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Single-Molecule Localization Microscopy
Olympus and Abbelight: A Collaboration of Innovators

Olympus is proud to partner with Abbelight to offer powerful multimodal nanoscopy solutions for our inverted microscopes.

Olympus and Abbelight have joined forces to provide researchers with advanced and intuitive nanoscopy imaging systems. Abbelight’s vast expertise in single-molecule localization microscopy (SMLM) and Olympus’ rich history in optical precision form the foundation of this collaboration. By combining Abbelight’s SAFe nanoscopes with our IX™ series microscopes, users can transform their Olympus inverted microscopes into multimodal powerhouses that feature SMLM, total internal reflection fluorescence microscopy (TIRFM), and multicolor dSTORM all in one system.

2D and 3D Single Molecule Imaging

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TIRF Microscopy

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Multicolor dSTORM

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Meet the SAFe Nanoscopes*

The three Abbelight nanoscopes include the same SAFe Light illumination module, which integrates ASTER technology for homogenous illumination over a 150 × 150 μm field of view and enables EPI, HiLo, or TIRF excitation modality.

SAFe 180
  • 2D single-molecule imaging
  • Sequential multicolor imaging
  • Capable of 3D SMLM via astigmatism
  • Ideal for beginners

SAFe 360
  • Dual camera detection for 3D single-molecule and simultaneous multicolor imaging
  • Extended axial range with nanometer accuracy

  • Dedicated platform for multicolor far-red spectral demixing
  • Cost-effective and robust solution

*Model availability may vary by region.

Modular Nanoscopy Solutions

The SAFe nanoscopes can be incorporated onto any Olympus inverted microscope that features a camera port, making it an easy and flexible  nanoscopy solution. Our IX83 microscope is well suited for nanoscopy applications due to its stable frame, TruFocus™ Z-drift compensation module, and open structure.

SAFe nanoscopes can also be combined with our FV3000 confocal laser scanning microscope and the IXplore™ SpinSR super-resolution microscope system, enabling researchers to maximize their  imaging  capabilities with confocal microscopy, TIRFM, and SMLM in one system.

SAFe 360 and IX83 CAD

How Single-Molecule Localization Microscopy Works

SMLM relies on the ability to stochastically activate only a subset of fluorescent molecules to distinguish them spatially. Repeating the process by acquiring consecutive images, the accumulated raw data are processed in real time to detect and localize every single molecule with nanometer precision (down to 10 nm).

Data quantification and analysis are then performed to resolve either structures or dynamics at the nanoscale level. SMLM is unique because it produces highly resolved images and the 3D coordinates of single molecules, thus creating new avenues for spatial and temporal quantitative analysis.

Raw image

Data processing

Image reconstruction

Unique Excitation System for High Localization Precision with a Wide Field of View

The SAFe nanoscopes all share the same unique excitation system, which is based on the Adaptable Scanning for Tunable Excitation Regions (ASTER) technology.1 ASTER generates homogenous illumination in TIRF, HiLo, and EPI modes while performing SMLM modalities such as PALM  STORM,  or PAINT with a localization precision reaching 10–15 nm in 3D on a field of view (FOV) of 150 x 150 µm.2

ExcitationFOV (100X obj.)Localization precision3D localizationMulti-color imagingSpectral demixing (SD)SPT PALM
SAFe 180 EPI, HiLo, TIRF

150 x 150 µm2

<15 nm YES Sequential NO YES
SAFe RedSTORM EPI, HiLo, TIRF 150 x 150 µm2
<15 nm YES Simultaneous YES NO
SAFe 360 EPI, HiLo, TIRF 150 x 150 µm2
<15 nm YES Sequential or simultaneous YES YES

The ASTER illumination method offers a novel capability to exploit the entire FOV of sCMOS cameras for SMLM and TIRF imaging.

ASTER uses two galvanometer mirrors to control illumination at the sample plane. While the excitation beam keeps its position in the back focal plane (BFP), an angular rotation of a galvanometer induces a similar angle in the BFP, corresponding to a different position in the sample plane.

By applying specific patterns, such as raster scanning, ASTER can provide uniform excitation on tunable FOVs of up to 150 × 150 µm2 for all excitation modes (EPI, HiLo, and TIRF).

Schematic of ASTER and the resulting illumation patterns.

Wide FOV TIRF Imaging with Homogenous Illumination

Olympus is a pioneer in the TIRF microscopy field, and our range of TIRF objectives is designed to provide tight control over the evanescent wave produced in TIRF imaging with magnifications ranging from 60X to 150X. The APON100XHOTIRF objective has the world’s highest NA of 1.7*, while the UPLAPO60XOHR and UPLAPO100XOHR are the world’s first plan apochromat objectives with a NA of 1.5.*

With Olympus’ optics and Abbelight’s ASTER illumination technology, users can achieve homogenous TIRF illumination over a wide FOV.

*As of November 2018. According to Olympus research.

Cultured hippocampal neurons stained for spectrin cytoskeleton and imaged in TIRF microscopy mode. Homogeneous TIRF over the entire field of view of a Hamamatsu Fusion sCMOS camera (larger than the camera port size) was achieved using Abbelight ASTER technology. Sample courtesy of C; Leterrier, NeuroCytoLab, Marseille, and images by Adrien Mau, ISMO, Orsay.

U2OS cells stained for microtubules (alpha-Tubulin antibody) CF660, mitochondria (anti-TOMM20) CF680, and chromatin (EdU) AF647. Simultaneous multicolor 2D dSTORM with spectral demixing.

Multicolor Imaging Using Far-Red Spectral Demixing

Although 3D nanoscopy has revolutionized the fluorescence microscopy field by attaining unprecedented resolutions, multicolor imaging remains challenging in SMLM. This difficulty is due to several factors, including chromatic aberrations, the choice of optimal buffers, and the choice of single-molecule-compatible dyes.

To provide a solution, Abbelight has implemented spectral demixing for SMLM. By separating far-red dyes using a combination of the appropriate dichroic cube and ratiometric algorithms, this technique elegantly enables simultaneous multicolor imaging in SMLM.

In practical terms, it enables users to easily image in multicolor with one buffer, one laser, and one live acquisition.

Powerful Software for SMLM Data Analysis

Unlike standard fluorescence microscopy, which generates pixel-based images, SMLM produces 2D/3D point clouds with millions of localizations and associated uncertainties. Thus, new computational methods are required for the quantification and interpretation of SMLM data to enable researchers to extract novel and groundbreaking biosignatures of biological structures and functions. Designed to be user-friendly and complete, NEO software simplifies data acquisition, providing real-time image reconstruction and quantitative feedback as the number of detections per frame, photons counting, ratio distribution for color assignment via spectral demixing, etc.

Abbelight’s NEO software also offers powerful tools to process SMLM data to study spatial and temporal distribution of localized single molecules. This includes cluster analysis using DBSCAN, Voronoi, and colocalization algorithms as CBC or single-particle tracking algorithms.

Personalized Support

Abbelight provides a unique support system of SMLM expert scientists involved in multidisciplinary research, education, and international training. A dedicated expert is assigned to support the user in each step of SMLM projects: sample preparation, imaging, and data analysis. Users can also acquire expertise and access online tools such as user guides, video tutorials, and best practices on the Abbelight Academy.

A good experiment starts with good sample preparation, so ready-to-use and optimized SMLM kits are also available:

  • Smart kit dedicated buffer for STORM imaging  
  • Blinking pad kit for nonadhesive cells for photo-activated localization microscopy (PALM) and STORM imaging

This comprehensive support means that even SMLM novices can achieve successful results during their first experiment.

Application Notes

Spectral Demixing


How to obtain simultaneous multicolor nanoscopy with one laser

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Single Particle Tracking


How to track single molecules in living cells

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How to Analyze Clusters in SMLM

How to analyze clusters in SMLM data.

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Blinking Pad Kit for Nonadherent Cells

How to immobilize non-adherent cells for single molecule imaging.

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DNA Replication

Imaging DNA replication sites at the nanoscale.

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How to Image Microbes

Imaging microbes at the nanoscale.        

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Neuroscience Week Webinar

How to obtain simultaneous multicolor nanoscopy with one laser


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Discovery Summit Webinar

This presentation focuses on the groundbreaking advantages that single molecule localization techniques have brought to biological imaging

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1: A. Mau, K. Friedl, C. Leterrier, N. Bourg, and S. Lévêque-Fort. Fast scanned widefield scheme provides tunable and uniform illumination for optimized SMLM on large fields of view. Nature Communication. May 21, 2020.

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IXplore SpinSR

High-Content Screeinng

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