1. Arranging and aligning the excitation path

  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.
  2. Insert multi-edge dichroic mirror DM4 into the filter cube turret of the microscope frame.
  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.
  4. 1.4.Before inserting any lenses into the excitation path, accurately define the optical axis for the system.
    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.
    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.
    3. 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
    4. 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.
    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.
  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.
  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.
  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.
  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.
  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.
  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.
  11. Expand and collimate the reference beam using a Keplerian beam expander.
    1. Mount the two lenses (L1 and L2) in a cage system for ease of adjustment.
    2. Centre the cage system on the reference beam by removing the lenses and replacing them with irises.
    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.
    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.
  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

  1. 2.1.Mount the LCVR with its fast axis at 45° to the incident polarization.
  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.
  3. 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

  1. 3.1.Coarsely position the camera using a stage micrometer slide and transmitted light.
    1. Focus on the reticle using the microscope oculars and fix the objective lens at this position.
    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.
  2. 3.2.Finely adjust the camera position using a fluorescent bead sample.
    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.
    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. 3.3.Generate the appropriate SIM binary grating patterns as bitmap files.
    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.
    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.
  4. 3.4.Upload the bitmap images to the SLM using the manufacturer's software (for example MetroCon).
    1. Load the SLM control software and click "Connect".
    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. Click "Send to Board" to upload the repertoire file to the SLM.
    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.
  5. 3.5.Display a windowed alignment grating on the SLM for the first orientation (for example 0°).
    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").
    2. Click "Select" to change the Running Order to the alignment grating.
    3. 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.
  6. 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.
  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.
  8. Repeat for the final orientation (120°, running order 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.
  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.
  11. Once the position of the sample plane has been set, keep the objective position fixed.
  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.
    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.
    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

  1. 4.1.Place the bead monolayer sample on the objective and bring into focus.
  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°).
    1. Using the SLM control software, switch to Running Order 4 of the example repertoire.
    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.
    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.
  3. Acquire a series of 3 images.
    1. In the "Sequence" pane, select "Hard Disk Record" as the scan type, and set the frame count to 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. 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.
    1. Load the LCVR calibration software.
    2. Enter 0 and 8 for the Minimum and Maximum Voltage respectively.
    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. Wait for the calibration process to complete, and note down the measured voltage.
  5. Repeat this calibration process for the remaining two pattern orientations (60° and 120°) and each of the excitation wavelengths.
  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.
  7. Due to axial chromatic aberration, for each wavelength, also apply a z-offset to the sample stage.
    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.
    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.
  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.
  9. Using the camera control software, acquire 9 images of the bead sample.
    1. In the "Sequence" pane of the camera software, select "Hard Disk Record" as the scan type, and change the frame count to 9.
    2. Click "Start" to acquire images.
    3. Save the acquired images as TIFF files by selecting "TIFF" as the image type in the "Save Buffered Images" window, and clicking OK.
  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.