Inverted microscope with TIRF unit
Van Noort group
Home built inverted microscope based on Nikon 100x
Single molecule microscopy, also single-pair FRET experiments
- Single-molecule sensitivity with S/B>50 at 100 Hz
- By means of dichroic wedges (Chroma, 'Schmidt wedge'), 2 images can be acquired simultaneously, essential for single-pair FRET experiments.
- Two color alternating excitation (515 and 636 nm) by Ar-laser (Coherent, Innova300, 5W all lines), fiber-coupled into the microscope (20mW) and a diode-laser at 635nm and output power of 30mW. Wavelength and excitation timing controlled by acousto-optical filter (AA-OptoElectronic, AOTFnC-VIS)
- NA 1.45 objective
- Temperature controlled sample stage (5-50oC, water bath)
- Positional accuracy <40nm in xy with 3D piezo scanner (Physic instrumente, P-733.3DD)
- Signal detection on back-illuminated electron-multiplying CCD camera (Photometrics, Cascade, 512x512 pixel, QE>0.92, noise<7cnt/pxl)
- Motor controlled switch between wide-field and TIR mode
- Setup is fully controlled by LabView.
- Analysis is performed offline using LabView.