Custom-built two-photon scanned light-sheet microscope (513b)

QBI's custom-built two-photon scanned light-sheet microscope uses near-infrared laser pulses from a Mai Tai HP Ultrafast Laser System (Spectra-Physics Lasers) with a tuning range of 690 - 1040 nm. The laser is scanned using 6-mm clear aperture scanning galvo mirrors (Cambridge Technology), utilising fast scanning of the y-mirror to create two-photon excitation light sheets orthogonal to each other and focused using two Olympus 4x/0.28 NA objective lenses with a working distance of 29.5 mm. The fluorescence signal is detected orthogonally from the light-sheet plane by an 82 % QE pco.edge 4.2 sCMOS camera with Camera Link and a 95 % QE Photometrics Prime BSI, using an electrically-tunable lens and relay lenses in a 4f configuration, and a range of detection objectives, notably the Zeiss 20x/1.0 NA 2 mm WD water-immersion objective, and the LaVision Biotec 12x/0.53 NA 8.5 mm WD MI PLAN objective for aqueous buffers (n=1.33-1.41), with dipping caps for DBE/BABB/DISCO/ECi (n=1.49-1.57, 10 mm WD) and CLARITY/CUBIC/ScaleS (n=1.42-1.48, 10.9 mm WD), and the Olympus 10x/0.6 NA 8 mm WD MPE-dedicated multi-immersion objective (n=1.33-1.52) . Hardware integration is achieved by using custom-written LabVIEW software. In addition to two-photon excitation with the infrared laser, the setup is also able to do single-photon light-sheet illumination made possible by the incorporation of Coherent OBIS 488 nm and 640 nm optically-pumped semiconductor lasers and a Lasos Helium-Neon 543 nm laser (Lasertechnik GmbH). Applications include whole-brain live 3D imaging of zebrafish larvae and fast volumetric imaging of cleared whole brains and organoids.

Features:

• Spectra-Physics Mai Tai HP Ultrafast tunable 2-photon laser (690 nm - 1040 nm) for exciting deep into thick tissues
• Photometrics Prime BSI 95% QE sCMOS camera for maximum sensitivity
• Electrically-tunable lens for fast focus adjustment while scanning lightsheet in Z
• Multiple imaging objectives designed for different sample preparations

Objectives:

Water:

• Zeiss 20xWI / 1.0 NA / 2 mm WD

Clearing:

• LaVision Biotec 12x MultiImmersion / 0.53 NA / 8.5-10.9 mm WD / RI 1.33-1.57
• Olympus 10x MultiImmersion / 0.6 NA / 8 mm WD / RI 1.33-1.52

Fluorescent Filter Sets For:

• GFP / Alexa 488 BP500-550
• Cy3 / Alexa 555/568 /BP570-640

Lasers:

• 690-1040nm tunable 2-photon laser
• 488nm, 561nm, 640nm 1-photon lasers

2-photon lightsheet calcium imaging in larval zebrafish with QBI's Custom-built Lightsheet Microscope

Whole-brain imaging of larval zebrafish with QBI's Custom-built Lightsheet Microscope