Automated imaging system, equiped for TIRF
The VisiTIRF Cell Explorer is a fluorescence imaging system combining total internal reflection fluorescence (TIRF) and epi-fluorescence microscopy. TIRF microscopy exploits the evanescent field generated at the interface between two substances with differing refractive indices on total reflection of excitation light to specifically observe fluorescence in a narrow section at the interface of the two media (mostly glass and water). Whilst microscopy with a spinning disk confocal yields optical sections of ~1 μm, the TIRF evanescent field only extends ~100 nm into a sample, providing high signal to noise ratio images of fluorescent molecules in and around cell membranes.
Our VisiTIRF system is a custom-modified Nikon Ti-E inverted microscope, built by Visitron Systems. Three illumination sources (mercury short arc lamp, laser and LED) permit both TIRF and epi-fluorescence. The system is equipped with two single photon application EM-CCD digital cameras for maximum sensitivity. Dual-Cam mode can be used for simultaneous acquisition of two channels. A third camera, a Hamamatsu Orca Flash4 V3 is coupled to the right port. This is a sensitive camera (~80% QE in green and red) with a large field of view. The system is (almost) fully automated, equipped with temperature and CO2 control, and Nikon’s Perfect Focus System (PFS).
- Nikon Ti-E inverted microscope
- TIRF condenser
- VS LaserModule with LaserMerge (405 nm (100 mW), 488 nm (50 mW), 561 nm (100 mW) and 640 nm (150 mW))
- Objectives: 20X air N.A. 0.75, 40X water N.A. 1.15, 40x oil N.A. 1.30 and the TIRF compatible 60X oil N.A. 1.49 and 100X oil N.A. 1.49
- High-power LED illumination system: 400, 470, 500 and 635 nm
- HXP 120X illumination system: 120 W mercury short arc lamp
- Microscope filter sets: DAPI, GFP, YFP, mCherry, Cy5 and a quad-band cube (DAPI, GFP, RFP and Cy5) for TIRF
- Perfect Focus System
- Motorized stage with high precision X-Y control and a piezo Z-Stage control for rapid optical sectioning
- Two Hamamatsu EM CCD 1024*1024 pixel digital camera systems: high sensitive frame transfer Electron Multiplication CCD, up to 10 frames/sec, readout noise of <10 electrons
- One Hamamatsu Orca Flash4 V3 sCMOS 2048*2048 pixel camera.
- Dual-Cam optical unit: Dual-Cam cubes for simultaneous GFP/RFP or CFP/YFP imaging
- Incubation system with temperature and CO2 environment control
- System integration and control with VisiView software