Advantages of Light Sheet Fluorescence Microscopy (LSFM)

1. Reduction in out of focus signal (since only fluorophores in the vicinity of the imaged plane are excited). This results in an increased contrast and signal to noise. If the light sheet is made thin enough (as is the case with the Lattice Light Sheet), this will also result in improvements in the axial resolution (i.e. resolution along the imaging axis). Importantly, also allows for sectioning (3D volume imaging) using a widefield detection modality and without the need of employing a confocal detection scheme (which comes with several disadvantages). Such widefield detection can allow for orders of magnitude faster volume imaging compared to typical point scanning approaches.

2. A second advantage of only illuminating the sample in the vicinity of the plane being imaged, is that one will vastly reduce potential phototoxicity since only a small fraction of the sample is illuminated for each image. In contrast all epi-detection schemes (confocal, spinning disk-confocal and epi-widefield) illuminate also a significant portion of the sample above and below the imaged plane. For long-term imaging or imaging of large samples this may lead to undesirable effects such as photobleaching of fluorophores or other adverse phototoxic effects on the sample. LSFM thus is also advantageous for volume imaging of samples over extended time periods in a minimally perturbative way.

3. As a result of the perpendicular excitation-detection scheme samples need to often be mounted in a manner that is distinct from more common epi-detection microscopy in order to assure optical axis from two perpendicular directions simultaneously. While desirable in the sense that this allows one to study samples in an often more physiological 3D environment (as opposed to on a cover slip – which is typically a less physiological environment), it can also prove to be an additional challenge and will in many cases will be sample dependent. Samples may e.g. be suspended in a gel, mounted on the tip of a pin or inside a transparent tube placed between the objectives, depending on the type of sample and the LSFM setup design.