SAFe 180 – SMLM & SPT Microscope / Add-On

SAFe 180 – SMLM & SPT Microscope / Add-On

Modality

2D & 3D SMLM (PALM, STORM, PAINT) & SPT

Configuration

Widefield & TIRF / Inverted

Lateral Resolution / Axial Resolution

10 nm / 13 nm (100x/1.49 lens)

Sensitivity

80-95% QE

FOV

150 µm x 150 µm (100x/1.49 lens)

Speed

100 fps (at 2304 x 2304)

SAFe 180 is an illumination system developed by abbelight for high-throughput 2D single-molecule and TIRF imaging.

It can be connected to any camera port of any inverted microscope to provide a very large homogeneous illumination for an affordable price.

The ultimate 2D nanoscope

  • Super-resolution imaging modes: PALM,dSTORM, PAINT, SPT…
  • Illumination modes; automatic switching from EPI, HiLo to TIRF
  • The largest homogeneous 2D Field Of View: 150 x 150 µm²
  • Low power laser needed: abbelight SAFe light technology
  • 3D option with astigmatism: capture range ~ 600nm

SMLM Principle

Breaking the diffraction limit

SMLM relies on the ability to randomly activate only a subset of fluorescent molecules to distinguish them spatially. By repeating the process in consecutive image acquisitions, accumulated raw data are processed to detect single molecules with a nanometric precision (down to 10 nm). Data quantification and analysis are then performed to resolve either structures or dynamics at the nanoscale level. To reconstruct a nanoscopy image, each molecule is detected and localized by specialized algorithms.

SAFe Light: see wider and faster!

ASTER technology

Single-molecule imaging experiments or TIRF microscopy are in many cases limited from the range of the field of view. This is due to the use of Gaussian excitation which does not allow a homogeneous field of illumination on the samples. To solve this issue, we innovated with a very versatile illumination scheme called “Adaptative Scanning for Tunable Excitation Rendering” (ASTER). This can create a uniform field of illumination up to from 25×25μm² to150x150μm²

  • 150×150 µm² field of view with 300 mW laser power
  • More intensity on the sample with lower laser power
  • Illumination adaptable to the sample
  • TIRF, HiLo or EPI illumination modes
  • 16-times more quantitative data
  • Homogeneous illumination, no interference patterns in the image

Description

SAFe 180 is an illumination system developed by abbelight for high-throughput 2D single-molecule and TIRF imaging.

This TIRF microscope add-on can be connected to any camera port of any inverted microscope to provide a very large homogeneous illumination for an affordable price.

The ultimate 2D nanoscope

  • Super-resolution imaging modes: PALM, dSTORM, PAINT, SPT…
  • Illumination modes; automatic switching from EPI, HiLo to TIRF
  • The largest homogeneous 2D Field Of View: 150 x 150 µm²
  • Low power laser needed: abbelight SAFe light technology
  • 3D option with astigmatism: capture range ~ 600nm

SMLM Principle

Breaking the diffraction limit

SMLM relies on the ability to randomly activate only a subset of fluorescent molecules to distinguish them spatially. By repeating the process in consecutive image acquisitions, accumulated raw data are processed to detect single molecules with a nanometric precision (down to 10 nm). Data quantification and analysis are then performed to resolve either structures or dynamics at the nanoscale level. To reconstruct a nanoscopy image, each molecule is detected and localized by specialized algorithms.

SMLM capabilities: from structure to dynamics

Current SMLM approaches only differ in how the fluorophores activation-inactivation is induced. Among them, STORM, PALM and PAINT resolve spatial structures with nanometric precision, while SPT reveals temporal dynamic processes in living cells.

Structures …

dSTORM

direct STochastic Optical Reconstruction Microscopy

dSTORM exploits the photo switching properties of some fluorophores. In certain conditions, fluorophores can be sent to an intermediary state (the “dark state”) from which they randomly cycle between ON and OFF states.

  • Standard organic fluorescent dyes (cyanines, rhodamines, oxazines…)
  • Specific imaging buffer containing a reducer, which induces the transition to a dark state, and an oxygen scavenging system to stabilize this state before returning to the ground state.

PALM

PhotoActivated Localization Microscopy

PALM uses specific fluorescent fusion proteins called “photoactivatable” or “photoconvertible”. The ON state of these proteins can be randomly activated with the proper laser excitation.

  • Photo-activatable or convertible fluorescent proteins (mEos3,2, Dendra2, PA-mCherry, mMaple,…);
  • No specific buffer, live-cell compatible

PAINT

Point Accumulation for Imaging in Nanoscale Topography

PAINT uses specific dyes that are ON only when they are transiently bound to their target. W ith the appropriate dye concentrations, it is possible to achieve a regime where only a few dyes are ON at the same time. DNA-PAINT uses short DNA single strands coupled to fluorophores. These probes are ON when they transiently hybridize to the complementary DNA strand coupled to an antibody targeting the structure of interest.

  • Specific fluorophores that have the ability to emit fluorescence only upon binding to their biological target (ex: Nile Red, which fluoresces only when interacting with membranes)
  • No specific buffer, live-cell compatible.

Dynamics…

SPT: Single Particle Tracking

SPT in SMLM combines Single Particle Tracking with SMLM (PALM or STORM) to obtain spatially and temporally highly resolved diffusion maps of single molecules.SPT need a precise algorithm to found and draw tracks from each molecule in the cell, state of the art algorithm are implemented in Neo Softwares.

SAFe Light: see wider and faster!

ASTER technology

Single-molecule imaging experiments or TIRF microscopy are in many cases limited from the range of the field of view. This is due to the use of Gaussian excitation which does not allow a homogeneous field of illumination on the samples. To solve this issue, we innovated with a very versatile illumination scheme called “Adaptative Scanning for Tunable Excitation Rendering” (ASTER). This can create a uniform field of illumination up to from 25×25μm² to150x150μm²

  • 150×150 µm² field of view with 300 mW laser power
  • More intensity on the sample with lower laser power
  • Illumination adaptable to the sample
  • TIRF, HiLo or EPI illumination modes
  • 16-times more quantitative data
  • Homogeneous illumination, no interference patterns in the image

Manufacturers website: https://www.abbelight.com/