Multi-mode Single Molecule Fluorescent Super-resolution System:
Uniform illumination TIRF
When a laser beam is focused 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 induced 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 blur uniformities such as fringes or rings patterns.
Ultrafast incidence angle / TIRF penetration motorization
The 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 several channels even at different penetration depths.
Total Internal Reflection Fluorescence
•360° Spinning TIRF and fast angle motorization
•Simultaneous multi-wavelength TIRF with penetration depth adaptation
•Unmatched illumination uniformity
Total Internal Reflection Fluorescence (360° TIRF) microscopy is the ideal technique for observations close the coverslip surface as it provides the highest axial resolution possible (between 60 to 300nm depending on the angle of incidence). This technique covers a large field of applications such as single molecule tracking, imaging secretion processes, interaction of cell membrane with matrix components or actin filament behavior.
•Lower illumination needed
•Close to coverslip optical sectioning
•No need for wide field light source
Using tilted illumination to lower background blur that results from out-of-focus planes and to enhance the excitation illumination (Dark field laser illumination). As a result, users maintain image quality and achieve less excitation power with less observational bleaching or faster acquisition rates. The oblique illumination sectioning is the extension of the dark field laser illumination. For high incident angles but smaller than the critical angle, starting the TIRF domain, the angle of the excitation beam going through the sample is so high that the illuminated thickness is very thin (around 2μm).
3.Super resoultion localization microscopy（PALM，STORM，...）
•TIRF or WF capabilities
•Lower backgroung for better event detection
•Remove artifacts coming from field non uniformity
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. The scanner 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.
The MetaMorph® Super-Resolution System from Molecular Devices® provides a means to control experimental hardware, capture image sequences, perform localization calculations and display the developing super-resolution image in real time. The MetaMorph Super-Resolution System currently works with many commercially available laser launches as well as TIRF optics, and can be enabled on previously installed imaging systems compatible with MetaMorph Software.
The MetaMorph Super-Resolution System allows:
•Wavelet filtering and Gaussian fitting
•3-D localization using astigmatism
•Real-time super-resolution image display at any CCD frame rate
•Drift correction using fiduciary markers
•Variable scaling of super-resolution image
•Automatic thresholding and splitting of closely spaced molecules
•Single molecule localization text file generation for data exportation
•Image stack acquisition
•Arbitrary acquired image size
•Galvo-based mirror scanning/ Vectorial mode at 20kHz
•Fast Multi ROI/Point targeting
Localized laser action techniques such as Fluorescence Recovery After Photobleaching (FRAP, FLIP), photoactivation, uncaging, photoablation 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 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, it 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.
Using 355nm pulse laser through direct coupling to the scanner for tissue microdisecction or cell ablation. Laser microdissection process does not alter or damage the morphology and chemistry of the surrounding cells.
•TIRF Imaging of membrane proteins or surface-bound molecules
•Molecular diffusion, binding and exchange studies
•Optogenetics, photoactivation and photoconversion
•DNA damage induction