TIRF
Total Internal Reflection Fluorescence microscopy (TIRF) (Axelrod, 2013) is the most suitable imaging method as it is offering an advantageous optical sectioning (up to a few hundred nanometers after the coverslip. Nevertheless, in practice, TIRF microscope remains quite difficult to preset for non-expert users. It requires perfect optical conjugation of the microscope and very precise positioning of the beam in the Back Focal Plane (BFP) of the objective (Mattheyses et al., 2010), resulting in experiments that are not reproducible from day to day.
What is Abbelight TIRF?
This technology is a hardware and software solution that provides an optically perfect TIRF imaging. Adaptable on any combination of microscopes, objectives, XYZ stages and accessories, our new product guarantees the automatically calibrated and reproducible positionned TIRF angles.
Coupled with a detection optical module, it offers an optimal and simultaneous multicolor imaging with the benefits of ASTER technology. This technology provides an ultra-widefield and speckle-free illumination of biological structures close to the coverslip such as membranes, focal adhesions, and much more.
Adaptable
The optical system installed in Abbelight SAFe excitation device is designed to obtain the optimal TIRF angle position and get the perfect TIRF illumination compatible with any combination of:
- microscopes
- objectives
- XYZ stages
- accessories
Very large and uniform FOV
Abbelight patented ASTER method offers homogeneous TIRF illumination of the sample over the largest FOV on the market.
ASTER TIRF illumination, co-developed with Sandrine Lévêque-Fort and published in Nature Communication (A. Mau et al., 2021), outperforms standard TIRF strategy because it breaks classical interference fringes like the Ring/Azimuthal TIRF method.
ASTER TIRF illumination also surpasses Ring/Azimuthal method because it is offering a >95% uniform excitation over the largest FOV, while Ring/Azimuthal TIRF is strongly limited by the gaussian shape of the laser irradiance.
Automatic, quantitative and reproducible
Our technology combined with a software application and a calibration sample allows automatic definition of the TIRF angles with <1,2% error, leading to reliable data over time.
- Software application integrated into AbbelightTM SAFe NEO aids in calculation and backup of TIRF angles based on image processing algorithm, that exploits image plane intensity as well as distribution and segmentation of your objective’s BFP
- Calibration sample based on nanometric beads embedded into a highly controlled and long life-time gel with refractive index of water. The calibration sample is supplied with our Support Program Abbelight Care 1
- Ultra stable TIRF stage based on a closed-loop long range piezo stage offering nanoscale repositionning of the beam at BFP
Reliable multicolor imaging
A robust optical design and software architecture leads to highly stable, multicolor imaging without chromatic aberration, optical artefact or misalignment over time.
Optics inside the illumination device are designed to reduce drastically chromatic aberration for the range 488-640 nm, thus offering TIRF excitation over a wide spectrum.
Special custom dichroic CUBES have been designed to remove ghost images and also to reduce crosstalk between channels, thus offering up to 4 colors simultaneous imaging.
Highly stable optomechanical components holding dichroic cubes have been carefully chosen to simplify the alignment of the 4 colors images. A software digital alignment tool has also been developed to offer < 0,7 pixel precision of alignment.
Multi-dimensional acquisition for TIRF and multicolor imaging
Multiple acquisition scenarios with adjustable parameters developed to best meet the needs of TIRF applications.
