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start [2018/07/22 13:56]
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Jon Daniels [The Implementation]
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 The diSPIM "head" can be mounted on various inverted microscopes including ASI's RAMM frame (shown here). diSPIM systems can be obtained from various [[docs:system integrators]].  Various open-source and proprietary software packages are available for both data acquisition and data processing. Most of the underlying microscope hardware is identical regardless of the system integrator and software used. The diSPIM "head" can be mounted on various inverted microscopes including ASI's RAMM frame (shown here). diSPIM systems can be obtained from various [[docs:system integrators]].  Various open-source and proprietary software packages are available for both data acquisition and data processing. Most of the underlying microscope hardware is identical regardless of the system integrator and software used.
  
-The choice of diSPIM objectives is limited because they must be co-focused without bumping into each other.  The most commonly-used objectives for diSPIM are 40x water-dipping objectives with a NA of 0.8 (Nikon CFI Apo 40XW NIR).  The Olympus 20x/0.5 objective is another possibility((According to the manufacturer's drawings the objectives co-focus exactly when they touch, which also implies zero gap between objective and coverslip. However, in practice they co-focus with a gap comparable to the 40x/0.8, with ~200um between the objective bottom and coverslip.)) as is the Nikon 10x/0.3.  ASI and Special Optics have co-developed an [[http://asiimaging.com/docs/cleared_tissue_objective|custom objective for cleared tissue]] that is suitable for the diSPIM and can image cleared tissue up to 5 mm deep in slab form or within a 12 mm spherical envelope.  Single-sided systems (iSPIM) have much more flexibility because the illumination objective can be a low-NA long-WD objective.+The choice of diSPIM objectives is limited because they must be co-focused without bumping into each other.  The most commonly-used objectives for diSPIM are 40x water-dipping objectives with a NA of 0.8 (Nikon CFI Apo 40XW NIR).  The Olympus 20x/0.5 objective is another possibility((According to the manufacturer's drawings the objectives co-focus exactly when they touch, which also implies zero gap between objective and coverslip. However, in practice they co-focus with a gap comparable to the 40x/0.8, with ~200um between the objective bottom and coverslip.)) as is the Nikon 10x/0.3.  ASI and Special Optics have co-developed two [[http://asiimaging.com/docs/cleared_tissue_objective|objectives for cleared tissue]] that are suitable for the diSPIM and can image cleared tissue up to 5 mm deep in slab form or within a 12 mm spherical envelope.  Single-sided systems (iSPIM) have much more flexibility because the illumination objective can be a low-NA long-WD objective.
  
-Most often sCMOS cameras are used for SPIM imaging.  There are working diSPIM systems with Hamamatsu Flash4, Andor Zyla, PCO Edge, and Photometrics Prime 95B cameras.+Most often sCMOS cameras are used for SPIM imaging.  There are working diSPIM systems with Hamamatsu Flash4 and Fusion, Andor Zyla, PCO Edge, and Photometrics Prime 95B cameras.
  
 ASI makes a compact fiber-coupled 2D galvo or "scanner" which is an integral part of the system. The original version of the scanner creates the light sheet by fast scanning in one axis and moves the sheet through the sample using the other axis ((i.e. the diSPIM is a digital light sheet microscope or DLSM in its original implementation)).  There is also a scanner version with a cylindrical lens for static light sheet generation available. The output of the excitation laser (or laser launch) simply is fed into the scanner; it is helpful to have a 2x1 optical switch or dual-output laser launch so the excitation can all be steered to the scanner in the active light path. ASI makes a compact fiber-coupled 2D galvo or "scanner" which is an integral part of the system. The original version of the scanner creates the light sheet by fast scanning in one axis and moves the sheet through the sample using the other axis ((i.e. the diSPIM is a digital light sheet microscope or DLSM in its original implementation)).  There is also a scanner version with a cylindrical lens for static light sheet generation available. The output of the excitation laser (or laser launch) simply is fed into the scanner; it is helpful to have a 2x1 optical switch or dual-output laser launch so the excitation can all be steered to the scanner in the active light path.
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 The bottom objective (the inverted microscope) typically has a lower-magnification objective and less expensive camera for locating the sample. Epi-illumination can easily be added. The bottom objective (the inverted microscope) typically has a lower-magnification objective and less expensive camera for locating the sample. Epi-illumination can easily be added.
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 +For dedicated cleared tissue imaging setups the ct-dSPIM variant is employed where the SPIM head is mounted on posts and the sample is placed on an XYZ stage (usually without an inverted microscope but it is possible to add one).  This has advantages especially for larger samples.
  
  

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