LightSheet Microscopy @ Biovis
Compared to many other imaging techniques LightSheet microscopy allows you to image living samples such as zebrafish embryos over a long time scale without compromising on details. You can discover fluorescently labelled 3D structures in high resolution within your specimen without harming the whole organism. The different ways of mounting and positioning your specimen offers you maximum flexibility of visualizing your sample in all possible angles. LightSheet Microscopy allows for fast and sensitive image acquisition.
Overview of a 2 day old zebrafish larvae mounted in agarose.
Zebrafish embryo (24 hpf) expressing two fluurescent proteins.
Ventral Z-stack of a Tg(col2a1a:GFP) zebrafish larvae.
Blood vessel sprouting - Time-lapse imaging
This movie shows the development of the intrasegemntal vessels in a fli1:GFP transgenic zebrafish larvae. Time-lapse imaging was started at the 20 somite stage and Z-stacks were recorded every 15 minutes. The gif animation to the left shows maximum intensity projections of the first 8 out of 20 hours.
LightSheet Microcopy - Technical details
The image to the left depicts schematically the principle of LightSheet micrcoscopy. LightSheet microscopy is a special technique within the field of Fluorescence microscopy. Ideally, the sample should be translucent and fluorescently labelled or having intrinsic fluorescence. As the name reveals – in LightSheet microscopy, a sheet of light is casted through the specimen perpendicular to the detection optics, so that an optical section is created per se. The light sheet is created by low numerical aperture (N.A.) objectives, resulting in a sheet of light of several micrometres with low photon intensity. The emitted light of the excited fluorophores will be collected by a high N.A. objective, resulting in high resolution images, which are finally detected by a sensitive camera with a high dynamic range. All those features will enable the user to image fast and with low light conditions, minimizing detrimental effects caused by light - a perfect tool for live cell/animal imaging.
The LightSheet microscope is able to optically zoom in or out to allow for visualization of larger or smaller field of view. During the imaging process the sample is hanging freely into a chamber filled with the preferred medium of refractive index (R.I.) 1.33 (waterlike). The chamber´s temperature can be maintained from 10-42°C. CO2 enrichment can be applied. The hanging sample is freely movable in X, Y and Z and can be rotated 360°. Those features will enable the user to find easily the best view for imaging, to perform Z-stack imaging, tiling and multiview imaging. Multiview imaged Z-stacks can be fused to gain further isotropic resolution throughout your sample for 3D reconstruction of your sample.
The sample can be mounted in different ways depending on the needs of the experiment: embedded in low melting agarose or methylcellulose, using glass capillaries or FEP tubes, or even free-hanging in the imaging chamber.
Detection objectives and Filter combinations available at BioVis´ Zeiss Lightsheet Z.1 seen in Image to the left. The set-up of the instrument also allows for reflection imaging and even bright field imaging.
Protocols for the LightSheet microscope as well as for preparation and mounting of zebrafish embryos are listed in the box to the right.
For help regarding zebrafish housing and care-taking contact Katarina Garpenstrand.
Microscopy: Light Sheet Sectioning (Ernst Stelzer)
Read more about LightSheet microscope Zeiss Z.1.