Spinning disc confocal on an inverted optical microscope (Zeiss Axiovert 200). The inverted microscope has halogen- and Hg-lamp illumination. A commercial atomic force microscope (Digital Instruments, Bioscope) can also be mounted on the axiovert 200.
The microscope is equipped with a 3D piezo scanner (Physik Instrumente, P-733.2DD & P-721.CDQ) allowing for nanometric position control of the sample. The sample stage is temperature controlled (5-50oC) by means of a water bath. The microscope is equipped with objectives ranging between 10x,NA0.3 to 100x, NA1.45. Fluorescence excitation is alternatively achieved by an Ar-laser (Coherent, Innova300, 5W all lines), fiber-coupled into the microscope (20mW) and a diode-laser at 635nm and output power of 30mW. The single-line power at the objective <20mW and the wavelength and excitation timing is controlled by an acousto-optical filter (AA-OptoElectronic, AOTFnC-VIS). The signals are detected on a back-illuminated electron-multiplying CCD camera (Photometrics, Cascade, 512x512 pixel, QE>0.92, noise<7cnt/pxl). The setup is controlled by LabView (National Instruments) and IQ (Andor). Analysis is performed offline using our Matlab package.
Live cell imaging