Overview of fluorescence light-sheet techniques in comparison with epifluorescence, confocal and two-photon microscopy
| Technique . | Unique features . | Advantages . | Disadvantages . | Ideal application . | References . | |
|---|---|---|---|---|---|---|
| Fluorescence microscopy | ||||||
| Epifluorescence microscopy | Uniform illumination of the sample | Easy to use, low cost | No optical sectioning | Screening, thin sections | ||
| Confocal microscopy | Point illumination (laser focus) and point detection (pinhole) | Optical sectioning, improved resolution (√2) | Slow scanning, low penetration | Medium-thick sections, surface of embryos | Pawley, 2006 | |
| Two-photon microscopy | True 3D imaging, infrared laser | Low scattering, excellent penetration, reduced bleaching | Slow scanning, low resolution, high cost, high power beam | Deep penetration in scattering tissue | Helmchen and Denk, 2005 | |
| Fluorescence light-sheet microscopy | ||||||
| Orthogonal-plane fluorescence optical sectioning (OPFOS) | Single-sided light-sheet illumination | Optical sectioning | Shadows, stripes | Fluorescently stained samples | Voie et al., 1993 | |
| High-resolution orthogonal-plane fluorescence optical sectioning (HROPFOS) | Like OPFOS, light-sheet translated | Thin light-sheet of constant thickness | Slow, shadows, stripes | Like OPFOS | Buytaert and Dirckx, 2007 | |
| 3D light scanning macrography | Extended depth of field | Large samples | Only surface | Surface scanning of flies | Huber et al., 2001 | |
| Thin light-sheet microscope (TLSM) | Light-sheet illuminates tank of medium from the side | Improved contrast in aqueous suspensions | Low resolution | Aquatic microbes | Fuchs et al., 2002 | |
| Selective plane illumination microscopy (SPIM) | Single-sided light-sheet illumination, sample rotation about vertical axis | Fast acquisition, low photo-bleaching, good penetration, MVR | Shadows, stripes | Extended time-lapse imaging of millimetre-sized embryos, high speed movies | Huisken et al., 2004 | |
| Objective coupled planar illumination (OCPI) microscopy | Single-sided light-sheet illumination, attached to detection lens | Fast stack acquisition, no sample movement, conventional sample | Shadows, stripes | Conventionally prepared samples, fast 3D imaging | Holekamp et al., 2008 | |
| Ultramicroscopy | Dual-sided light-sheet illumination, upright detection path | Sealed chamber with clearing solution | No index-matched optics, stripes | Large fixed and cleared specimens | Dodt et al., 2007 | |
| Digital scanned laser light-sheet fluorescence microscopy (DSLM) | Like SPIM, light-sheet generated by beam scanning | Like SPIM, uniform, adjustable light-sheet | Like SPIM | Like SPIM | Keller et al., 2008 | |
| Multidirectional SPIM (mSPIM) | Like SPIM, pivoting light-sheet, two alternating illumination arms | Like SPIM, reduced stripes, reduced shading | Space constraints | Like SPIM, especially scattering and absorbing tissue | Huisken and Stainier, 2007 | |
| Highly inclined and laminated optical sheet (HILO) microscopy | Single lens for oblique light-sheet illumination and detection | Single lens | Very narrow FOV | Single cell microscopy close to the coverslip | Tokunaga et al., 2008 | |
| Oblique plane microscopy (OPM) | Like HILO, matching, tilted image plane | Like HILO, extended FOV | Extensive optics | Like HILO | Dunsby, 2008 | |
| Technique . | Unique features . | Advantages . | Disadvantages . | Ideal application . | References . | |
|---|---|---|---|---|---|---|
| Fluorescence microscopy | ||||||
| Epifluorescence microscopy | Uniform illumination of the sample | Easy to use, low cost | No optical sectioning | Screening, thin sections | ||
| Confocal microscopy | Point illumination (laser focus) and point detection (pinhole) | Optical sectioning, improved resolution (√2) | Slow scanning, low penetration | Medium-thick sections, surface of embryos | Pawley, 2006 | |
| Two-photon microscopy | True 3D imaging, infrared laser | Low scattering, excellent penetration, reduced bleaching | Slow scanning, low resolution, high cost, high power beam | Deep penetration in scattering tissue | Helmchen and Denk, 2005 | |
| Fluorescence light-sheet microscopy | ||||||
| Orthogonal-plane fluorescence optical sectioning (OPFOS) | Single-sided light-sheet illumination | Optical sectioning | Shadows, stripes | Fluorescently stained samples | Voie et al., 1993 | |
| High-resolution orthogonal-plane fluorescence optical sectioning (HROPFOS) | Like OPFOS, light-sheet translated | Thin light-sheet of constant thickness | Slow, shadows, stripes | Like OPFOS | Buytaert and Dirckx, 2007 | |
| 3D light scanning macrography | Extended depth of field | Large samples | Only surface | Surface scanning of flies | Huber et al., 2001 | |
| Thin light-sheet microscope (TLSM) | Light-sheet illuminates tank of medium from the side | Improved contrast in aqueous suspensions | Low resolution | Aquatic microbes | Fuchs et al., 2002 | |
| Selective plane illumination microscopy (SPIM) | Single-sided light-sheet illumination, sample rotation about vertical axis | Fast acquisition, low photo-bleaching, good penetration, MVR | Shadows, stripes | Extended time-lapse imaging of millimetre-sized embryos, high speed movies | Huisken et al., 2004 | |
| Objective coupled planar illumination (OCPI) microscopy | Single-sided light-sheet illumination, attached to detection lens | Fast stack acquisition, no sample movement, conventional sample | Shadows, stripes | Conventionally prepared samples, fast 3D imaging | Holekamp et al., 2008 | |
| Ultramicroscopy | Dual-sided light-sheet illumination, upright detection path | Sealed chamber with clearing solution | No index-matched optics, stripes | Large fixed and cleared specimens | Dodt et al., 2007 | |
| Digital scanned laser light-sheet fluorescence microscopy (DSLM) | Like SPIM, light-sheet generated by beam scanning | Like SPIM, uniform, adjustable light-sheet | Like SPIM | Like SPIM | Keller et al., 2008 | |
| Multidirectional SPIM (mSPIM) | Like SPIM, pivoting light-sheet, two alternating illumination arms | Like SPIM, reduced stripes, reduced shading | Space constraints | Like SPIM, especially scattering and absorbing tissue | Huisken and Stainier, 2007 | |
| Highly inclined and laminated optical sheet (HILO) microscopy | Single lens for oblique light-sheet illumination and detection | Single lens | Very narrow FOV | Single cell microscopy close to the coverslip | Tokunaga et al., 2008 | |
| Oblique plane microscopy (OPM) | Like HILO, matching, tilted image plane | Like HILO, extended FOV | Extensive optics | Like HILO | Dunsby, 2008 | |