors.
LONG ABSTRACT:
Optical super-resolution imaging
with structured illumination microscopy (SIM) is a key technology for the visualization
of processes at the molecular level in the chemical and biomedical sciences.
Although commercial SIM systems are available, systems that are custom designed
in the laboratory can outperform commercial systems, the latter typically
designed for ease of use and general purpose applications, both in terms of
imaging fidelity and speed. This article presents an in-depth guide to building
a SIM system that uses total internal reflection (TIR) illumination and is
capable of imaging at up to 10 Hz in three colors at a resolution reaching 100
nm. Due to the combination of SIM and TIRF, the system provides better image
contrast than rival technologies. To achieve these specifications, several
optical elements are used to enable automated control over the polarization
state and spatial structure of the illumination light for all available
excitation wavelengths. Full details on hardware implementation and control are
given to achieve synchronization between excitation light pattern generation,
wavelength, polarization state, and camera control with an emphasis on
achieving maximum acquisition frame rate. A step-by-step protocol for system
alignment and calibration is presented and the achievable resolution
improvement is validated on ideal test samples. The capability for video-rate
super-resolution imaging is demonstrated with living cells.
INTRODUCTION:
Over
the last half a decade, super-resolution microscopy has matured and moved from
specialist optics labs into the hands of the biologist. Commercial microscope
solutions exist for the three main variants for achieving optical
super-resolution: single molecule localization microscopy (SMLM), stimulated
emission depletion microscopy (STED), and structured illumination microscopy (SIM)1,2. SMLM such as
photoactivated localization microscopy (PALM) and stochastic optical
reconstruction microscopy (STORM) have been the most popular techniques,
largely due to the simplicity of the optical setup and the promise of high
spatial resolution, readily down to 20 nm. However super-resolution microscopy via single molecule localization comes
with an intrinsic trade-off: the spatial resolution attainable is dependent on
accumulating a sufficient number of individual fluorophore localizations, hence
limiting the temporal resolution. Imaging dynamic processes in live cells
therefore becomes problematic as one must adequately sample the movement of the
structure of interest to prevent motion artifacts while also acquiring enough localization
events in that time to reconstruct an image. In order to meet these
requirements, live cell SMLM demonstrations have obtained the required increase
in fluorophore photoswitching rates by greatly
increasing the excitation power, and this leads in turn to phototoxicity and
oxidative stress, thereby limiting sample survival times and biological
relevance3.
A clear
advantage of STED over both SIM and SMLM is that it can image with
super-resolution in thick samples, for example lateral resolution of around 60
nm was achieved in organotypic brain slices at depths up to 120 μm4. Imaging at such depths
with single objective implementations of SMLM or SIM is unfeasible, but becomes
possible with either single-molecule light sheet or lattice light sheet microscopy5. Video-rate STED has
also been demonstrated and used to map synaptic vesicle mobility, although so
far this has been limited to imaging small fields of view6.
For
applications in cell biology and molecular self-assembly reactions7–12 that
require imaging with high temporal resolution over many time points, structured
illumination microscopy (SIM) can be well-suited as it is not dependent on the photophysical properties of a particular fluorescent probe.
Despite this inherent advantage of SIM, up to now its use has been mainly
confined to imaging fixed cells or slow moving processes. This is due to the
limitations of commercially available SIM systems: the acquisition frame rate of
these instruments was limited by the rotation speed of the gratings used to
generate the required sinusoidal illumination patterns as well as the polarization
maintaining optics. The newest generation of commercial SIM instruments are
capable of fast imaging but they are
prohibitively expensive to all but central imaging facilities.
This protocol presents a guide to
the construction of a flexible SIM system for imaging fast processes in thin
samples and near the basal surface of living cells. It employs total internal
reflection fluorescence (TIRF) to generate an illumination pattern which
penetrates no deeper than approximately 150 nm into the sample13 which vastly reduces the out of focus background
signal. The idea of combining SIM with TIRF is almost as old as SIM itself14 but was not realized experimentally before 200615. The first in
vivo images obtained with TIRF-SIM were reported in 200916 achieving frame rates of 11 Hz to visualize
tubulin and kinesin dynamics, and two color TIRF-SIM systems have been presented17,18. Most recently, a guide for the construction and
use of a single color two-beam SIM system was presented featuring frame-rates
of up to 18 Hz19,20.
The set-up presented here is
capable of SIM super-resolution imaging at 20 Hz in three colors, two of which
can be operated in TIRF-SIM. The whole
system is constructed around an inverted microscope frame and uses a motorized xy translation
stage with a piezo-actuated z stage. To
generate the sinusoidal excitation patterns required for TIRF-SIM, the system
presented uses a ferroelectric spatial light modulator (SLM). Binary grating
patterns are displayed on the SLM and the resulting ±1 diffraction orders are
filtered, relayed and focused into the TIR ring of the objective lens. The
necessary phase shifts and rotations of the gratings are applied by changing
the displayed SLM image. This protocol describes how to build and align such
an excitation path, details the alignment of the emission path, and presents
test samples for ensuring optimal alignment. It also describes the issues and
challenges particular to high speed TIRF-SIM regarding polarization control and
synchronization of components.
Design Considerations and Constraints
Before
assembling the TIRF-SIM system presented in this protocol, there are several
design constraints to consider which determine the choice of optical
components. All abbreviations of optical components refer to Figure 1.
Spatial Light Modulator (SLM)
A
binary ferroelectric SLM is used in this setup as it is capable of
sub-millisecond pattern switching. Grayscale nematic
SLMs may be used but these offer greatly reduced switching times. Each on or
off pixel in a binary phase SLM will impart either a π or 0 phase offset
to the incident plane wavefront, therefore if a
periodic grating pattern is displayed on the SLM it will operate as a phase
diffraction grating.
Total internal reflection (TIR)
To
achieve TIR and produce an evanescent field, the incident angle of the
excitation beams at the glass-sample interface must be greater than the
critical angle
Reconstruction
of TIRF-SIM images requires the acquisition of a minimum of three phase shifts
per pattern rotation therefore the SLM pattern period must be divisible by 3
(see Fig 1). For example, a period of 9 pixels for 488 nm illumination and 12
pixels for 640 nm illumination. For a comprehensive discussion of SLM pattern
design, including sub-pixel optimization of pattern spacing using sheared
gratings, see the previous work of Kner et al16 and Lu-Walther et al20. The position of the two
excitation foci must be inside the TIR ring for all wavelengths, however the
diffraction angle of the ±1 orders from the SLM is wavelength dependent. For
standard SIM, multicolor imaging can be achieved by optimizing the grating
period for the longest wavelength, and tolerating a loss in performance for the
shorter channels. For TIRF-SIM however, optimizing for one wavelength means
that the other wavelength foci are no longer within the TIR ring. For example,
using a grating period of 9 pixels is sufficient to provide TIRF for 488 nm, as
the foci are at 95% of the diameter of the back aperture and within the TIR
ring, but for 640 nm this period would position the foci outside the aperture.
For this reason different pixel pattern spacings must be used for each
excitation wavelength.
The
alignment of the TIRF-SIM excitation path is extremely sensitive to small
changes in the position of the dichroic mirror (DM4 in Fig 1) in the microscope
body, much more so than in conventional SIM. Use of a rotating filter cube
turret is not recommended, instead use a single, multi-band dichroic mirror,
which is kept in a fixed position and designed specifically for the excitation
wavelengths used. It is essential that only the highest quality dichroic
mirrors are used. These require thick substrates of at least 3 mm, and are
often designated as “imaging flat” by manufacturers. All other substrates lead
to intolerable aberration and image degradation in TIRF-SIM.
To
achieve TIRF-SIM it is
essential to rotate the polarization state of the excitation light in
synchronicity with the illumination pattern such that it remains azimuthally
polarized in the objective pupil plane with respect to the optical axis (i.e. s-polarized).
Alignment of the polarization control optics will depend on the specific
optical element employed, for example a Pockels cell21, or a half wave plate in a motorized rotation
stage22. In this protocol a custom liquid crystal variable
retarder (LCVR) is used, designed to provide full-wave (2π) retardance
over the wavelength range 488 to 640 nm as it allows fast (~ ms) switching. If
using a liquid crystal retarder it is essential to use a high quality
component: standard components are typically not stable enough to give a
constant retardance over the length of the camera exposure time which leads to
a blurring out of the illumination pattern and low modulation contrast. Liquid
crystal retarders are also strongly temperature dependent and require built in
temperature control.
Synchronization
The lasers must be synchronized
with the SLM. Binary ferroelectric SLMs are internally balanced by switching
between an on state and off state. The pixels only act as half wave plates in
either their on or off state, but not during the interframe
switching time. Therefore the lasers should only be switched on during on/off
states via the LED Enable signal from the SLM to prevent a reduction in pattern
contrast due to the intermediate state of the pixels. An acousto-optic
modulator (AOM) could alternatively be used as a fast shutter if the lasers
cannot be digitally modulated.
Choice of lenses
Based
on these constraints, the required demagnification of the SLM plane onto the
sample plane to produce the desired illumination patterns can be determined.
This allows calculation of the focal lengths of the two lenses L3 and L4 in the
image relay telescope and the excitation tube lens L5. In this system a
100X/1.49NA oil immersion objective lens is used with 488 nm and 640 nm
excitation, hence uses focal lengths of 300 and 140 mm for L4 and L3, and 300
mm for L5, giving a total demagnification of 357x, equivalent to an SLM pixel
size of 38 nm at the sample plane. Using this combination of lenses, SLM
grating periods of 9 for 488 nm illumination and 12 pixels for 640 nm give
pattern spacings of 172 and 229 nm at the sample, corresponding to angles of
incidence of 70° and 67° respectively. For a glass-water interface, the
critical angle is 61°, and is independent of wavelength, therefore these two pattern
spacings allow TIRF excitation for both wavelengths. An objective lens equipped
with a correction collar is useful for correction of spherical aberrations
introduced by variations in coverslip thickness, or if operating at 37°C.
Image Reconstruction
Once raw SIM data has been
acquired it is a matter of computational effort to generate super-resolved
images in a two-step process. Firstly, the illumination pattern has to be
determined for every image and secondly, the components of the SIM spectrum must
be separated and recombined appropriately as to double the effective OTF
support (see Figure 6 insets).
Precise knowledge of the
projected illumination patterns is paramount, as the super-resolved frequency
components have to be unmixed as accurately as possible to prevent artifacts
caused by the residual parts of overlapping components. We determine the
illumination pattern parameters a posteriori from the raw image data
following the procedure introduced by Gustafsson et
al.23. In short, a set of illumination parameters that
describes a normalized two-dimensional sinusoid has to be found for each of the
Hereby
As the phase step between shifted
patterns is
PROTOCOL:
1. Arranging and aligning the
excitation path
1.1.
Mark the positions of the
components on the optical table (see Figure 1 for an overview of the optical
setup). Separate the objective, lenses L3, L4, L5, and the
SLM each by the sum of their respective focal lengths such that the SLM surface
will be relayed onto the focal plane of the objective.
1.2.
Insert
multi-edge dichroic mirror DM4 into the filter cube turret of the microscope
frame.
1.3.
Insert the second
dichroic mirror DM3 into a 1” square kinetic mirror mount, and position it one
focal length away from the tube lens L5.
Note: This excitation path design incorporates two identical dichroic
mirrors DM3 and DM4 which are taken from the same production batch to ensure
identical optical properties. The dichroic mirror (DM4) is positioned such that
the s- and p- axes are switched compared to the dichroic located in the
microscope (DM3) thereby cancelling any polarization ellipticity introduced by
its birefringence (Figure 1). This compensation works equally well for each illumination
wavelength. This step is essential for maintaining high modulation contrast.
1.4.
Before inserting any lenses into the excitation
path, accurately define the optical axis for the system.
1.4.1. Remove the objective lens (OB) from the turret and instead screw in an
alignment tool. This
consists of a 300 mm long optical cage system with two alignment disks at both
ends.
1.4.2. Use the dichroic mirror DM3 and a temporary alignment mirror positioned
at the approximate later location of the SLM to steer a collimated reference
beam from Laser 1 through the center of the holes in the two alignment disks. Direct
the beam from Laser 1 to the temporary mirror as depicted in Figure 1 using
three mirrors and dichroic mirror DM2. The temporary mirror at the SLM
position must be close to perpendicular to the optical axis.
Note: Use Laser 1 as the reference beam, as the other lasers can be
subsequently aligned once the excitation path is in place.
1.4.3. Remove the alignment tool once the coarse optical axis has been
determined. Insert an iris into the beam
path before it enters the microscopy body and center it on the beam. Attach a
piece of white card with a small hole centered on the iris. Reinsert the objective lens
(OB).
Note: The beam leaving the objective will now be highly divergent, but
there will be a very weak reflection from the back surface of the lens that
will be visible on the white card. All lenses, even if they are anti-reflection
coated, will have weak back reflections that can be used to ensure coaxial
alignment. If the beam is exactly perpendicular to the lens then the back
reflection will go back through the center of the iris.
1.4.4.
Make iterative angular adjustments to the two
mirrors (DM3 and alignment mirror at the SLM position) to center the back
reflection on the card with the incoming beam. Temporarily remove the objective
lens (OB) and mark the laser spot on the ceiling to create a reference
position.
1.4.5. Insert a pair of irises at the
height of the reference beam along the threaded holes of the table. The beam
should be parallel to the surface of the optical table. The optical axis is now
defined.
1.5.
Insert the tube lens (L5) roughly one focal length
away from the objective. Mount this lens on a linear translation stage set to
translate along the direction of the reference beam.
1.6.
Adjust the tube lens position and angle such that
the beam leaving the objective is collimated and hits the reference spot on the
ceiling. Check that the lens is perpendicular to the beam by again checking the
back reflection with the iris and white card. Remove the objective lens (OB)
and insert the second lens of the image relay telescope (L4).
Note: Ensuring proper collimation and non-deflection of the beam is made
easier when there is an even number of lenses in the beam path.
1.7.
Adjust the position and angle of this lens using a
linear translation stage to maintain collimation and to ensure the reference
beam still hits the marked spot on the ceiling.
1.8.
Replace the objective lens (OB) and insert the
first lens of the telescope (L3). Adjust the position and angle of this lens to
ensure collimation and non-deflection, as described in previous steps.
1.9.
Mount the
SLM chip on a gimbal mount which provides rotation without translation about
the center of the chip surface.
Note: The specific mounting design depends on the SLM used. If the SLM
is supplied without a mount, it should be fixed to a custom machined aluminum
plate which is then attached to a lens gimbal mount.
1.10.
With the lenses aligned, insert the SLM in place of
the mirror. Adjust the position of the SLM such that the reference beam is
located at the center of the SLM chip, and adjust the angle such that the beam
passes through the two relay lenses (L3 and L4). Check that the reference beam
is still centered on the marked spot.
1.11.
Expand
and collimate the reference beam using a Keplerian beam expander.
1.11.1. Mount the two lenses (L1 and L2)
in a cage system for ease of adjustment.
1.11.2. Centre the cage system on the
reference beam by removing the lenses and replacing them with irises.
1.11.3. Insert the two lenses and adjust
the axial position of L2 to collimate the expanded beam using a shearing
interferometer. L2 should be one focal length away from the surface of the SLM.
1.11.4. Check that the expanded beam is
still collimated after the two relay lenses L3 and L4. Use the shearing
interferometer just after DM3 to check for collimation.
1.12.
Once the
excitation path has been aligned for a single wavelength, couple the other two
lasers into the beam path. Steer each beam through two irises centered on the
excitation path using the beam combining dichroic mirrors (DM1 and DM2).
2.
Alignment of polarization rotator
2.1.
Mount the LCVR with its fast axis at 45° to the
incident polarization.
2.2.
Fine tune
polarization angle of the beam incident to the LCVR using an achromatic half
wave plate (HWP) by inserting the HWP and the LCVR between crossed polarizers.
Rotate the HWP to minimize the transmitted power.
Note: In order to act as a variable polarization rotator, the fast axis
of the liquid crystal retarder (LCVR) must be precisely aligned at 45°
to the incident vertical beam polarization. The LCVR is physically mounted at
45° but this is only a coarse alignment. The HWP is used to ensure perfect 45°
alignment of the incident polarization with respect to the LCVR fast axis. The
quarter wave plate (QWP) converts the tilted elliptical polarization induced by
the LCVR back to linear polarization at an angle controlled by the applied
voltage24.
2.3.
Insert
the QWP after the LCVR and rotate it to align its slow axis to the incoming
polarization by minimizing the transmitted power between crossed polarizers.
3.
Alignment of the emission path
3.1.
Coarsely position the camera using a stage
micrometer slide and transmitted light.
3.1.1.
Focus on the reticle using the microscope oculars
and fix the objective lens at this position.
3.1.2.
Roughly center the camera and move the camera
position to bring the image of the reticle into focus by observing the image on
screen.
Note: If an external filter wheel is used then the filter cube will not
contain an emission filter, therefore the oculars must not be used when lasers
are switched on.
3.2.
Finely
adjust the camera position using a fluorescent bead sample.
3.2.1.
Prepare a monolayer of fluorescent beads by
spreading a drop of 100 nm multicolor beads on a #1.5 coverglass. Leave to dry
to adsorb the beads to the coverglass and then re-immerse in water.
3.2.2.
Place the bead sample onto the objective with
immersion oil. Finely adjust the position of the camera such that the
fluorescent bead layer is in focus. Do not adjust the objective lens
position once the focus has been found.
Note: As the SLM must be in a
plane conjugate to the sample plane, the position of the SLM, relay lenses, and
objective must be fixed. To adjust the focus, move the sample axially instead
of the objective using a piezo z-stage.
3.3.
Generate
the appropriate SIM binary grating patterns as bitmap files.
3.3.1. For 2D/TIRF-SIM, generate a
series of 9 binary grating images: 3 pattern orientations each with 3 equally
spaced phase shifts. Generate these numerically (using MATLAB for example) from
a rotated 2D sinusoid with a phase offset applied, then thresholding to produce
a binary image. See Supplemental Code Files for example code.
3.3.2. For alignment purposes, also
generate grating patterns that have been windowed by a small circular aperture
for each of the 3 orientations, as shown in Figure 2. The windowed alignment
gratings do not need to be externally triggered but can be manually switched by
the user via the SLM’s software.
Note: See references for a
discussion of the optimal rotation angles and an example of grating pattern
generation code 16,20.
3.4.
Upload
the bitmap images to the SLM using the manufacturer’s software (for example MetroCon).
3.4.1.
Load the SLM control software and click “Connect”.
3.4.2.
In the “Repertoire” tab, click “Load” to open the
repertoire file and check the number of Running Orders contained in the file. In the example repertoire file
given there are five Running Orders.
3.4.3.
Click “Send to Board” to upload the repertoire file
to the SLM.
3.4.4. Wait for the bitmap images to
upload and for the device to automatically reboot.
Note: An example repertoire file,
which contains grating bitmap images and a file defining the order, is included
as a Supplemental Code File. The “.repz” file may be
opened using ZIP file archiver software.
3.5.
Display a
windowed alignment grating on the SLM for the first orientation (for example
0°).
3.5.1.
In the SLM control software, select the “Status”
tab, enter the number of the Running Order (in the case of the example file,
this is Running Order “1”).
3.5.2.
Click “Select” to change the Running Order to the
alignment grating.
Note: This will illuminate a small circular region in the sample plane.
If the SLM surface is correctly conjugated to the sample plane then the edges
of this region will be sharply in focus. The grating pattern will produce
multiple diffraction orders at the focus of L3: the zero order reflection from
the reflective backplane of the SLM, the -1 and +1 orders corresponding to the
grating, and also weaker higher orders that arise from diffraction of internal
elements specific to the SLM device (e.g. reflections of the internal wirings
of the SLM pixels and irregularities at the pixel edges). All but the -1 and +1
orders must be filtered out.
3.6.
Insert a spatial mask (SM) mounted in an x,y stage into the beam path at
the focal position of L3, and translate its position with respect to the
optical axis such that only the desired first orders are passed. Directly after
the spatial filter, only two circular beams will be visible.
Note: The spatial mask is fabricated by punching 6 holes into aluminum
foil using a needle. The holes should be large enough to pass the first order
beams for all laser wavelengths. A detailed analysis of the spatial mask is
given in reference20.
3.7.
Display
the next orientation of the alignment grating (60°, running order 2) and again
ensure that only the first orders are let through the spatial mask, adjusting
its position if required.
3.8.
Repeat
for the final orientation (120°, running order 3).
3.9.
Check the image of the fluorescent bead layer on
the camera. If the two circular beams are not overlapping as depicted in Figure
2 then reposition the sample plane by iteratively adjusting the objective lens
and camera position.
3.10.
Adjust
the objective position to overlap the two beams which will bring the image out
of focus. Reposition the camera to bring the image back into focus and fine
tune the objective in case two circles are still visible. Repeat this process
until the two beams overlap and a single circular region is in focus.
3.11.
Once the
position of the sample plane has been set, keep the objective position fixed.
3.12.
To confirm TIRF illumination, image a solution of
fluorescent dye, for example for a 488 nm excitation wavelength, use a solution
of 10 μM rhodamine 6G.
3.12.1.
Bring the dye sample into focus. If the two beams
are incident at the correct TIRF angle then single molecules will be visible
without high background, and the edges of the circular aperture will be in
focus. See
Figure 2B-D for examples of aligned and misaligned TIRF beams.
3.12.2.
Display each orientation of the windowed gratings
in turn and ensure that all three orientations provide TIRF illumination and
that the two beams overlap at the sample plane. Fine adjustments to the
position of the beams can be made by adjusting dichroic mirror DM3.
Note: Although different wavelengths are focused at slightly different
positions due to axial chromatic aberration, this is not critical and may be
corrected by applying a constant z-offset to the sample position prior to excitation
with the second wavelength.
4.
System synchronization and
calibration
4.1.
Place the bead monolayer sample on the objective
and bring into focus.
4.2.
Program the SLM using its control software to
display each of the 3 phase shift images in turn, for the first pattern
orientation (0°).
4.2.1.
Using the SLM control software, switch to Running
Order 4 of the example repertoire.
4.2.2.
Configure the camera using its acquisition software
(for example HCImage) to output two signals: one
positive and one negative TTL trigger signal during the global exposure period.
In the camera software, under “Advanced Camera Properties”, set Output Trigger
Kind 1 and 2 to “Exposure”, and Output Trigger Polarity 1 and 2 to “Positive”
and “Negative” respectively.
4.2.3. Connect Output 1 and 2 of the
camera to the “Trigger” and “Finish” inputs of the SLM respectively, using coaxial
cables. The SLM is now synchronized to the camera.
4.3.
Acquire a
series of 3 images.
4.3.2.
Click “Start” to acquire 3 frames. The SLM pattern
will change upon each exposure. The fluorescent beads in the image will appear
to blink on and off between each of the 3 images. The amount of blinking is a
read out of the modulation contrast of the sinusoidal illumination pattern.
4.4.
Rotate
the polarization of the excitation laser with the LCVR using custom software in
order to achieve azimuthal polarization and therefore the highest modulation contrast
for the given pattern orientation.
4.4.1. Load the LCVR calibration
software.
4.4.2. Enter 0 and 8 for the Minimum and
Maximum Voltage respectively.
4.4.3. Click “Sweep LCVR Voltage” to
rotate the polarization.
Note: The LCVR retardance is a function of temperature and can drift
day-to-day even with temperature control. In this step, optimal azimuthal
polarization is found empirically by sweeping the applied voltage between its
minimum and maximum voltage which has the effect of rotating the polarization
incident at the sample. The modulation contrast is calculated for each voltage25 and the
voltage that achieves peak contrast is used in the following steps.
4.4.4. Wait for the calibration process
to complete, and note down the measured voltage.
4.5.
Repeat this
calibration process for the remaining two pattern orientations (60° and 120°) and
each of the excitation wavelengths.
4.6.
Synchronize
the camera exposure with the LCVR, lasers, emission filter wheel and piezo
z-stage26. To accomplish this, use a high speed data
acquisition (DAQ) board as the master clock source for the system, and use the
SLM’s LED Enable output signal to modulate the lasers (see Figure 3B).
Note: The specific implementation
is dependent on the components used but the use of a high speed DAQ board for
digital trigger synchronization and control of the LCVR using an analog
voltage, controlled via software, is recommended. The control software used in
this protocol is available upon request.
4.7.
Due to
axial chromatic aberration, for each wavelength, also apply a z-offset to the
sample stage.
4.7.1. Determine the offset
experimentally by focusing on a multicolor bead monolayer sample at the first
wavelength (e.g. 488 nm) then switching to the second (e.g. 640 nm). The beads
will now be out of focus.
4.7.2. Refocus the beads and measure the
change in z position that was needed. This offset can then be applied to the piezo
z-stage every time the excitation wavelength is changed.
4.8.
Using the SLM control software, switch the SLM
Running Order to the full series of 9 binary grating images required for
TIRF-SIM. This is Running Order 0 in the example repertoire.
4.9.
Using the
camera control software, acquire 9 images of the bead sample.
4.9.2.
Click “Start” to acquire images.
4.9.3.
Save the acquired images as TIFF files by selecting
“TIFF” as the image type in the “Save Buffered Images” window, and clicking OK.
4.10.
Reconstruct
a super-resolution image from the raw TIFF images using commercial or custom
software to validate the improvement in resolution over standard TIRF.
Note: For our microscope we use custom reconstruction code developed both
in-house and by Dr Lin Shao27.
REPRESENTATIVE RESULTS:
Multicolor 100nm diameter
fluorescent beads were imaged to compare standard TIRF to TIRF-SIM and quantify
the attainable improvement in lateral resolution (Figure 4A-B). Reconstruction
of raw frames into super-resolution images was performed using standard
algorithms as outlined in the literature27,28. It can be seen that TIRF-SIM clearly has
significantly higher lateral resolution compared to TIRF. The point spread
function (PSF) of a microscope is well approximated by the image of a single
sub-diffraction sized fluorescent bead, therefore the PSF and the resolution
can be quantified by fitting 2D Gaussian functions to individual beads for each
wavelength. The estimated resolution of the microscope based on the mean value
of the full width half maximum (FWHM) is 89 nm and 116 nm for 488 and 640 nm
TIRF-SIM respectively (Figure 4C). This corresponds to a two-fold improvement
in lateral resolution for both wavelengths compared to the theoretical
diffraction limited case. Fluorescently
labelled amyloid fibrils are also an excellent test sample for demonstrating
doubled resolution (Figure 4D). Amyloid fibrils were formed in vitro by incubating β-amyloid
labelled with 10% rhodamine derivative dyes (488 nm excitation) for 1 week and
subsequently imaging with TIRF-SIM. See reference12 for more information.
Subcellular structures with high
contrast such as emGFP labelled microtubules (Figure
5B, G) or LifeAct-GFP (Figure D) are ideal for
TIRF-SIM imaging and yield high contrast super-resolution images. TIRF-SIM
imaging using the setup detailed in this protocol enables observation of a sub-population
of microtubules located in the vicinity of the basal cell cortex, and microtubule
polymerization and depolymerization can be seen over
time (Animated Figure 1). Not all samples are amenable to imaging with TIRF-SIM,
in particular, low contrast samples without discrete structures. Cells
expressing cytosolic GFP lack high resolution information aside from at the
edges of the plasma membrane (Figure 5 F, H and Animated Figure 2) and are hence
sub-optimal for TIRF-SIM imaging as the resulting reconstructions are
essentially TIRF images overlaid with artifacts. In such samples, the increase
in contrast can often be attributed to the deconvolution step of the
reconstruction algorithm.
High modulation contrast is
essential for successful SIM imaging. The Fourier transform of the
reconstructed image allows visualization of the SIM optical transfer function
(OTF) (Figure
6A, inset). Without maximizing the modulation contrast for
each orientation by ensuring azimuthal polarization with a polarization rotator,
there is very little modulation of the high-resolution information in the
sample leading to a low signal-to-noise ratio in the SIM passbands.
Reconstruction algorithms which use the standard Wiener filter approach will
simply amplify the noise in the SIM passbands and yield an image which is
essentially a standard TIRF image overlaid with hexagonal (or “honeycomb”)
ringing artifacts (Figure 6A, right panel). A possible enhancement might be the
use of iterative29,30 or blind reconstruction algorithms31,32 to reduce these artifacts depending on the type of
sample. We recommend the use of the ImageJ plugin SIMcheck
to check the quality of SIM data before and after reconstruction33.
Figure 1: Layout of the multicolor
TIRF-SIM setup. The
TIRF-SIM microscope consists of three main parts, the beam generation unit, the
pattern projection unit, and the detection unit. In the beam generation unit,
three different lasers are aligned onto the same beam path via dichroic mirrors
(DM1 and DM2) and directed through four optical elements for polarization
control. First, a polarizer (P) ensures the purity of the linear polarization
state of each of the laser beams. The following three optical elements are
needed to rotate the polarization in a fast, automated manner as described in
detail in the text. Afterwards, two lenses (L1 and L2) in a telescope
configuration expand the beam to match the active surface of the spatial light
modulator (SLM) and are diffracted into three beamlets
by the SLM’s projected binary grating patterns (examples are shown in tiles
1-9). The polarization state of the illumination light relative to the SLM
pattern is shown as an arrow. A second telescope (L3 and L4) de-magnifies the
pattern and offers access to the Fourier plane of the SLM pattern. In this
plane a spatial mask (SM) is used to filter out the central component and other
unwanted diffraction components from the pixelated structure of the SLM and its
internal wiring. Before the two remaining beams are focused onto the back focal
plane of the objective (OB) via the tube lens (L5), two dichroic mirrors (DM3
and DM4) are included in the setup. DM4 acts as a conventional dichroic mirror
in fluorescence microscopy to separate illumination from emission light.
However, this mirror unavoidably induces ellipticity in the polarization state
of the illumination light which can be compensated for by DM3, a dichroic
mirror from ideally the same batch as DM4. The oil immersion TIRF objective has
a large enough NA to directly launch two counter-propagating waves onto the
coverslip that are reflected totally and give rise to a structured evanescent
field in the coverslip. The sample is mounted on an x-y-z translation stage.
Detection is performed through the same objective and DM4 in transmission, plus
an additional filtering by bandpass emission filters, mounted in a computer
controlled filter wheel (EFW). Finally, the image is projected onto a sCMOS camera by the internal microscope tube lens (L6).
Figure 2: Alignment of overlapping beams. (A) An SLM grating pattern windowed with a circular aperture is useful
for alignment. If two non-overlapping beams are visible on the camera (left),
then the position of the sample plane must be repositioned by iteratively
adjusting the axial positions of the objective lens and the camera to give a
single circular illumination spot (right). The beams must overlap in order to
produce the sinusoidal excitation pattern required for TIRF-SIM. If the beams
do not fully overlap this reduces the field of view over which the interference
pattern is formed. (B and C) The precise angle of incidence of the beams is
important for TIRF-SIM. If the angle is incorrect, one of the beams will not be
at the required angle for TIRF and this is easily visible when imaging a
fluorescent dye solution. One beam has an angle of incidence greater than the
critical angle which yields the circular spot, and the other does not, which
leads to the bright streak on the left of the image in (B). (D) Adjusting the
angle of mirror DM3 ensures both beams are incident at the same angle, and this
can be validated by defocusing the objective: if correctly aligned, the xz projection of
a z stack of a fluorescent dye sample
should show two symmetrically intersecting beams with negligible background at
the focus.
Figure 3: Synchronization dependencies of the different system
components. (A) For
fast SIM acquisition, synchronization of the system components using a hardware
based solution is essential. (B) A data acquisition board (DAQ) should be used as
a master trigger. A TTL signal from the DAQ board is sent to the sCMOS External Input and used to trigger the camera
exposure. The camera Global Exposure output then triggers the SLM to display a
grating pattern, and the SLM LED Enable output is used to digitally modulate
the laser excitation such that the laser is only emitting when the SLM pixels
are in the “on” state. After the exposure is complete, the camera Global
Exposure output is used to advance the SLM pattern on to the next grating phase
or angle. The DAQ board also outputs an analog voltage to the LCVR controller
to control the linear polarization state of the illumination beam. This voltage
is switched after acquisition of the 3 phase images for each pattern angle. After
acquisition of 9 images for a single wavelength, the DAQ board outputs a signal
to the emission filter wheel controller, and switches to the next wavelength.
The DAQ board also applies a z-offset to the sample by outputting an analog
voltage to the z-stage piezo controller.
Figure 4: TIRF-SIM imaging of test samples of 100 nm multicolor beads
and fluorescently labelled amyloid fibrils. (A and B) Comparison
of standard TIRF compared to TIRF-SIM reconstructions for 488 nm and 640 nm
excitation. (C) Histogram of full-width
half-maximum (FWHM) of Gaussian fits to the TIRF-SIM beads showing the expected
resolution improvement. (D) TIRF versus TIRF-SIM of β-amyloid fibrils
labelled with 10% rhodamine derivative dye (488 nm
excitation). Scale bars 1 µm.
Figure 5: Live cell TIRF-SIM imaging. Comparison of conventional TIRF
and TIRF-SIM images of (A, B) microtubules (emGFP-tubulin)
in a HEK293 cell, (C, D) filamentous actin (LifeAct-GFP)
in a COS-7 cell and (E, F) cytosolic GFP in a HEK293 cell. Images in B and F
are single time points from the movies. Boxed areas are shown magnified in (G,
H). Scale bars
3 μm.
Figure 6: Influence of polarization rotator on reconstructed bead images.
(A)
Without the use of a polarization rotator such as an LCVR, the signal-to-noise
ratio in the SIM passbands is low which results in characteristic hexagonal
artifacts in the reconstructed SIM images (right), (B) In
2D-SIM, the structured illumination patterns are directly visible in the
Fourier transform of the raw images (left, excitation spatial frequency
highlighted) as they fall within the radius of the emission OTF support,
however in TIRF-SIM, they are outside the OTF support and therefore not visible
(right). In this case, the pattern modulation contrast must be assessed
using a sparse bead monolayer, as outlined in the protocol.
Figure 7: Spatial light modulator based pattern generation allows
implementation of other imaging modalities such as multifocal SIM. (A) In MSIM, a lattice of square
points displayed on the SLM (inset)
yields a lattice of diffraction limited foci at the image plane. A thin layer
of low concentration rhodamine 6G is imaged to visualize the foci. The pattern
is translated across the sample (B) and the acquired raw image z-stack is
reconstructed to generate an image with reduced out-of-focus light (C). Scale
bars 5 μm.
Animated Figure 1: Time series movie of emGFP-tubulin
in a HEK293 cell. Rapid
polymerization and depolymerization of emGFP labelled microtubules can be observed using TIRF-SIM.
Images acquired using 50 ms exposure time per raw frame (450 ms per SIM frame)
spaced at intervals of 0.5 s. Exposure time used was limited by the brightness
of the fluorophore, not by the speed of the camera or SLM.
Animated Figure 2: Time series movie of cytosolic GFP in a HEK293 cell. Samples with low contrast such as
this are not ideal samples for TIRF-SIM imaging. Retrograde membrane flow can
be seen in the TIRF images but TIRF-SIM does not provide any additional information
apart from at the cell edges. TIRF-SIM images were acquired using 50 ms
exposure time per raw frame (450 ms per SIM frame) spaced at intervals of 5 s.
Supplemental Code File: Example SLM repertoire file (48449 300us 1-bit Balanced.seq3).
Supplemental Code File: Example SLM repertoire file (period9_001.bmp).
Supplemental Code File: Example SLM repertoire file (period9_002.bmp).
Supplemental Code File: Example SLM repertoire file (period9_003.bmp).
Supplemental Code File: Example SLM repertoire file (period9_004.bmp).
Supplemental Code File: Example SLM repertoire file (period9_005.bmp).
Supplemental Code File: Example SLM repertoire file (period9_006.bmp).
Supplemental Code File: Example SLM repertoire file (period9_007.bmp).
Supplemental Code File: Example SLM repertoire file (period9_008.bmp).
Supplemental Code File: Example SLM repertoire file (period9_009.bmp).
Supplemental Code File: Example SLM repertoire file (period9_mask_1.bmp).
Supplemental Code File: Example SLM repertoire file (period9_mask_2.bmp).
Supplemental Code File: Example SLM repertoire file (period9_mask_3.bmp).
Supplemental Code File: Example SLM repertoire file (TIRF-SIM_example.rep).
Supplemental Code File: Example grating generation code (1 of 2) (generate_gratings.m).
Supplemental Code File: Example grating generation code (2 of 2) (circular_mask.m).
Supplemental Code File: Example code to calculate modulation contrast (calculate_contrast.m).
DISCUSSION:
Custom-built
TIRF-SIM systems such as the setup detailed in this protocol are capable of multicolor
super-resolution imaging at high speed compared to commercially available
microscopes. The
inherent advantage of SIM as a super-resolution technique is that the temporal
resolution is not limited by the photophysics of the
fluorophore, compared to other methods such as single molecule localization
microscopy (SMLM) or point scanning methods such as stimulated emission
depletion microscopy (STED). Unlike these other techniques, SIM does not
require photoswitchable or depletable
fluorophores so multicolor imaging is straightforward. Non TIRF-SIM systems,
such as optical sectioning SIM and multifocal SIM can usually achieve
resolution improvements of 1.7 times or less in practice as opposed to the
factor of 2 improvement reported here, and commercial systems are also often
slower and less flexible than the system presented in this protocol.
The two main difficulties in
implementing this technique are firstly the necessity for precise positioning
of the six SIM beams within the TIR zone of the objective’s back aperture,
which requires a laborious and time consuming optical alignment procedure.
Secondly, to produce high pattern contrast at the sample, polarization rotation
is essential. For low NA 2D-SIM systems, polarization rotation can be avoided
by careful choice of the linear polarization orientation, but this becomes
impossible for TIRF-SIM25. For high-speed multicolor imaging, electro-optical
polarization control is necessary and this increases the complexity and expense
of the system.
Limitations of the technique
TIRF-SIM, like conventional TIRF,
is naturally limited to observation of biological structures and processes located
at the basal cell membrane that can be illuminated by the 150-200 nm
penetration depth of the evanescent field. While SIM is often quoted as being
less photodamaging to cells than either STED or SMLM,
lateral resolution doubling does still increase the required number of photons
by at least 4-fold5 compared to conventional TIRF microscopy. For
imaging at high frame rates with short exposures times, this photon increase
necessitates use of increased illumination intensities. While any fluorophore
may be used for SIM imaging of fixed or slow moving samples, high brightness
fluorescent proteins or next generation synthetic dyes with enhanced
photostability are recommended for live cell imaging.
Although this implementation is
capable of imaging a single color at SIM frame rates in excess of 20 Hz, multicolor
imaging in the presented system is limited by the switching time of the
motorized emission filter wheel. Due to the large size of the sCMOS camera chip, the use of a multiband emission filter
and image splitting optics would be possible and permit simultaneous imaging
with multiple wavelengths at no speed penalty. Another possibility would be to alternate
the different excitation lasers and use a multiband notch filter to reject the
excitation light. The use of a binary ferroelectric SLM in this implementation
also is not optimal. The diffraction efficiency of such an SLM is very low, so
most of the incident light is in the zero order reflection, which is filtered
out by the spatial mask. For applications requiring very high frame rates, the
imaging speed is therefore limited by the output power of the laser diodes. The
SLM also introduces some ellipticity in the polarization for wavelengths away
from the 550 nm design wavelength where the pixels do not operate as ideal half
wave plates. Although this could be compensated for by using an additional
LCVR, the ideal solution may be the use of a digital micro-mirror device (DMD)
as a pattern generator.
Possible modifications
The setup
presented here is flexible and more easily modified than commercial instruments
so other imaging modalities such as 3D-SIM, fast 2D-SIM, multifocal SIM (MSIM)
and non-linear SIM (NL-SIM) can be implemented21,34,35.
2D-SIM can be well
suited for imaging relatively flat, fast moving structures such as the
peripheral endoplasmic reticulum. The peripheral ER lies deeper within the cell
than can be illuminated using a TIRF evanescent field but due to its flat
structure can be imaged using standard 2D-SIM with negligible out-of-focus background.
Additionally, the use of improved optical sectioning reconstruction algorithms to
suppress out-of-focus light extend the use of 2D-SIM to optically thick samples,
albeit where axial resolution doubling is not required21.
In MSIM, the
sample is illuminated by a sparse lattice of excitation foci36. This modality can be implemented by
simply removing the spatial mask (SM) and replacing it by a polarizer. The SLM
now operates as an amplitude modulator. The binary SIM gratings displayed on
the SLM can be replaced by a 2D lattice of spots, with the size of the spots
chosen to be equal to the size of a diffraction limited focus in the image
plane. In Figure 7A, a lattice of 4x4 pixel squares is displayed on the SLM
(inset) which when demagnified onto the sample
generates diffraction limited foci of 150 x 150 nm, given the physical SLM
pixel size of 13.62 μm. The excitation foci can then be translated by
shifting the lattice pattern on the SLM and this is repeated multiple times in
order to illuminate the entire field of view. Images are acquired for each translated
pattern position and the stack is post-processed to yield a reconstructed image
with improved resolution of up to a factor of
Finally, the setup
can be modified to enable either high-NA linear TIRF-SIM or patterned
activation non-linear SIM (PA NL-SIM), as presented recently by Li et al, by use of an ultrahigh 1.7 NA
objective or addition of a 405 nm photoactivation
laser and careful optimization of the SLM grating patterns35.
Future Applications
SIM is still a rapidly evolving
technique and many applications in the life sciences will be enabled in the
future. The speed, resolution, and contrast enhancements of the technique and
the capability of using standard fluorophores mean that for bioimaging, SIM is
set to replace conventional many microscope systems, such as confocal and wide
field platforms. Commercial SIM systems are already available today with
outstanding technical specifications, however, they are beyond the financial
reach of many research laboratories, and, crucially, they are inflexible to be modified
and developed to implement the latest research developments in the field. They
also lack the essential capability to ‘be adapted for the experiment at hand’,
often a critical bottleneck in cutting edge life science research. The system
described here will be particularly well suited to study dynamic processes near
the cell surface, for in vitro studies
of reconstituted bilayer systems, to study surface chemistry in the materials
and physical sciences, e.g. of 2D materials, and many other applications.
ACKNOWLEDGMENTS:
This work was supported by grants
from the Leverhulme Trust, the Engineering and
Physical
Sciences Research Council
[EP/H018301/1, EP/G037221/1]; Alzheimer Research UK [ARUK-EG2012A-1]; Wellcome Trust [089703/Z/09/Z] and Medical Research Council
[MR/K015850/1, MR/K02292X/1]. We thank E. Avezov and M. Lu for transfection of
the LifeAct-GFP and cytosolic-GFP cells respectively,
and W. Chen for preparation of the HEK293 culture. We also thank K. O’Holleran
for assistance with the design of the microscope, and L. Shao and R. Heintzmann for useful discussions and suggestions.
DISCLOSURES:
The authors have nothing to
disclose.
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