The optical sectioning capability is fundamental for three-dimensional imaging. One of the very few microscopes, which claim this property is light sheet-based fluorescence microscopy (LSFM). In general, FM provides a high contrast, since only specifically labelled cellular components are observed while all other structures remain “dark”. However: 1) Excitation light degrades endogenous organic compounds and bleaches fluorophores. 2) Only a finite number of excitable fluorophores is available, which limits the quantity of collectable emitted photons. 3) Organisms are adapted to the solar flux of 1.4 kW/m2.
LSFM decouples the excitation and emission light pathways. The optical axis of the illumination objective lens is aligned with the focal plane of the perpendicularly arranged detection objective lens. By design, only the fluorophores around the focal plane are excited for each acquired two-dimensional image. Hence, each fluorophore is exposed only once to the illumination light when a three-dimensional image is recorded. The significance of the illumination-based optical sectioning property is that the viability and the fluorescence signal of a living specimen are retained while millions of images are recorded for days or even weeks.
Further benefits: (i) a good axial resolution, (ii) imaging along multiple directions, (iii) deeper tissue penetration due to the low numerical aperture of the illumination objective lens, (iv) a high signal-to-noise ratio, (v) an unrestricted compatibility with fluorescent dyes and proteins, (vi) reduced fluorophore bleaching and (vii) photo-toxicity at almost any scale, (viii) millions of pixels are recorded in parallel and (ix) a dramatically improved viability of the specimen.
Ernst Hans Karl Stelzer, "Light sheet microscopy revolutionizes dynamic multi-dimensional light microscopy of three-dimensional specimens (Conference Presentation)," Proc. SPIE 10505, High-Speed Biomedical Imaging and Spectroscopy III: Toward Big Data Instrumentation and Management, 105050H (Presented at SPIE BiOS: January 30, 2018; Published: 15 March 2018); https://doi.org/10.1117/12.2296845.5752187611001.
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