Your Next Confocal Should Be a Light-Sheet Microscope

Light-Sheet Fluorescence Microscopy

Light-Sheet Fluorescence Microscopy is the method of choice for long-term, high interval (minutes to days) live sample imaging. Most of the models used in confocal microscopy are suitable for light-sheet microscopy. Due to its unique capabilities, additional challenging specimens are included in the spectrum of samples that can be acquired with a light sheet microscope.

 

Compared to confocal laser scanning and spinning disk confocal microscopy, light-sheet microscopy enables fast, high resolution, true volume, and in-depth imaging with the following major advantages:

Low Photobleaching

Photobleaching refers to the permanent loss of ability to fluoresce due to light-induced damage of the fluorophore molecules in a sample. Long-term exposure to light, especially in time-lapse studies, induces photobleaching, hindering the detection of the fluorescent molecules.

References

Harms, G.S., et al. (2001). Autofluorescent proteins in single-molecule research: applications to live-cell imaging microscopy. Biophys. J. 80: 2396-2408.

Im, K.B., et al. (2013). Diffusion and binding analysed with combined point FRAP and FCS. Cytometry A 89: 876-889.

Parameters

  • InVi SPIM: 62x/1.1NA, 2048 × 2048 pixel, pixel size 100 × 100 nm, light-sheet thickness 2 µm, illumination time per voxel 25 µs
  • Spinning disk confocal: 60x/1.2NA, 2048 × 2048 pixel, pixel size 100 × 100 nm, pinhole diameter 50 µm or 1.5 Airy units, pinhole distance 250 µm, illumination time per voxel 10 µs
  • Point confocal: CLSM with 10k resonant scanner; 60x/1.2NA, 200 × 200 pixel, pixel size 250 nm, pinhole diameter 50 µm or 1.5 Airy units, illumination time per voxel 0.5 µs
  • Fluorescence lifetime: 2.5 ns
  • Intersystem crossing rate: 2.5·106 s-1
  • Triplet lifetime: 5 µs
  •  Bleach rate: 100 s-1 at 1 kW cm-2

A comparison of the photobleaching rates of light-sheet microscopy, spinning disk, and confocal microscopy reveals a reduction in photobleaching when working with light-sheet microscopy. The effect is already visible when imaging a single plane @ 100 fps, but the difference becomes particularly astonishing when comparing imaging of a stack of 40 µm (1 µm steps) @ 100 fps.

 

 

Low Photo-Toxicity

Long-term imaging can have phototoxic effects on the sample, altering the normal behavior of the cells and the whole specimen.

Light-sheet microscopy stands out for its effective use of excited photons, which minimizes phototoxic effects. This contributes to prevent the generation of misleading and artificial results.

The study from Jemielita et al. (2013) brings out seemingly imperceptible phototoxic effects induced by long-term exposure to light. The comparison of light-sheet microscopy and spinning disk microscopy images revealed inappropriate bone development in zebrafish due to photo-damage in spinning disk microscopy.

Source article

Jemielita, M. et al. (2013) Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light-sheet fluorescence microscopy techniques. Biophotonics 6 (11-12): 920-8

High Temporal and Spatial Resolution

Light-sheet microscopy enables high imaging speed and the possibility to capture a higher number of events. This is of particular relevance in fast occurring dynamic processes.

A study by Reichmann et al. (2018) carried out at EMBL serves as an example. It shows that during the first cell division in mouse embryos, the maternal and paternal chromosomes remain separated. Only light-sheet microscopy made these findings possible.

Source article

Reichmann, J. et al. (2018) Dual-spindle formation in zygotes keeps parental genomes apart in early mammalian embryos. Science, published online.

Optical Sectioning

Optical sectioning refers to the generation of clear images of specific focal planes within a 3D structure. Good Z resolution enables the 3D reconstruction of a sample.

Fluorescence microscopy, e.g. confocal microscopy, spinning disk confocal and light-sheet microscopy, enable optical sectioning. Confocal Microscopy and Spinning Disk Microscopy image a specific focal plane by point scanning the sample and rejecting out of focus fluorescent signal with a pinhole(s). These techniques enable high-resolution image acquisition at the expense of photo-damaging effects and/or high time consumption.

Light-Sheet Microscopy offers intrinsic optical sectioning by the specific illumination of one particular focal plane. This is achieved by the orthogonal arrangement of the illumination and detection objective lenses as well as the projection of a thin light-sheet on the sample. Intrinsic optical sectioning significantly reduces photo-bleaching and phototoxic effects offers high acquisition speed and the possibility to perform long-term experiments.

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