A revolution in the imaging technology

Over the last two decades a revolution in the capabilities of life imaging and massive parallel data acquisition has occurred. Early single trajectory or particle measurements were often only possible under the well defined in vitro circumstances and for only a few sample paths ^121,128. Unlike these classical experiments, today in vivo single particle tracking in ∝1nm resolution in

small volumes is possible⁷, and, on larger scales, the development of whole organisms can be imaged in single cell resolution and with minimal experimental interference. Movies in single cell resolution have been recorded for the embryonic development ofDrosophila^92,165(Fig. 2.1a) and the early stages of zebrafish⁸³(Fig. 2.1b) and C.elegans²⁹(Fig. 2.1c) development. For the visualization of such complex and rapidly changing biological systems, large improvements in the available optical microscopy imaging technology were necessary.

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4:53 Dorsal view

Figure 2.1: Progress in the microscopy techniques enables time-lapse imaging of develop-mental processes in 3d and all cell resolution. (a): Time-lapse imaging of nuclei in earlyDrosophila development, recorded with a multiview lightsheet setup⁴(reproduced from Amat et. al.⁴). The time inset denotes hours and minutes after egg laying. Scale bar, 50µm. (b): Early zebrafish development, visual-ized with a GFP-Histone marker¹²⁷ and recorded with multi-view lightsheet microscopy with recordings taken every 3min and over a course of 12h¹⁷⁶(reproduced from Weber et. al.¹⁷⁶). The time inset denotes hours post fertilization (hpf). Scale bar, 150µm. (c): First division ofC. elegans oocyte, recorded with a lattice lightsheet and with respect to its actin dynamics²⁹(reproduced from Chen et. al.²⁹). Time in minutes witht= 0min placed during pseudocleavage ingression. Scale bar, 5µm.

The imaging revolution began with the discovery of the green fluorescent protein (GFP), and the ability to specifically tag proteins of interest^26,101. Early experiments illuminated the entire fluorescence tagged system, while simultaneously recording from it¹²². This wide field microscopy termed technique, faces two problems when recording living systems¹⁶⁴. (ii) Illumi-nating all focal planes simultaneously yields a diminished resolution both due to background noise and light scattering from out of focus planes and directions. (i) For time lapse recordings, illuminating the whole system simultaneously leads to fast photo-bleaching and faces the prob-lem of photo-toxicity. The first probprob-lem is addressed in confocal microscopy, which due to an illumination pinhole in front of the light source and the confocal pinhole in front of the camera, only illuminates and records from fluorophores in one spot of the focal plane^122,164. In confocal laser scanning microscopy (LSM), this spot is then scanned over the probe^162,164. Due to the high scanning frequencies, the laser intensity must be high to excite enough fluorophores during

Detector

Light source Specimen Emitted light

Illumination beam

Dichroic

Illumination pinhole Confocal

pinhole

Spinning disc with microlenses

Confocal

spinning disc Lightsheet

Lightsheet Spinning Disk Confocal

Scanning Confocal Widefield

Figure 2.2: Schematic overview of different fluorescence based microscopy techniques. Shown are the illumination (green) and recording (blue) beams including the name giving and essential compo-nents (Figure adapted from Stephens and Allan¹⁶²). (Left): Widefield microscopy records from the full specimen simultaneously¹²². (Middle left): In laser scanning confocal microscopy (LSCM) a laser beam is scanned over the probe and a confocal pinhole prevents out of focus light from entering the detector^162,164. (Middle right): In spinning disc confocal microscopy (SDCM), a Nipkow disk parallelizes the concept of a confocal pinhole from LSCM. Often, a second in sync spinning disk, placed in the incoming lightpath and equipped with microlenses, is used to realize SDCM as a fully parallelized version of a LSCM⁶¹. (Right): Selective plane imaging microscopy (SPIM) replaces the illumination beam with a lightsheet orthogonal to the detection pathway, which only illuminates the current focal plane¹³². Lightsheets have been realized optically⁷⁶, as digital laser scanning beam⁸³, or as an interfering lattice of bessel beams²⁹. Today, they are often realized with two orthogonal lighsheets to allow for true 3d recordings^92,132,165.

the short illumination time per spot⁶¹. While the resolution is increased compared to widefield microscopy, photo-toxicity and photo-bleaching stays a relevant problem^61,164.

Spinning disc confocal microscopy (SDCM) addresses the problem of slow single beam scan-ning and its high illumination intensities per spot^61,162,164. In SDCM the single excitation and emission pinhole in the LSM is replaced by up to 1000 pinholes on a rotating disc, which, in current designs, serve both as illumination and confocal pinholes⁶¹. Instead of illuminating one spot at a time up to 1000 spots are simultaneously illuminated and recorded from⁶¹. This allows for a drastic increase in the obtained frame rates. Compared to single beam LSM, it allows to illuminate each individual spot longer, and thus with less intensity, which is believed to reduce both bleaching and photo-toxicity¹⁶⁴. To enhance the resolution of spinning disc microscopy, and avoid losing most of the incoming light at the spinning disc, the incoming light is often guided through another disc equipped with micro-lenses which rotates in sync with the pinhole disc⁶¹. Modern spinning disc microscopes are thus parallel confocal microscopes⁶¹.

In general, confocal imaging techniques can be combined with two-photon approaches^75,152. This technique exploits the spectroscopic properties of the used fluorophores. Instead of one-photon excitation, fluorophores are excited by 2 (or more) one-photons, with longer wavelength¹²². This switch to longer wavelengths then allows for a deeper penetration into the tissue with less scattering and as it seems increased resolution^122,152. However, as the likelihood of simultane-ously photo absorption is relatively low, high laser peak intensities are required in two-photon microscopy, which is associated with increased photo-toxicity^83,122.

The relatively low light efficiency of confocal microscopy and in consequence, resulting in-creased bleaching and photo-toxicity, has been addressed with selective plane imaging microscopy (SPIM)⁷⁶. In this already 1902 by Zsigmondy invented¹⁵⁵ and decades later recovered tech-nique^75,76,173, a light-sheet illuminates the probe perpendicular to the optical axis, and thus almost only the plane in focus. The pinhole used in convocal microscopy to confine the recorded light in z-direction becomes obsolete⁷⁵. Additionally, the whole plane defined by the light-sheet can be recorded simultaneously. Sequentially illuminating and recording from several planes

has been used to reconstruct 3d images^76,83. Combining two perpendicular light-sheets, it is now possible to record a developing organism in 3d and with equal resolution in all spatial directions^92,132,165.

The currently standard SPIM microscopy relies on light-sheets with a relatively thick Gaus-sian intensity profile inz-direction^29,132. This problem has been addressed with lattice light-sheet microscopy. This technique replaces the lightsheet by a lattice of Bessel beams, which through interference, yield an adjustable light-sheet²⁹. This constructive version of a light-sheet now al-lows to combine super resolution techniques with 3d all cell long term imaging in one conceptual setup^54,102.