Elyra 7 Lattice SIM²

Super-resolution · Zeiss

Our flagship super-resolution platform. Combines Lattice SIM², single-molecule localization microscopy (SMLM/STORM/PALM), and TIRF in one system — with full live cell environmental support for dynamic super-resolution imaging.

Instrument Features

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Lattice SIM²

Second-generation Lattice SIM provides ~60 nm lateral resolution

📍

SMLM — Single Molecule Localization

Supports dSTORM, PALM, and PAINT modalities for nanoscale imaging down to ~20 nm resolution. Thousands of single molecule events are localized and reconstructed into a super-resolution image. Ideal for precise structural analysis of protein assemblies.

🌊

TIRF — Total Internal Reflection

Variable angle TIRF limits excitation to a ~100–200 nm evanescent field above the coverslip. Dramatically improves signal-to-background for membrane-proximal structures, receptor dynamics, vesicle fusion, and cytoskeletal events at the cell surface.

🌡️

Live Cell Incubation

Full environmental control (37°C, 5% CO₂, humidity) enables live super-resolution imaging. Lattice SIM² is gentle enough for time-lapse live cell super-res — far less phototoxic than STED or SMLM for extended acquisitions.

⚡

High-Speed sCMOS Camera

Back-illuminated sCMOS camera provides high sensitivity, large field of view, and fast frame rates essential for live Lattice SIM acquisition and SMLM blinking event capture.

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Multimode Flexibility

Switch seamlessly between Lattice SIM², TIRF, and SMLM modes within a single session. Combine widefield, TIRF, and super-resolution channels on the same sample for correlative experiments.

Startup Guide

1

Before you start

Book time in iLabs, complete training with Adeela and bring your sample on a No. 1.5H coverglass bottom dish. All new users need a training session before independent access

Prepare your sample as described in the OBC Sample Preparation Guide. The Elyra 7 requires EM grade fixation, nuclei stained before mounting, and #1.5H glass

Book instrument time in iLabs

For new researchers, come during regular office and let Adeela know so she can be available to assist

2

Power ON & Launch ZEN Black (Elyra)

  1. Live imaging only: turn on the incubation chamber before any other switch. Set up CO₂ by following the instructions posted on the wall next to the CO₂ tank. Skip this step for fixed samples.
  2. Press microscope switches 1 and 2.
  3. Press switch 3 for the HXP lamp. The HXP unit sits on the desk shelf beneath the monitor.
  4. Login to computer using OBC credentials
  5. Open ZEN (Black edition) on the computer. It always starts on the Locate tab.
3

Load your sample and find it in Locate

Always find your sample on the 10x objective first, then change to your final imaging objective.

  1. Choose the magnetic stage insert that fits your imaging dish and fit it to the stage.
  2. Place the dish in the insert while the 10x objective is in position.
  3. In the Locate tab, choose BF for brightfield or FL for fluorescence.
  4. Focus on your sample with the 10x and center the area you want to image.
  5. Move to your final imaging objective in ZEN. Add the immersion medium that objective requires, oil or water, before you refocus.
  6. Refocus in Locate with the new objective, then go to the Acquisition tab.

Use only the immersion medium designated for each objective, and clean the objective with lens paper when you finish

4

Lattice SIM and Apotome imaging

Start from settings saved with Adeela when possible, because the light path, grid and filters are already validated for your sample. Only build new settings if your fluorophores or mode differ.

  1. Click the Acquisition tab.
  2. Reuse saved settings: load the experiment or settings file made with Adeela, or select a previous image and click Reuse to copy its parameters. Then skip to the checks in step 5.
  3. Create new settings: open Imaging Setup and choose the mode for your experiment:
    • Apotome for optical sectioning of thicker or lower signal samples.
    • Lattice SIM for super resolution.
    • Laser WF for SMLM and TIRF (see the SMLM and TIRF section).
  4. For Lattice SIM or Apotome, build the light path:
    • Add one track per fluorophore combination in the Channels window and tick the cameras you need. Start from longest to shortest excitation
    • Pick the emission filter and beam splitter for each track. Do not change the filter between tracks within one acquisition.
    • Select the 1.6x lens option.
    • Use the laser blocking filter that covers your lines. Mostly we just use the 405/488/561/642 blocking filter for Lattice SIM
    • The grid is picked automatically from the active laser.
    • IF USING BOTH Cameras, but sure to run “Camera Alignment” before imaging
  5. In the Channels window, switch on only the lasers your labels need, then set laser power and exposure:
    • Start low, around 0.2% laser power, and raise it only if the signal is weak.
    • Cameras on the same track share one exposure time, so adjust brightness with laser power.
    • Set bit depth to 16 bit.
  6. Choose the number of phases in the Acquisition Mode window. The 13 phase option is recommended by one facility as the best balance of resolution, time and bleaching. Option to switch to 9 phases for live imaging
  7. Set the field of view (frame size) or select “crop”. It changes the area imaged, not the pixel size.
  8. Optional extras: tick Positions to visit several fields, Time Series for time lapse (the interval must be longer than the time for one full image, and ZEN will not warn you if it is not)
  9. SAVE all data to the “SWAP” drive for post imaging processing. See below for more instructions

Lattice SIM delivers more light than widefield, so bleaching and phototoxicity are higher. Keep laser power as low as gives a usable image, because strong bleaching causes reconstruction artefacts

5

SMLM and TIRF imaging (Laser WF)

Follow the power on and Locate steps above, then use these steps instead of the Lattice SIM steps. SMLM gives localisation precision of roughly 20 nm, but it needs a suitable blinking sample and imaging buffer. Settings below come from the Elyra PS.1 guides at other facilities and the Elyra 7 manual,

  1. In the Acquisition tab choose Laser WF in Imaging Setup. The camera is selected here, and the filter and beam splitter are set per track as for widefield.
  2. Select the 1.6x lens option and the high NA oil objective your trainer assigns for SMLM.
  3. Set the frame size to the full chip or a cropped region, and the bit depth to 16 bit.
  4. In Channels, switch on the laser for your dye (for example 642 nm for Alexa 647) and start at low power. Use the Range Indicator: red pixels are saturated and blue are underexposed.
  5. Choose the illumination mode: EPI, HILO or TIRF
  6. For TIRF, adjust the angle until the field is evenly bright from left to right and background is lowest, then save the angle. One guide notes the uHP TIRF setting is not evenly illuminated, so check with staff which option to use
  7. Turn on Definite Focus for long runs, after Find Surface reports Reflex found.
  8. Drive the dye dark first. Run a time series of at least 20,000 frames with high laser power so most molecules bleach or go to the dark state, then raise power stepwise without overexposing the camera. Wait until single molecules are distinguishable before recording.
  9. If the fluorophore does not blink well, add the 405 nm or 488 nm activation laser at very low power, typically 0.001 to 1% in the cited guide.
  10. Set the exposure, commonly 30 to 50 ms in the cited guide, and the same exposure for every camera in the track. If the exposure value turns red it is not compatible with the frame size.
  11. Set a Time Series with the shortest interval. Published guides record about 20,000 to 50,000 frames.
  12. Click Start Experiment, then save the raw data to the location your trainer gives you.

Plain TIRF imaging uses the same Laser WF setup: set the mode to TIRF, find and save the angle, set exposure, check saturation with the Range Indicator, then click Snap or Start Experiment.

6

Processing SMLM data

  1. Open the Processing tab, choose PALM, click Select to use the displayed image.
  2. In Settings choose how to treat overlapping molecules: discard them, ignore overlap (more molecules but lower precision), or account for overlap (multi object fit, slowest and most accurate).
  3. Set the Peak Mask Size and Peak Intensity to Noise in Peak Finder. Too small a mask misjudges the baseline, too large creates overlaps
  4. Under Localizer choose the x,y 2D Gauss Fit. Do not tick Average before Localization. Click Apply.
  5. Correct drift with PAL-Drift, using fiducial beads or the model based method
  6. Merge repeat detections with PAL-Grouping by setting Max On Time, Off Gap and Capture Radius, then Group.
  7. Remove poor localisations with PAL-Filter. Precision above 100 nm is removed by default, so tighten the range if needed.
  8. Render the image in PAL-Rendering. Set the pixel size to no less than half the mean localisation precision, then choose a display mode such as Gauss or Centroid.
  9. Right click a data table to save it, and use Convert To Image to save the rendered image.
7

Processing in ZEN Black Apotome/SIM images. This is best done on Workstation

Raw Lattice SIM data must be processed before analysis. Process a few files on the microscope computer, and move large datasets to a processing computer if your trainer offers one [4]. Move data off shared computers when finished [4].

SIM and SIM² for a single image

  1. Open the raw image, then go to the Processing tab and select SIM in the method list. Method Parameters stay grey until an image is open and selected.
  2. Click Select to use the image you have open.
  3. ZEN recognises Lattice SIM Z stacks and leap data and sets 3D automatically. For 2D data choose 2D or 2D+. 2D+ suppresses out of focus planes for better sectioning.
  4. Choose the Sharpness preset: Weak, Standard or Strong. Stronger keeps more high frequency detail but also more noise. Leave Auto on, or untick it to fix a value from 1 to 15 across a whole dataset
  5. For SIM², tick the SIM² box and choose Weak, Standard or Strong for a Live or Fixed sample. Use Low Contrast for large homogeneous structures such as DAPI. Raise strength only if signal to noise is good, otherwise signal is lost and noise rises.
  6. Test SIM and SIM² on your data and adjust each channel if needed. Use one method for all channels within a dataset
  7. For quantitative work, tick Scale to Raw Image. Leave it off only for presentation images.
  8. Click Apply. The result opens as a new image named with _SIM or _SIM2 added. Save it manually.

SIM² preset values from the manual:

PresetSampleIterationsRegularization weight
WeakLive120.15
StandardLive160.065
StrongLive200.02
WeakFixed150.065
StandardFixed200.025
StrongFixed250.007
Low Contrast (best for DAPI0Any50.8

A higher regularization weight softens the image and a lower one increases resolution. Choose the lowest weight that gives no artefacts. Processed SIM images have half the pixel size and four times the XY pixel count of the raw data, so files are larger.

Reuse settings and batch processing

  1. Click Save to store the settings as a .par file, and Load to reuse them. Check the Info tab of a processed image to confirm which settings were recorded.
  2. Tick Use settings for batch (named Use Current for Batch in some versions) and click Run Batch, then select the raw files in the folder. Output is saved beside the raw data and does not open automatically .
  3. SIM² batch output uses the _SIM name and can overwrite earlier SIM results, so check date and time stamps
8

Shutdown

  1. Clean oil off all objectives and go back to the 10x objective.
  2. If incubation chamber was on, turn “off” and close CO2 tank per instructions on wall
  3. Make sure all data is saved to the SWAP drive for processing
  4. Exit out of ZEN and logoff
  5. Check iLabs for when the next user is coming in.
    1. If someone is coming in within an hour, leave everything on
    2. If someone is coming in within 24 hours, turn off “3” and leave 1, 2 on
    3. If no one is scheduled to come in within 24 hours shutoff 1, 2& 3
9

References used for this guide

  1. Max Perutz Labs, Elyra: Setting up Lattice SIM
  2. Max Perutz Labs, Elyra: Data processing manual
  3. Institut Jacques Monod, Manuel ELYRA 7
  4. UTHSC Advanced Imaging Core, Elyra 7: Taking a SIM image
  5. Queensland Brain Institute, Elyra PALM/STORM guide (2018)
  6. Cornell Biotechnology Resource Center, 2D PALM/STORM on the Elyra
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