Ernst H.K. Stelzer

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Light sheet-based Fluorescence Microscopy 

In Light Sheet-based Fluorescence Microscopy (LSFM), optical sectioning in the excitation process minimizes fluorophore bleaching and phototoxic effects.  This allows biological specimens to survive long-term three-dimensional imaging at high spatiotemporal resolution and along multiple directions.  The two canonic implementations, i.e. Selective/Single Plane Illumination Microscopy (SPIM) and Digital Scanned Laser Light Sheet Microscopy (DSLM), have become indispensable tools in developmental biology, three-dimensional cell biology and plant biology. 

LSFM was the result of developing confocal, 4Pi and Theta fluorescence microscopies for more than twenty years.  It is an excellent example for the achievements of those with an interdisciplinary spirit.  My patents in 1993 and 2003 and my seminal papers in 2004 and 2008 document the intensity and the determination that are necessary.  My further work and the refinements of my technology enable the imaging of live biological samples in close-to-natural conditions for several days, leading to breakthroughs in, among other fields, histopathology and drug development.  The initial work was done at EMBL.  However, many of my former students and postdocs have independently pushed the technology to new levels. 

Short description and impact  

Light sheets are fed into the specimen from the side and overlap with the focal plane of a wide-field fluorescence microscope.  In contrast to an epi-fluorescence microscope, the azimuthally arranged light sheet-based fluorescence microscope (LSFM) uses at least two independently operated lenses for illumination and detection.  A single/selective plane illumination microscope (SPIM) employs a cylindrical lens to generate a light sheet.  A collimated laser beam is focused into the plane of the detection lens along one direction while the other direction remains collimated.  A digital scanned laser light sheet-based fluorescence microscope (DSLM) generates light sheets by moving a focused beam that overlaps with the focal plane of the detection lens.  It relies entirely on cylindrically symmetric optics.  In general, optical sectioning and no phototoxic damage or photo bleaching outside a small volume close to the focal plane are intrinsic properties of LSFM.  It takes advantage of modern camera technologies and can be operated with laser cutters as well as in conjunction with fluorescence correlation spectroscopy (FCS).  We have successfully evaluated the application of structured illumination.  We also designed and implemented a SPIM-based frequency domain fluorescence lifetime imaging setup and developed new scanning schemes based on novel optical arrangements that allow us to take full advantage of very high-resolution light microscopy.  During the past decade, we applied LSFM, e.g. in the investigation of three-dimensional microtubule dynamics, the development of Drosophila melanogaster and Danio rerio embryos as well as many different aspects of three-dimensional cell biology.  During the past four years, we placed a particular emphasis on plant biology, three-dimensional cell biology and emerging model organisms such as Tribolium castaneum. LSFM have been commercialized by several companies. They have become the state-of-the-art instruments in developmental biology, three-dimensional cell biology as well as in plant biology and have generated more than 500 publications and more than 7,000 citations. 

Basic concept 

LSFM has revolutionized fluorescence microscopy.  It is based on an extremely simple and yet ingenious optical arrangement that provides true optical sectioning over an extended field of view.  LSFM refers to a technology that observes a specimen with at least one microscope objective lens whose focal plane is illuminated azimuthally by at least one sheet of light.  In LSFM, only a thin volume wrapped around the focal plane of the detection microscope objective lens is illuminated.  Therefore, endogenous organic molecules or fluorophores in the volumes in front and behind the light sheet do not receive any light and are not subject to photo-damage.  These fluorophores cannot contribute to image blurring by out-of-focus light.  In physical terms, LSFM provides true optical sectioning and an axial resolution.  Three-dimensional image stacks are generated by moving the light sheet and the specimen relative to each other.  Hundreds of planes in different locations along the optical axis of the detection lens are illuminated.  Using cameras, tens to hundreds of images, which consist of millions of picture elements, are recorded every second.  Since the optical sectioning capability of LSFM is provided in its illumination process, it exposes a specimen to up to five orders of magnitude less energy than confocal fluorescence microscopy. 

Light sheets have been known for more than 100 years, but so have light spots. Until lasers became available in 1960 (Maiman TH (1960) Stimulated Optical Radiation in Ruby. Nature, 187(4736):493–494.), neither light spots nor light sheets were diffraction limited. Hence, optically sectioning instruments (I. J. Cox, “Scanning optical fluorescence microscopy,” J. Microsc., vol. 133, no. 2, pp. 149–154, 1984.) could not be built. A confocal fluorescence microscope, which illuminates a specimen sequentially with a diffraction-limited spot of light, can only be operated with a laser. Laser light sheet–based devices, including macroscopes (A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc., vol. 170, no. 3, pp. 229–236, 1993 & E. Fuchs, J. Jaffe, R. Long, and F. Azam, “Thin laser light sheet microscope for microbial oceanography,” Opt. Express, vol. 10, no. 2, pp. 145–154, 2002), had been built several times, but their capability to perform at a microscopic level was not known until, starting around 2002, my group (LMG, Light Microscopy Group), then at the European Molecular Biology Laboratory (EMBL) in Heidelberg, described the SPIM. The group applied Light Sheet Microscopes to observe live biological specimens (Huisken, Swoger, Del Bene, Wittbrodt, & Stelzer, 2004), (Keller, Schmidt, Wittbrodt, & Stelzer, 2008), determined its optical properties (Engelbrecht & Stelzer, 2006), evaluated its applicability for multiple-views imaging (Swoger, Huisken, & Stelzer, 2003; Verveer et al., 2007)and assessed its applicability at the molecular level with FLIM/FRET- (Greger, Neetz, Reynaud, & Stelzer, 2011)as well as FCS-SPIM (Wohland, Shi, Sankaran, & Stelzer, 2010). The LMG had systematically evaluated diffraction-limited microscopes with two to four lenses, both in theory and in practice, since the early 1990s, selected papers (Hell & Stelzer, 1992; Huisken, Swoger, & Stelzer, 2007; Lindek & Stelzer, 1996; E.H.K. Stelzer & Lindek, 1994). The impact of LSFM was recognized in 2015, when Nature Methods announced “Light Sheet-based Fluorescence Microscopy” Method of the Year 2014 (Ernst H K Stelzer, 2015).

The past eight years

My move from EMBL to the Goethe University in Frankfurt am Main allowed me to re-consider, to which scientific topics I could contribute.  A major goal of the Physical Biology Group is to pursue experiments in the life sciences under close-to-natural conditions.  Hence, its members favour primary cell lines and try to avoid experiments with cultured cell lines, they favour three-dimensional cell cultures over two-dimensional cell monolayers that are cultivated on hard and flat surfaces and they try to maintain the three-dimensional context of plants, cell clusters, tissues sections and small animal embryos.  In consequence, many specimens are relatively large, optically dense and require advanced methods in terms of preparation, maintenance, visualization, data handling and data analysis.

We apply our LSFM to investigate the development of Arabidopsis thaliana’s root system (Maizel, von Wangenheim, Federici, Haseloff, & Stelzer, 2011).  Close-to-natural growth conditions (e.g. upright position, roots in liquid and leaves in air, maintenance of a 16 hrs / 8 hrs diurnal cycle) are essential for observations lasting multiple days.  We have successfully established a complete plant imaging pipeline.  The careful sample preparation and fluorescence light microscopy under close-to-natural conditions allow us to image large volume elements in plants with subcellular resolution.  Our image processing applications identify cells in three dimensions and as a function of time.  A careful analysis allows us to track nuclei movements over time, to determine when cell divisions occur, to measure the orientation of daughter cells in three dimensions and to generate the complete lineage of all cells (Berson et al., 2014; Lucas et al., 2013; Rosquete et al., 2013; Vermeer et al., 2014; von Wangenheim, Daum, Lohmann, Stelzer, & Maizel, 2014).

Most group members establish and apply tissue and spheroid technologies for both fundamental and applied as well as translational, research.  We develop three-dimensional cultures of cell spheroids for drug and toxicity screening purposes.  We collaborate with industrial partners as well as with academic partners and the University’s Clinics.  Spheroids serve as models for the development of healthy tissues and their maintenance in the living organism but also as models for tumour growth (Francesco Pampaloni, Reynaud, & Stelzer, 2007).  We currently evaluate the therapeutic benefits as well as the toxicity of drug candidates in spheroids by means of conventional fluorescence microscopy as well as LSFM and many different biochemical and molecular biology-based assays (Ansari, Müller, Stelzer, & Pampaloni, 2013; F Pampaloni & Stelzer, 2010; Wenzel et al., 2014).

We established the red flour beetle Tribolium castaneum as an EvoDevo model organism in our group and developed a non-invasive long-time fluorescence live imaging protocol for Tribolium embryos for our LSFMs.  By using a new mounting technique, we became able to observe Tribolium embryogenesis for up to 50 hours along multiple directions, documenting all major embryogenic events such as gastrulation, germ band elongation, germ band retraction and dorsal closure continuously in the same specimen.  The embryo survives the imaging process, develops into an adult and produces fertile progeny.  Our long-term observations allowed us to find correlations between morphogenic processes that happen more than 24 hours apart, for example the serosa scar, a transiently formed structure that links serosa window closure during gastrulation and serosa opening during dorsal closure (F. Strobl, Schmitz, & Stelzer, 2015; F. Strobl & Stelzer, 2016; Frederic Strobl & Stelzer, 2014).

Two further major projects are the development of image processing pipelines and the mathematical modelling of our results.