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docs:comparison_light_sheet_methods [2015/08/05 23:11] 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 | ||
| + | | diSPIM | ||
| + | | Zeiss Z.1 (similar to OpenSPIM) | ||
| + | | Leica TCS SP8 DLS | 1 fixed | Dish with media | Single proprietary | ||
| + | | 3i Lattice Light Sheet | 1 fixed | Small coverslip in dish | Single proprietary | ||
| + | | LaVision BioTec Ultramicroscope | ||
| + | |||
| + | In general the diSPIM approach is ideal for cells or small groups of cells (e.g. c. elegans embryos). | ||
| + | |||
| + | 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/ | ||
| + | |||
| + | ==== Detailed Comparison ==== | ||
| * [[: | * [[: | ||
| - | * sample mounting like inverted microscope with water/ | + | * sample mounting like inverted microscope; light sheet objectives lowered into open chamber |
| - | * two fixed orthogonal views | + | * 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 | + | * 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 NA 0.8 water-dipping objectives most common (others possible) | + | * 40x 0.8 NA water-dipping objectives most common (others possible) |
| - | * yields | + | * yields |
| * 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:// | * [[http:// | ||
| * OpenSPIM can be thought of as simplified " | * OpenSPIM can be thought of as simplified " | ||
| * sample is suspended from glass capillary into special chamber which holds dipping media | * sample is suspended from glass capillary into special chamber which holds dipping media | ||
| - | * sample can be rotated to see all different sides (unlimited views around Z axis) | + | * sample can be rotated to see all different sides (unlimited views around Z axis) but requires time to rotate |
| * different views can be combined computationally for isotropic resolution | * different views can be combined computationally for isotropic resolution | ||
| - | * scattering | + | * scattering mitigated by multiple views; typically ~2x deeper than diSPIM |
| * 20x water-dipping objective most common for imaging (others possible); usually imaging objective has large NA and illumination objective has low NA | * 20x water-dipping objective most common for imaging (others possible); usually imaging objective has large NA and illumination objective has low NA | ||
| * [[http:// | * [[http:// | ||
| * usually used for larger samples, including cleared tissue | * usually used for larger samples, including cleared tissue | ||
| * available magnifications 1.26x - 12.6x (" | * available magnifications 1.26x - 12.6x (" | ||
| - | * one fixed view => relatively poor axial resolution (" | + | * one fixed view => relatively poor axial resolution, e.g. >4 um (" |
| * [[http:// | * [[http:// | ||
| * add-on module for Leica confocal microscope | * add-on module for Leica confocal microscope | ||
| * 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 | ||
| + | * uses structured light sheet " | ||
| + | * 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 | ||
| + | * objectives dipped in media at angle from above ([[http:// | ||
| + | * 25x 1.1 NA detection objective and custom excitation objective for fixed single-view; | ||
| + | * commercialized by [[https:// | ||
| - | In general the diSPIM approach | + | Note: if anything here is inaccurate please simply create an account |
