The Andor Revolution XD spinning disk confocal system is capable of performing high speed live cell imaging in 3-D as well as on multiple fields of view. This will allow users to increase their experimental throughput for long-term live cell imaging.
The system is equipped with several key features that will facilitate a wide variety of experimental designs:
1. Nikon Ti PerfectFocus microscope
The active mechanism for compensating focal drift due to thermal instability is key to keeping the user-defined focal plane during long term live cell imaging. Employing a near-IR laser that totally interanlly reflects at the coverslip-liquid interface, the system uses this reflection angle to maintain the user-defined reference point, thus keeping the desired focal plane constant through long-term imaging.
2. FRAPPA photomanipulation laser
The Andor FRAPPA unit allows user to perform several types of photomanipulation experiments including photoablation, photoactivation, photoconversion and photo-uncaging.
The FRAPPA unit can deliver the following laser wavelengths in a Galvo-controlled manner.
405nm, 445nm, 488nm, 515nm, 561nm
This means that users can draw any (and multiple) irregularly shaped regions of interest, to be targeted for rapid photobleaching, photoconversion or photoactivation, simultaneously using multiple laser lines if needed. This set-up is crucial as it will allow users to perform several advanced live cell imaging techniques including fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), photoconversion (with Dendra, Kaede etc), or photoconversion (PA-GFP), and also photostimulation of signaling molecules.
3. Total Internal Reflection Fluorescence (TIRF) module
The system is also equipped with a TIRF module for high resolution studies of cell-substrate interaction. Employing the evanescent wave that penetrates into the substrate following total light internal reflection, the TIRF microscope allows users to study fluorescent molecules at 70-100 nm past the coverslips, which is significantly thinnner than the confocal axial resolution of ~1µm. Our Andor system is designed to perform TIRF imaging for all five wavelengths as stated above.
4. Automatic X-Y stage
Equipped with an ASI motorized X-Y stage, this system can easily perform time lapse microscopy at multiple user-defined XY positions, thus increasing the experimental throughput with simultaneous acquisition of multiple time-lapse videos.
5. Tokai HIT stage-top incubator
The stage top incubator is CO2-buffered heating stage, allowing users to perform long term live cell imaging without having to worry about continual perfusion of culture medium.
6. Neo sCMOS cameras
Our Andor system is uniquely equipped with two neo sCMOS cooled CCD cameras, which have 6.5µm pixels (2560X2160 active pixels), and is cooled to -40 C for exceptionally low read noise of 1 e- rms. sCMOS camera also has a frame rate of 100 fps at full frame capturing speed. These cameras will allow users to perform sensitive and rapid image acquisition, thus reducing the light intensity needed to acquire images, and also minimizing photobleaching. The duplication of this camera allows us to bypass the spinning disk confocal head for low light imaging work such as TIRF, and also allow us to offer significantly better DIC imaging.
This instrument is purchased with the support of an S10 Shared Instrumentation Grant S10RR031680-01 from the National Center for Research Resources (NCRR), awarded to Dr. Vladimir Gelfand (Cell & Molecular Biology).
