In our theoretical opinion and experience, the best way to generate TIRF images through a microscope objective is to induce the evanescent wave from as many points as possible around the circumference of the lens, so as to average out ring and fringe artefacts. By using a pair of fast galvanometer mirrors to flexibly scan the back focal plane of the objective within each camera frame, the ILAS 2 system achieves this and also allows oblique illumination, multiwavelength depth correction, and widefield imaging from the same laser source.
Fast Scanning Multi-Depth TIRF
Systems built around the ILAS2 with MetaMorph software are extremely flexible as the ILAS head has provision for an additional pair of galvo scanners for FRAP / OptoGenetics / Photolysis and there is a further bypass port for standard (non-laser) widefield imaging. We configure systems with EM or sCMOS cameras, or sometimes both using our TwinCam adapter.
The powerful control interface in MetaMorph allows automatic adjustment of TIRF angles for different colours and fast interchange between widefield, TIRF, and oblique illumination. This is also the preferred illumination source for our realtime 2D and 3D STORM systems using a Cairn zoom attachment to achieve the high intensities needed from standard diode lasers.
- “Circular” TIRF removes fringe and ring artefacts
- Large field of view with high uniformity
- Multiwavelength and multidepth in realtime
- Oblique illumination for 3D STORM
- Single molecule localisation & tracking
- Kinetic studies of single molecule interactions (i.e. ligand binding, protein:protein and protein:DNA interactions)
- Kinetic studies of proteins (i.e. actin filaments & microtubules)
- Super-resolution techniques (i.e. PALM/STORM) for subdiffraction localization of single molecules
- Imaging cellular dynamics (i.e. secretion processes & vesicle trafficking, plasma membrane dynamics, cytoskeletal dynamics)
- Imaging organelles close to the plasma membrane (i.e. mitochondria, ER, Lysosomes)
- Imaging ion channel activity in the plasma membrane and ER (i.e. Calcium signalling)
- Imaging cellular signalling (movement and recruitment of ion channels, receptors, G-proteins etc.)