Zeiss Lightsheet

Zeiss LightSheet

The Zeiss LightSheet Z.1 allows fast 3D fluorescence imaging of live or fixed and cleared specimens up to 1 x 1 x 2 cm with reduced phototoxicity and photobleaching.

LightSheet Fluorescence Microscopy (LSFM)

LSFM is based on the Planar Illumination Microscopy (PIM) technique which works by illuminating a specimen with a thin sheet of light that passes through the whole focal plane in the direction of illumination, and gathers the emitted photons in the orthogonal plane. The Zeiss Z.1 microscope illuminates the specimen from both the left and the right side of the specimen and detects the emitted fluorescence orthogonally through a single detection objective. In addition, the thin light sheet is scanned along the focal plane to cover the whole field of view and provide a more homogeneous field of illumination.

The Micro Imaging Facility’s LightSheet Z.1 is coupled with an Environics 4000 gas mixing system to fully control the levels of O₂, CO₂ and N₂ and a tightly regulated thermo chamber, allowing long term live imaging.

System Specifications

• Dual side illumination
• Ultrafast light sheet Pivot scan
  4 axis sample positioning system with XYZ and α-stage: motorized
• Overview camera with LED illumination for easy specimen positioning
• Fully equipped for live imaging, temperature and O2, CO2 and N2 controlled
• Software: ZEN Black 2014

Detectors

2x 16-bit sCMOS PCO.Edge cameras for simultaneous dual colour acquisition (1920 x 1920 pixels)
Frame rate from 10 to 30 fps at 1000 x 1000 pixels

Illumination objectives

• Zeiss LSFM 5X/0.1 (in combination with 5X and 10X detection objectives
• Zeiss LSFM 10X/0.2 (in combination with 20X objectives)
• Light sheet thickness 2 μm - ~14 μm

Detection Objectives

Lasers

Filters for emission selection

Laser Blocking Filters (LBF)
Filter Module LBF 405/488/561/638
Filter Module LBF 445/515/561
Filter Module LBF 445/515/638

Environics 4000

The Environics 4000 instrument, fully controlled by a standalone software, allows tight regulation of gas concentration, flow and schedule throughout the imaging session.

Environics 4000 Applications

Live imaging

Developmental processes such as cell migration, gene spatio-temporal expression pattern, organ morphogenesis.
Biological dynamic processes such as blood flow, calcium flux.
3D Cell Culture: organotypic and organoid culture, 3D cell culture proliferation

Fixed/cleared imaging

Fast 3D imaging of in toto specimen fixed and fluorescently labelled for volume analysis, atlas preparation. Larger specimen can now also be imaged after clearing, saving hours of slicing, microscopic slide preparation and classic confocal imaging and reconstruction, which are time consuming methods.

Acquisitions post-processing

ZEN Black dual-side imaging fusion
Huygens Multiview fusion and LSFM-dedicated deconvolution
Arivis Vision4D mosaic reconstruction, 3D/4D visualisation, movie recording.

Citation

[Live] Three-dimension images of [specimen] were acquired on a LightSheet Z.1 (Zeiss). The light sheet was focus onto the specimen with a [nX/NA] illumination objective on both the left and the right sides and images were acquired with a [detection objective description], with a z-step of [… μm] and a pixel size of [… nm]. [precise Multiview angle, mosaic’s tile number…]. Dual-side fusion [+ other postprocessing] was run [online/offline] using [Zen Black/Huygens Fuser].

Micro Imaging Facility Support

When you book in time to use the Innovation Centre's Zeiss Lightsheet, you will receive the following support from the Micro Imaging Facility team:

• Specimen preparation and mounting
• X-Clarity based or passive methods specimen clearing
• Full training for fixed and live imaging
• Full training for image processing including Huygens Fuser based deconvolution and Multiview alignment, Zeiss based dual-side imaging alignment, Arivis based mosaic reconstruction, 3D/4D observations.
• Fee for service

Enquiries

Dr Scott Page, BSc, PhD
Head, Micro Imaging Facility
s.page@victorchang.edu.au