Inverted microscope (Zeiss, Axiovert 100TV) with halogen-lamp illumination. 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). By means of dichroic wedges (Chroma, 'Schmidt wedge'), 2 images can be acquired simultaneously. The insertion of controlled astigmatism into the detection beampath the 3-dimensional position of objects are determined at nanometer accuracy. The setup is controlled by LabView (National Instruments) and WinView (Photometrics). Analysis is performed offline using our Matlab package.
Single molecule microscopy, 3D tracking