3-dimensional single molecule detection (3D-SMD) can be performed using the Highly Inclined and Laminated Optical (HILO) method using a modified illumination method of Total Internal Reflection Fluorescence Microscopy. Compared to TIRF the angle of incidence is slightly reduced thus the excitation beam does not suffer total reflection but penetrates the sample and illuminates a thin slice of pattern providing a contrasting image in deeper sample planes. The optical axis movement of the sample is performed by a piezo Z-stage thus allowing 3-dimensional imaging of the sample.
Microscope: Olympus IX83
Lasers: 488 nm, 639 nm
Objectives: UMPLFLNW 20x, UAPON OTIRF 100x OIL,
Camera: Hammamatsu Orca-Flash 4.0
Z-stage: Physik Instrumente, PI E-709 piezo Z-stage