Super-resolution light microscopy methods
| Near-field | Far-field | |||||||
| Principle | Small aperture scanning (no lens) | Evanescent wave illumination | Wide-field + deconvolution | Confocal laser scanning | Moiré effect with structured illumination | PSF shaping with saturated emission depletion | Photoswitching and localization of single molecules (pointillism) | |
| Acronym | SNOM/NSOM | TIRFM | CLSM | SIM (HELM, PEM) 3D-SIM | SSIM (SPEM) | STED/CW-STED | PALM/FPALM/STORM/dSTORM/PALMIRA | |
| Illumination-emission dependence | Linear | Linear | Linear | Linear | Linear | Non-linear | Non-linear | Linear |
| Detector | Scanning PMT/APD | Wide-field CCD/CMOS | Wide-field CCD/CMOS | Scanning PMT/APD | Wide-field CCD/CMOS | Wide-field CCD/CMOS | Scanning PMT/APD | Wide-field CCD/CMOS |
| XY-resolution | 20–120 nm | 200–300 nm | 180–250 nm | 180–250 nm | 100–130 nm | 50 nm | 20–100 nm | 20–50 nm |
| Z-resolution | 10 nm (near-field range) | 100 nm (near-field range) | 500–700 nm | 500–700 nm | 250–350 nm | N.D. | 560 nm (CW-STED) to 700 nm (100 nm with z-phase mask) | 100 nm (TIRF) 20–30 nm (3D-STORM, TIRF) 75 nm (BP-FPALM, in plane) |
| Serial z-sectioning | No | No | Yes | Yes | Yes | Yes | Yes | Yes |
| Z stack range | N.A. | N.A. | 100 µm | 100 µm | 10–20 µm | N.A. | >20 µm | 100 nm – few µm (BP-FPALM) |
| Dyes | Any | Any | Any | Any | Most conventional dyes (photostable) | Dyes require special characteristics | Dyes require special characteristics (CW-STED works with many conventional dyes) | Dyes require special characteristics |
| Simultaneous colors | 2 | 3 | >3 | >3 | 3 | 1 | 2 | 2 |
| Temporal resolution for 512 × 512 image | s-min | ms | ms | ms-s | ms-s | s-min | ms-min | s-min |
| Energy load/intensity | Low | Low | Low | Medium | Medium | High | Medium-high | Medium-high |
| Live-cell imaging | Yes | Yes | Yes | Yes | Restricted (2D-TIRF) | No | Restricted | Restricted |
| Postprocessing required | No | No | Yes | No | Yes 9–25 raw images per slice | Yes z100 raw images per slice | No | Yes >1,000 raw images per slice |
| Notes | No intracellular imaging | Restricted to region near the coverslip | Risk of artifacts; better for sparse samples | Reconstruction bears risk of artefacts | High excitation required; reconstruction bears risk of artefacts | Complex instrumentation; photobleaching | May require TIRF setup for best performance; labeling density is critical; performs better on particles and filaments as on volume stains | |
| Dual lens implementation | I5M | 4Pi | I5S | 4 Pi-STED/iso-STED | iPALM | |||
| Z-resolution | 70 nm | 80 nm | 100 nm | 20–100 nm | 10 nm (depth ∼200 nm) | |||
| Near-field | Far-field | |||||||
| Principle | Small aperture scanning (no lens) | Evanescent wave illumination | Wide-field + deconvolution | Confocal laser scanning | Moiré effect with structured illumination | PSF shaping with saturated emission depletion | Photoswitching and localization of single molecules (pointillism) | |
| Acronym | SNOM/NSOM | TIRFM | CLSM | SIM (HELM, PEM) 3D-SIM | SSIM (SPEM) | STED/CW-STED | PALM/FPALM/STORM/dSTORM/PALMIRA | |
| Illumination-emission dependence | Linear | Linear | Linear | Linear | Linear | Non-linear | Non-linear | Linear |
| Detector | Scanning PMT/APD | Wide-field CCD/CMOS | Wide-field CCD/CMOS | Scanning PMT/APD | Wide-field CCD/CMOS | Wide-field CCD/CMOS | Scanning PMT/APD | Wide-field CCD/CMOS |
| XY-resolution | 20–120 nm | 200–300 nm | 180–250 nm | 180–250 nm | 100–130 nm | 50 nm | 20–100 nm | 20–50 nm |
| Z-resolution | 10 nm (near-field range) | 100 nm (near-field range) | 500–700 nm | 500–700 nm | 250–350 nm | N.D. | 560 nm (CW-STED) to 700 nm (100 nm with z-phase mask) | 100 nm (TIRF) 20–30 nm (3D-STORM, TIRF) 75 nm (BP-FPALM, in plane) |
| Serial z-sectioning | No | No | Yes | Yes | Yes | Yes | Yes | Yes |
| Z stack range | N.A. | N.A. | 100 µm | 100 µm | 10–20 µm | N.A. | >20 µm | 100 nm – few µm (BP-FPALM) |
| Dyes | Any | Any | Any | Any | Most conventional dyes (photostable) | Dyes require special characteristics | Dyes require special characteristics (CW-STED works with many conventional dyes) | Dyes require special characteristics |
| Simultaneous colors | 2 | 3 | >3 | >3 | 3 | 1 | 2 | 2 |
| Temporal resolution for 512 × 512 image | s-min | ms | ms | ms-s | ms-s | s-min | ms-min | s-min |
| Energy load/intensity | Low | Low | Low | Medium | Medium | High | Medium-high | Medium-high |
| Live-cell imaging | Yes | Yes | Yes | Yes | Restricted (2D-TIRF) | No | Restricted | Restricted |
| Postprocessing required | No | No | Yes | No | Yes 9–25 raw images per slice | Yes z100 raw images per slice | No | Yes >1,000 raw images per slice |
| Notes | No intracellular imaging | Restricted to region near the coverslip | Risk of artifacts; better for sparse samples | Reconstruction bears risk of artefacts | High excitation required; reconstruction bears risk of artefacts | Complex instrumentation; photobleaching | May require TIRF setup for best performance; labeling density is critical; performs better on particles and filaments as on volume stains | |
| Dual lens implementation | I5M | 4Pi | I5S | 4 Pi-STED/iso-STED | iPALM | |||
| Z-resolution | 70 nm | 80 nm | 100 nm | 20–100 nm | 10 nm (depth ∼200 nm) | |||
N.A., not applicable; N.D., not described.