Resources - Advanced Microscopy Facility

Welcome to the Advanced Microscopy Facility’s Resources hub. Whether you are a first-time user trying to figure out how booking works, a seasoned super-user looking up acknowledgement text, or simply curious what on earth a “depletion beam” is, there is something here for you.

Access and the working model

Booking, fees, and the three-tier support model that keeps the facility running.

How the facility runs

Imaging SOPs

Quick-start guides per instrument. Abbelight is live; ONI and Abberior STED guides are on the way.

Operating procedures

Image analysis

Freeware pointers, commercial software per instrument, and where to ask for help.

Analysis pointers

Super-resolution primer

STED, STORM, PAINT, MINFLUX. What they are, how they differ, and when to use which.

Read the primer

Choose a microscope

A short decision tree to match your sample and your question to the right instrument.

Find your microscope

Learn more

Vendor learning hubs, open educational resources, foundational papers and books.

Reading list

Data handling

All raw and processed image data should be managed via the TU/e research data platform. For storage allocation, retention rules, backup procedures, and data-use guidance, see cockpit.research.tue.nl.

The microscope workstations are not for permanent storage. Please move your data off after each session so the next user starts with a clean disk. The workstations are working benches, not graveyards.

Acknowledgement and citation

If your work used the ICMS Advanced Microscopy Facility, please acknowledge the facility and cite the founder paper(s) for the technique you used. Below is a general facility acknowledgement, followed by per-instrument blocks you can copy directly into your manuscript.

General facility acknowledgement

We acknowledge the ICMS Advanced Microscopy Facility, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, for access to advanced microscopy infrastructure and technical support.

ONI NanoImager

The ONI NanoImager is a single-molecule localisation microscopy (SMLM) platform. Work using the NanoImager builds on the foundational PALM and STORM papers, please cite both.

We acknowledge the ICMS Advanced Microscopy Facility, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, for access to the ONI NanoImager and technical support with single-molecule localisation microscopy. Founding references for SMLM: Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642-1645 (2006). https://doi.org/10.1126/science.1127344 Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793-795 (2006). https://doi.org/10.1038/nmeth929

Abberior STED ExpertLine

The Abberior STED ExpertLine implements stimulated-emission-depletion microscopy. Please cite the original STED proposal.

We acknowledge the ICMS Advanced Microscopy Facility, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, for access to the Abberior STED ExpertLine and technical support with stimulated-emission-depletion (STED) microscopy. Founding reference for STED: Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Optics Letters 19, 780-782 (1994). https://doi.org/10.1364/OL.19.000780

Abbelight SAFe-360

The Abbelight SAFe-360 (on a Nikon Ti2-E body) supports multi-colour SMLM including STORM, PALM, and DNA-PAINT. Cite the relevant founder paper(s) for the modality you used.

We acknowledge the ICMS Advanced Microscopy Facility, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, for access to the Abbelight SAFe-360 system and technical support with single-molecule localisation microscopy. Founding references for SMLM: Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642-1645 (2006). https://doi.org/10.1126/science.1127344 Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793-795 (2006). https://doi.org/10.1038/nmeth929 For DNA-PAINT acquisitions, also cite: Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nature Methods 11, 313-318 (2014). https://doi.org/10.1038/nmeth.2835

Glossary

A short field guide to the vocabulary you will hear around the facility. Definitions are kept plain. When in doubt, ask the super-user.

Fluorophores

Bleaching: Irreversible loss of fluorescence as the dye is photochemically destroyed. Eventually it happens to every fluorophore. The question is just how fast.
dSTORM: Direct STORM. Single organic dyes are switched between bright and dark states in a reducing thiol buffer; no second activator dye required.
Fluorophore: A molecule (organic dye, fluorescent protein, quantum dot) that absorbs light at one wavelength and re-emits it at a longer one.
PALM: Photo-Activated Localization Microscopy. SMLM using genetically encoded photoactivatable fluorescent proteins.
Photoactivation: Switching a fluorophore from a dark to a fluorescent state using a (typically UV/violet) activation pulse, the trigger that drives PALM and many STORM variants.
Photoswitching: Reversible transition between fluorescent ON and dark OFF states. The chemistry that makes single-molecule blinking possible.

Optics

Confocal: Point-scanning fluorescence microscopy with a pinhole that rejects out-of-focus light. Gives optical sectioning and a modest resolution boost over widefield.
Depletion beam: The donut-shaped laser in a STED microscope. Its intensity drives stimulated emission everywhere except the central zero, only molecules at that zero are allowed to fluoresce.
Numerical Aperture (NA): NA = n·sin(θ). Sets both how much light the objective collects and the diffraction-limited resolution (Δr ≈ 0.61 λ / NA). Bigger is sharper.
Point Spread Function (PSF): The 3D blur that a single point emitter produces in the image. The microscope’s impulse response, and the reason resolution is finite.
Structured illumination: Illumination with a sinusoidal interference pattern. The resulting moiré fringes encode high-frequency sample information that an algorithm decodes for ~2× resolution gain.
TIRF: Total Internal Reflection Fluorescence. Light totally-internally-reflects at the coverslip, and only the evanescent field (~100–200 nm deep) excites fluorophores. Gold-standard background suppression for SMLM.
Widefield: Whole field-of-view epi-illumination with a camera detector. The baseline mode for most fluorescence microscopes.

Acquisition

Blinking: Stochastic ON/OFF flickering of a fluorophore, from photoswitching, transient binding, or photophysical dark states. The raw signal that SMLM is built on.
Coverslip thickness: Standard high-NA objectives are designed for #1.5 (≈0.17 mm) coverslips. Wrong thickness introduces spherical aberration and silently degrades resolution.
Drift correction: Computational removal of stage and sample drift over long acquisitions. Usually based on fiducial markers or image cross-correlation.
Fiducials: Bright, photostable point markers (e.g. 100 nm gold beads, FluoSpheres) embedded in the sample for drift correction and channel registration.
Fixed-cell imaging: Imaging on chemically fixed (typically paraformaldehyde-treated) samples. No live dynamics, but compatible with the harshest buffers and longest acquisitions.
Live-cell imaging: Imaging on cells kept alive on the stage, temperature, humidity, and CO₂ controlled, light dose minimised. Watch for phototoxicity.
Point cloud: The list of (x, y, [z, t, intensity]) localisations that constitutes an SMLM dataset before any image is rendered.

Analysis

Deconvolution: Computational sharpening that inverts PSF blur. Standard post-processing for confocal, STED, and SIM data.
Diffraction limit: The classical resolution floor (~λ/2, or ~200–250 nm in the visible). Super-resolution methods sidestep, not break, this limit by exploiting fluorophore photophysics.
DNA-PAINT: SMLM by transient hybridisation of dye-labelled imager strands to docking-strand-tagged targets. Enables multiplexed (Exchange-PAINT) and quantitative (qPAINT) imaging.
Localisation precision: Standard deviation of repeated single-molecule position estimates; scales as σ ∝ 1/√N (Thompson formula). Not the same thing as image resolution.
MINFLUX: Single-molecule localisation by iteratively placing a donut-excitation minimum on the emitter. Reaches ~1 nm precision with roughly 22× fewer photons than centroid fitting.
SMLM: Single-Molecule Localization Microscopy, the umbrella term for STORM, PALM, dSTORM, fPALM, DNA-PAINT, and related point-by-point reconstruction methods.