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docs:comparison_light_sheet_methods [2015/12/28 17:38]
Jon Daniels 		docs:comparison_light_sheet_methods [2017/07/20 20:11] (current)
Jon Daniels [Commercial Light Sheet Microscopes]
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 ===== Comparison of Light Sheet Microscopy Methods ===== ===== Comparison of Light Sheet Microscopy Methods =====
 +
 +==== Commercial Light Sheet Microscopes ====
 +^ Type                             ^ # Views                ^ Mounting                      ^ Software                           ^ Comments                                                                ^
 +| diSPIM                           | 2 fixed (isotropic)    | Coverslip or dish with media  | Free/open   + various proprietary  | Modular/flexible configuration, allows simultaneous photo-manipulation  |
 +| Zeiss Z.1 (similar to OpenSPIM)  | Unlimited (isotropic)  | Capillary with agarose        | Single proprietary                 | Rotating sample allows imaging scattering samples from both sides       |
 +| Leica TCS SP8 DLS                | 1 fixed                | Dish with media               | Single proprietary                 | Add-on to existing Leica confocal                                       |
 +| 3i Lattice Light Sheet           | 1 fixed                | Small coverslip in dish       | Single proprietary                 | Lattice illumination allows for improved axial resolution for thin samples |
 +| LaVision BioTec Ultramicroscope  | 1 fixed                | Dish with media               | Single proprietary                 | Optimized for large fixed samples (low mag, low res)                    |
 +
 +In general the diSPIM approach is ideal for cells or small groups of cells (e.g. c. elegans embryos).  For thicker samples (e.g. Drosophila embryos) where the light sheet cannot penetrate across the sample the Zeiss/OpenSPIM approach has the advantage that all sides of the sample can be directly seen via rotating the sample.  For sub-diffraction resolution on thin samples lattice light sheet gives better resolution (though much of the advantage can be gained simply by using the lattice light sheet objectives on the diSPIM).  The LaVision BioTec system is optimized for large fixed samples, though such samples can also be imaged on the diSPIM with an appropriate [[http://www.asiimaging.com/index.php/cleared-tissue-objective|objective for cleared tissue]].
 +
 +Instruments which rely on a single fixed view lead to relatively poor axial resolution and poor imaging of scattering samples.
 +
 +The diSPIM sample mounting is easy and extremely flexible compared with Zeiss/OpenSPIM.  Compared with other commercial light-sheet solutions a diSPIM system is quite inexpensive and extremely flexible/customizable.
 +
 +==== Detailed Comparison ====
  
   * [[:start|diSPIM]]   * [[:start|diSPIM]]
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       * two fixed orthogonal views without moving the sample (ideal for fast-moving samples)       * two fixed orthogonal views without moving the sample (ideal for fast-moving samples)
       * two views can be combined computationally for isotropic resolution       * two views can be combined computationally for isotropic resolution
-      * scattering slightly mitigated slightly by having two views; typically >100 um imaging depth+      * scattering slightly mitigated slightly by having two views; typically 30-300 um imaging depth depending on sample
       * inverted microscope objective allows simultaneous photomanipulation or other techniques       * inverted microscope objective allows simultaneous photomanipulation or other techniques
       * 40x 0.8 NA water-dipping objectives most common (others possible)       * 40x 0.8 NA water-dipping objectives most common (others possible)
-          * yields 330 um field of view with standard sensor/tube lens+          * yields >400 um diagonal field of view with standard sensor/tube lens
           * yields 380 nm lateral resolution at 500 nm wavelength (improved slightly by combining views computationally)           * yields 380 nm lateral resolution at 500 nm wavelength (improved slightly by combining views computationally)
   * [[http://www.zeiss.com/microscopy/en_de/products/imaging-systems/lightsheet-z-1.html|Zeiss Z.1]] / [[http://openspim.org/|OpenSPIM]]   * [[http://www.zeiss.com/microscopy/en_de/products/imaging-systems/lightsheet-z-1.html|Zeiss Z.1]] / [[http://openspim.org/|OpenSPIM]]
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       * uses axially-arranged objectives with special mirrors to create orthogonal light sheet       * uses axially-arranged objectives with special mirrors to create orthogonal light sheet
       * one fixed view => relatively poor axial resolution, scattering not mitigated       * one fixed view => relatively poor axial resolution, scattering not mitigated
 +      * light sheet parallel to dish bottom, so flat samples must be tilted with respect to dish
   * lattice light sheet   * lattice light sheet
       * uses structured light sheet "lattice" from interfering Bessel beams       * uses structured light sheet "lattice" from interfering Bessel beams
-      * scattering strongly affects imaging; typically <20 um imaging depth+      * scattering strongly affects lattice formation as well as imaging; typically <20 um imaging depth
       * can obtain sub-diffraction images like SIM by combining multiple exposures with shifted lattice; cost is extra time/dose like SIM       * can obtain sub-diffraction images like SIM by combining multiple exposures with shifted lattice; cost is extra time/dose like SIM
-      * objectives dipped in media from above ([[http://aicblog.janelia.org/?p=304|details]]) similar to diSPIM +      * objectives dipped in media at angle from above ([[http://aicblog.janelia.org/?p=304|details]]) similar to diSPIM 
-      * 25x 1.1 NA detection objective and custom excitation objective for fixed single-view => relatively poor axial resolution but better than typical diSPIM because of objective with higher NA +      * 25x 1.1 NA detection objective and custom excitation objective for fixed single-view; axial resolution is improved beyond the objective's capability (but not quite isotropic) because of the optical sectioning of the lattice illumination 
-      * beginning to be commercialized by [[https://www.intelligent-imaging.com/systems.php#lattice|Intelligent Imaging]] +      * commercialized by [[https://www.intelligent-imaging.com/systems.php#lattice|Intelligent Imaging]] under license from Zeiss
- +
-In general the diSPIM approach is ideal for cells or small groups of cells (e.g. c. elegans embryos).  For thicker samples where the light sheet cannot penetrate across the sample the Zeiss/OpenSPIM approach is probably better so that all sides of the sample can be directly seen.  For sub-diffraction resolution on thin samples lattice light sheet is preferred.  Compared with Zeiss/OpenSPIM the diSPIM sample mounting is easy and extremely flexible.  Instruments which rely on a single fixed view lead to relatively poor axial resolution and problems with scattering samples.  Compared with other commercial light-sheet solutions a diSPIM is quite inexpensive.+
  
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