The three-dimensional single molecule detection (3D-SMD) is conducted by HILO methods. HILO is the abbreviation for Highly Inclined and Laminated Optical sheet, which is derived from TIRF method. In this case the angle of incidence is slightly less than the critical angle. The laser beam penetrates into the sample exciting a thin (approximately 10 µm) optical section providing an image with relatively high signal to noise ratio. A piezo Z stage shift the sample in the direction of the optical axis making possible the three dimensional scanning of sample identifying single molecules in it.
- 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