Plenary Lecture

Ernst Hans Karl Stelzer
Buchmann Institute for Molecular Life Sciences, Goethe Universität Frankfurt am Main, Frankfurt am Main, Germany

In light sheet–based fluorescence microscopy (LSFM), optical sectioning in the excitation process minimizes fluorophore bleaching and phototoxic effects. Since biological specimens survive long-term three-dimensional imaging at high spatiotemporal resolution, light sheet-based microscopes (LSM) have become an indispensable tool in developmental, three-dimensional cell and plant biology. LSFM is based on two main optical paths. The detection path consists of a microscope objective, a spectral filter, a tube lens and a camera. The excitation path is perpendicular to the detection path and directs a light sheet into the side of the specimen. The thin light sheet and the focal plane of the detection objective overlap. LSFM provides at least three important degrees of freedom, which are usually not available in an epifluorescence microscope: a) the axial and b) lateral locations of the light sheet and c) the axial location of the focal plane. Further, probably less important, degrees of freedom are the tilt and the incline of the light sheet. LSFM takes full advantage of modern cameras, massively parallelizing the data acquisition process and recording ten to one hundred images per second with a high dynamic range. LSFM does not rely on traditional features that are required for ergonomic reasons. A powerful multiple-sensors-based image processing pipeline is, therefore, an inherent feature. Traditional fluorescence microscopy enforces specimen preparation schemes that rely on hard and flat surfaces. LSFM places the specimen in the center and arranges the optics around it. Specimens can be prepared in new ways, their three-dimensional integrity is maintained, and they can be used in experiments hitherto regarded as impossible. Fluorescence microscopy has several basic limitations. First, the excitation light is absorbed not only by fluorophores but also by many endogenous organic compounds, which are degraded much like fluorophores and thus unavailable for vital metabolic processes. Second, the number of fluorophores in any volume element at any given time is finite, and fluorophores can degrade upon excitation. As a consequence, the number of photons that are retrieved from a fluorophore-labeled specimen is limited. Finally, life on Earth is adapted to the solar flux, which is less than 1.4 kW/m2. This might not be a hard limit, but it indicates that irradiance should not exceed 1 nW/µm2 = 100 mW/cm2 when dynamic biological processes are observed. When imaging living biological samples, these challenges must be addressed. LSFM is perhaps the best technology we have so far, which makes a sincere and honest effort to address these challenges: 1) it provides optical sectioning, 2) a true axial resolution, 3) reduces fluorophore bleaching and 4) phototoxicity at almost any scale, 5) allows one to record millions of pixels in parallel and 6) dramatically improves the viability of the specimen. Stelzer, E. H.K. (2015). Light-sheet fluorescence microscopy for quantitative biology. Nature Methods, 12(1), 23–27.

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