Sepia 2 multichannel processor

Freely swimming sperm were tracked using an inverted microscope (IX71; Objective × 20, 0.75 numerical aperture, UPLSAPO; Olympus). Coherent illumination was achieved with a laser light source (LDH-D-C-510, PicoQuant GmbH) and the corresponding controller (Sepia II Multichannel Processor, PicoQuant GmbH). Laser light was coupled into a multimode fibre. A custom-made adapter was used to position the fibre parallel to the optical axis of the objective. The illumination intensity was adjusted to use the dynamical range of the camera (12-bit; PCO Dimax HD). Movies were collected at 600 frames per second; each frame represents a holographic image containing the complete 3D information of the sperm cell.
Caged compounds were photolyzed using a 365-nm LED (M365L2-C; Thorlabs). The ultraviolet light was coupled into a liquid guide (77566; Newport) followed by two Plano-convex lenses (LA 1951-A f=25.4 mm; LA 1509-A f=100 mm; Thorlabs) and coupled to the imaging optical path with a dichroic filter (ff 495-Di03; Semrock). The irradiation power (0.8 mW) was measured with a power meter (detector PowerMax and head model PS19Q; Coherent). The light spectrum was recorded with a spectrometer (51024 DW; Ocean Optics). Photolysis and data acquisition were synchronized using a wave generator (33500B; Agilent).

DIHM was performed in an inverted microscope (IX 71; Olympus) with an oil-immersion objective (40x, UPlanFL N, NA 1.3; Olympus) together with 1.6x magnifying lens in the microscope. A laser (510 nm, LDH-D-C serials, PicoQuant GmbH) driven by the corresponding controller (Sepia II Multichannel Processor, PicoQuant GmbH) was used as a coherent light source. The laser light was coupled into a multi-mode fiber and guided into the microscope. The fiber was held using a custom-made adapter onto the position of the bright light condenser. Sperm cells were placed in a custom-made observation chamber with a depth of $150\mathrm{\mu }\text{m}$ . The hologram was recorded by a fast monochrome camera (pco.dimax; PCO AG) at 1000 frames per second.