CBIS Confocal: 3I diSPIM


Marianas LightSheet

Live imaging directly involves inevitable tradeoffs of spatial resolution, temporal resolution and phototoxicity. This is especially true when interrogating imaging in three dimensions and/or three-dimensional samples over time (3D and/or 4D imaging) to obtain a complete picture of many dynamic subcellular processes. Selective Plane Illumination Microscopy (or Light Sheet Microscopy) uses a separate excitation lens perpendicular to the widefield detection lens to illuminate only the plane of interest within sheet of light. Its low photo toxicity, photo bleaching, high signal to noise ration and high imaging speed make it the most favorite tool for live imaging.

LocationCBIS Confocal Microscopy Laboratory
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About Marianas LightSheet

Marianas LightSheet™ from Intelligent Imaging Innovations, Inc. (3I, USA) merges dual inverted selective plane illumination (diSPIM) with the power and flexibility of a live-cell microscopy system. It employs two orthogonal objectives positioned at 45° above the specimen plane. By alternating between the objectives for imaging and excitation, diSPIM captures two volumes that are fused and deconvolved to achieve isotropic resolution. Unlike capillary-based light sheet methods, diSPIM allows for standard specimen preparation in standard dishes and standard media.

The 3I diSPIM at CBIS/DBS confocal core, offers a single- (iSPIM) or dual- (diSPIM) sided light-sheet arrangement on an inverted microscope with custom built OKO environment chamber for live cell imaging and photon manipulation unit for FRAP. 3D isotropic resolution of 360 nm in x-y-z is achievable (by beads). Samples are prepared on conventional glass coverslips and agarose gel for monolayer cell culture and small organisms imaging respectively.

  • Modalities: Single and dual inverted SPIM (iSPIM and diSPIM)
  • iSPIM x-y-z resolution: 360 x 360 x 760 nm
  • diSPIM x-y-z resolution: 360 x 360 x 360 nm
  • SPIM objectives: Nikon water dipping objectives: CFI NIR Apo 40X/0.8 NA WD 3.5 mm, x2; CFI Plan Fluor 10x/0.3NA WD 3.5mm, x2.
  • Finder objectives: Zeiss 40x and 10x
  • Sample platform: Rectangular coverslips (1.5#, 24 x 50 mm) or agarose on petri dishes
  • Excitation sources: 405nm (100mW), 488nm (50mW), 561nm (50mW) and 640 nm (100mW)
  • Dichroic mirror: Shemrock410/504/582/669 nm BrightLine® quad-edge dichroic beamsplitter
  • Emission filters Shemrock440/521/607/700 nm BrightLine® quad-band bandpass filter
  • Light-sheet camera: Twin Hamamatsu ORCA-Flash4.0 V3 sCMOS
  • Environmental control: diSPIM custom designed OKO incubator with temperature, CO2 and humidity control
  • Software: Slidebook 6
  • Operating system: Windows 10
  • Data transfer: 10 GB network connection – no external hard drives allowed
  • Usage: Staff-assisted and self-use allowed (following training)
  • 3D-5D Live cell imaging: multi-channel, 3D and time lapse imaging for isotropic resolution
  • 3D-5D Live small organism imaging
  • Advanced F-techniques: FRAP