Table 4. Summary of major imaging techniques
| Imaging technique | Features | Notesa | General references |
|---|---|---|---|
| Wide-field microscopy | Best sensitivity, but limited image quality resulting from the contribution of blurring from above and below the focal plane. | Most useful with thin tissues or culture cells or with low-NA, low-magnification dry objectives. | Davis 2000; Wallace et al. 2001 |
| Deconvolution Wide-field microscopy (3D imaging) |
Post-image acquisition processing of 3D x-y-z data. Constrained iterative deconvolution algorithms act by reassigning out-of-focus light to its point of origin to deliver increased signal-to-noise, resolution, and contrast. Requires multiple z sections. | The most commonly applied imaging technique for high-resolution analysis of structure and dynamic processes. | Parton and Davis 2005 |
| Deconvolution Wide-field microscopy (2D imaging) |
Limited deblurring or deconvolution approaches applied to 2D x-y-t data to sharpen image detail. | Useful increase in signal-to-noise and contrast when speed requirements/tissue sensitivity preclude z-stack collection. | MacDougall et al. 2003; Parton and Davis 2005 |
| OMX fast live wide-field deconvolution | Wide-field imaging system with increased sensitivity, temporal resolution, and extremely low background. Has the ability to image up to four fluorescence channels simultaneously. | Useful for fast live imaging of multiple components interacting in sensitive biological processes. | Dobbie et al. 2010 |
| Point-scanning confocal microscopy (or LSCM) | An optical arrangement that eliminates the contribution of out-of-focus light to the image by a physical pinhole arrangement to produce sharp optical sections. Images one spot at once, and slowly builds an image from these spots. | Useful in brightly labeled thick, hazy, or scattering material. Very good for high-resolution structural studies but suffers from speed limitations and is significantly less sensitive than wide-field techniques. | Pawley 2006 |
| Slit-scanning confocal microscopy | Similar to LSCM but increases speed by imaging one line rather than one spot at a time. Some compromise in optical sectioning. | An alternative to spinning disks when simultaneous multichannel imaging is essential. | |
| Spinning-disk or multifocal confocal microscopy | Similar to LSCM and slit-scan confocal but increases speed and sensitivity by imaging multiple points simultaneously and detecting with a CCD camera. Some compromise in optical sectioning and flexibility. | A compromise between wide-field and confocal microscopies for fast imaging in sensitive tissues. Much faster than confocal microscopy but less sensitive than wide-field microscopy. | Basto et al. 2006 |
| Multiphoton imaging | Achieves optical sectioning by the principle of multiple low-energy photon absorption (at 740-1100 nm) that occurs at very high illumination intensity, limiting excitation to a <1-μm-thick focal plane. | Is able to image deeper in thick, hazy, or scattering material with improved cell viability. Limited application so far in Drosophila. | Amos 2000; Diaspro et al. 2006 |
| SPIM (or Theta imaging, DSLM) | Alternative to confocal optical sectioning with improvements in 3D imaging and tissue viability. | Useful for detailed 3D mapping of dynamic processes in living specimens. Significantly reduces photodamage compared with either confocal or wide-field microscopy. | Huisken et al. 2004; Keller et al. 2008; Keller and Stelzer 2010 |
| STED (and STED 4Π) | True optical super-resolution imaging in x, y (and z with 4Π) | Useful to resolve fine structures and interactions of components at beyond conventional optical resolution. | Willig et al. 2006 |
| aAbbreviations: 2D: two-dimensional; 3D: three-dimensional; CCD: charge-coupled device; DSLM: digital scanned laser light sheet microscopy; LSCM: laser-scanning confocal microscopy; NA: numerical aperture; OMX: optical microscope experimental; SPIM: single-plane illumination microscope; STED: stimulated emission depletion. | |||
