The iLas MODULAR system is a unique multi-application device that offers complete control over any laser illumination. Its evolutive design allows researchers to choose and simultaneously combine :
- 360° TIRF imaging
- Single molecule imaging
- oblique illumination imaging
- Laser ablation
- Fast spinning disk confocal imaging
- Widefield imaging
Uniform illumination TIRF
Classical TIRF systems suffer from non uniform illumination and interference patterns the image. When a laser beam is focussed at the back of an objective and spins to describe a circle, each point of that circle creates a parallel beam which has the same incidence angle onto the coverslip. Thus, in TIRF and for a given wavelength, the evanescent wave resulting from each spot has the same penetration depth. However, interference patterns depend on the azimuth of the beam. Being able to spin the Laser beam very rapidly during the exposure time of the camera will removes fringes or rings patterns.
Ultra fast incidence angle / TIRF penetration motorization
The iLas² galvonometer based motorization enables to change the TIRF penetration depth in less than a millisecond, making it compatible with "overlap" streaming acquisition. Even complex multicolor Widefield/TIRF experiments can be carried out.
Multiple wavelengths / Wavelength correction
The fast motorization can be used to correct the penetration depth for its wavelength dependency. Advanced acquisition functions are also available to image simultaneously severals channels even at different penetration depths.
Targeted laser illumination (FRAP/PA/Ablation)
Localized laser action techniques such as Fluorescence Recovery After Photobleaching (FRAP, FLIP), photoactivation, uncaging, photo-ablation are very powerful tools to photo-manipulate tissues or to analyze intracellular dynamics of proteins and other macromolecular complexes. For example, FRAP permits perturbation of the steady state fluorescence distribution by bleaching fluorescence in selected regions. After the bleaching step, researchers can observe and analyze how the fluorescence distribution returns to the same or a different steady state, giving appraisal on the spatiotemporal half life of molecule of interest within one particular site of a living sample. Photo-activation or photo-conversion make use of photo-convertible probes, allowing morphological “pulse and chase” experiments.
The iLas² system provides an easy-to-use interface to manage the lasers, set-up ROIs and plan the experiment. In order to lighten the acquisition process and enhance steering speed, iLas² is driven by its own electronic. Vectorial scanning and live action mode provide the ability to measure the fastest phenomena. The user can bleach fast-moving structures and analyze their recovery as they continue to move with the help of tracking algorithm.
Hela cells transfected with GFP-tubulin. The laser nano-ablation @355nm has been done in live stream mode at 10 fps with 100ms exposure time. The arrows point the minus-end behavior following the ablation. Several microtubules were cut during the same acquisition without compromising cell viability. Dr. Ilya Grigoriev, Dept. of Cell Biology, Erasmus MC, Rotterdam.
Single molecule super resolution
Single molecule detection and tracking are very demanding techniques. Both require high performance imaging capabilities and the premium optical quality at the excitation and at the emission. iLas² provides the ability to produce wide-field laser illumination (either wide-field, oblique or TIRF) while it significantly improves the illumination uniformity. Thus, the probalitity to excite and to detect are not modulated by random fringe patterns and artifacts are avoided on high resolution reconstructed images.
Image courtesy JB Sibarita IINS Bordeaux
Images of a fluorescent layer made with TIRF illumination. In stationary mode (left), diffraction rings and fringes of various frequencies can be observed. In "spinning" mode those modulations disappear.
Precise motorisation enables fine TIRF angle tuning. Hela cells transfected with GFP Lifeact were imaged at 400/150/70 nm TIRF penetration depth.
iLas MODULAR user interface
HeLA cells expressing (mRFP-LCa clathrin lightchain). Several single-point ROIs werebleached at once to enables multiple quantifications.Image taken by G. Montagnac @InstitutCurie, Paris.
In-vitro actin polymerization imaged with TIRF modality. FRAP experiments have been realized to investigate the filaments polarity and growth mechanism from the imposed nucleation geometry. Image courtesy of L.Blanchoin, iRTSV/LPCV, CEA Grenoble.
Cells transfected with Lifeact-mCherry in which areas of actin filaments are disrupted during mitosis before chromosome segregation, using laser ablation at 355nm over the red rectangles. Fink et al., Nature Cell Biology.
Wide-field images of living M10 cells, expressing YFP-Langerine. Left image has been illuminated with perpendicular laser illumination. Right image has been illuminated with 50° tilted illumination using the samepower and acquisition settings. Background went down from 157 to 76 gray levels (white square region). Image courtesy of J.Salamero, Institut Curie, Paris.