Add versatility to your research
Complementing your STELLARIS 5 or STELLARIS 8 system with DLS renders your research more versatile, enabling you to easily image different types of samples in three dimensions with straightforward sample handling and multi-position experiments.
The DLS module is designed to improve your live cell imaging applications, increasing cell viability thanks to single plane illumination and fast imaging with a sCMOS camera. In addition to live specimens, DLS also allows to image cleared sample, providing more flexibility to reach your research goals.
The Vertical Turn – How is light-sheet brought to a confocal?
Light-sheet microscopy usually requires a dedicated optical setup on an independent system, where the illuminating and detecting objective are perpendicular to each other. DLS makes light sheet microscopy easily accessible. The unique TwinFlect mirror device deﬂects the illuminating light sheet at a 90° angle. This improvement allows the integration of the illumination and detection beam path into the vertical axis of every STELLARIS system with inverted microscope stand. All this enhancement without compromising confocal functionality.
Low phototoxicity and specimen imaging in 3D: Development of Drosophila melanogaster over six hours. Probe: Light-sensitive RFP. 3D rendering. 150 µm z stack, 30sec/stack.
Conduct and document long-term observations
Imaging requires light, but too much light can damage your cells. Light-sheet microscopy is the most gentle imaging method to date, as it reduces the overall photodamage from phototoxicity and bleaching. This automatically increases the viability of your specimen. Particularly developmental biology beneﬁts from light- sheet imaging. The combination of low light illumination and high speed acquisition allows you to follow sensitive, developing organisms like a Drosophila embryo over long periods of time and to understand how tissue and organs form in real time and 3D.
Principle of light-sheet imaging
Light-sheet microscopy is a highly suitable way of imaging sensitive samples or fast biological processes by illuminating the specimen only in a single plane.
Because there is no out-of-focus excitation, phototoxic eﬀects can be reduced to the focal plane. Also, by moving the sample through the light-sheet, you automatically have optical sectioning and can image specimens in 3D.
High speed imaging: Beating zebraﬁsh heart, transgenic line ﬂik1::EGFP. Post-acquistion alighment after xytz recording. Recording speed: 120 frames/second. Courtesy of Emily Steed, Vermot Lab, IGBMC Strasbourg, France.
Fast image acquisition with excellent resolution
Observing fast and periodic biological processes in 3D over an extended period of time, like the beating of a zebrafish heart, can be challenging without the right image speed and resolution. With the DLS module, you can choose between two cutting-edge sCMOS cameras for fast image acquisition with excellent resolution, both fully integrated into the LAS X LightSheet Wizard.
7.5 h time-lapse recording of mammary epithelial micro spheroid cultured in 3D. Data courtesy of intelligent imaging group (B. Eismann/C. Conrad) at BioQuant/DKFZ Heidelberg
Exploring 3D cell cultures, multiple positions at a time
Effective imaging of 3D cell cultures, such as organoids or spheroids, poses a new set of challenges due to their large volumes. Organoids can be fixed, immunolabeled, and studied using clearing techniques but also utilized live to study dynamic processes. Investigating these samples with DLS is simple thanks to defined workflows and provides meaningful information about cellular and molecular processes. DLS is well suited to address needs for both cleared and live preparations, giving you the benefit to study 3D cell cultures that offer conditions which are more physiological relevant compared to 2D cell culture on adherent cells.
Discover the complete 3D Cell Biology Workﬂow with Digital LightSheet Microscopy.
A light-sheet system for live and cleared samples
Deep tissue microscopy is difficult due to the opacity of biological tissues. This is where the ever growing number of tissue clearing protocols comes into play, but they can be a challenge for many imaging systems.
The DLS module not only delivers outstanding results with live samples, but can also image samples that have undergone a variety of different clearing techniques. With DLS, you can choose between several detection objectives and mounting frames that allow the visualization of structural details within cleared tissues and organisms.
Video acquisition conditions:
- Detection objetive HC APO L10x/0.30 W DLS
- image size: 1.39mm x 2.04mm (2x3 tiling)
- Z stack:1.5mm thickness (2um step size, 745 frames)
- Acquisition time:5 min 37 sec
- Emission: LP514nm long pass, 6 msec exposure time
Cerebral cortex and hippocampus of adult Thy1-YFP-H mouse brain slice cleared with SeeDB2G. Images courtesy of Dr. Meng-Tsen Ke and Dr. Takeshi Imai, RIKEN Center , Kobe, Japan.
This example demonstrates the advantage of confocal combined with DLS. A transgenic Zebrafish embryo was labelled to visualize macrophages involved in immune responses. To investigate their behavior upon wounding, a confocal laser was used to create a wound (arrow) and afterwards, a time-lapse movie was recorded. The video shows the dynamic reaction of the activated cells that are now migrating to the wound triggering the healing process. For this case, switching to DLS allows gentle live cell imaging of the Zebrafish.
Beneﬁt from many additional applications
Our light-sheet module is more than a functional add-on to your confocal. STELLARIS and DLS synergize to expand the options for your research. For example, you can manipulate your specimens using the confocal technology and then image with DLS.
It is simply done by switching between confocal and light-sheet mode in LAS X software. This way, photoconversion or wounding experiments with subsequent gentle long-term observations become easy and convenient.
Familiar sample preparation
Due to the vertical experimental setup of DLS, you can keep your familiar sample preparation. The specimens are mounted in conventional glass bottom petri dishes and are directly accessible. You even have the possibility to screen several samples in multi-positioning experiments. Making use of the confocal stage automation, several specimens can be imaged in one experimental setup. What is the only prerequisite? Some space for the TwinFlect mirrors on each side of the sample.
Additional sample handling Tools
Using U-shaped glass capillaries for sample mounting
U-shaped glass capillaries come in two different sizes. This way they fit with different TwinFlect mirrors and can serve as optimal scaffolds for positioning your sample(s).
Using a rotation device for DLS sample mounting
With a refractive index of 1,338, FEP tubes are often used for mounting specimens that are immersed in aqueous solutions. In order to optimally align the sample for imaging, a rotation device has been designed to adjust the viewing angle.
Using mounting frames for sample preparation
The Leica mounting frames shown here deliver a major benefit for the sample preparation needed to do DLS imaging: They allow preparation of a higher number of specimens They can be used together with potentially harmful reagents like BABB (Benzyl Alcohol Benzyl Benzoate).
Light-sheet with confocal benefits
Thanks to the seamless integration of DLS, your light-sheet imaging benefits from the technical innovations of your STELLARIS system.
Always the right laser
All visible lasers of your STELLARIS confocal are ready to be used for light-sheet imaging. A high flexibility in choosing the right dye for your light-sheet experiments is possible with the optional diode laser lines as well as the STELLARIS next generation White Light Lasers. With STELLARIS 8, you can now even unlock the imaging of near-infrared dyes.
Always the right scanner
On STELLARIS systems equipped with a tandem scanner, you can choose between the resonant scanner or the field-of-view scanner (at 1400Hz) to generate the scanned lightsheet. Generating the lightsheet with the resonant scanner produces shorter pixel dwell times, which is beneficial to perform even more gentle imaging.
Workﬂow-oriented Software Design
The LAS X software guides users step by step through data recording and evaluation.The workﬂow-oriented design helps you to use the instrument more eﬃciently. A convenient calibration routine establishes the light sheet precisely.
Dual-sided illumination of the sample comes by design: each of the two opposing mirrors of the TwinFlect can be targeted by the scanner to overcome darker areas. For crisp images with a large ﬁeld of view, you can merge the two images using the online or oﬄine fusion option of the LightSheet Wizard in the LAS X software.
Tailor LAS X to your needs with additional software packages. The LAS X 3D Visualization module provides novel ways to interact with your 3D data by intuitive clipping, fast rendering, and stereo display. Tile scanning experiments enable you to observe large areas. Mark & Find experiments allow you to observe several regions of interest in a multi-position set up.
Superior objectives for a wide range of applications is one of Leica Microsystems’ hallmarks. The heart of our system turning the light sheet vertically is formed by the objectives and the TwinFlect mirror device.
With the choice of diﬀerent illumination objectives, you can shape the light-sheet depending on your experiments’ requirements.
In order to reveal the ﬁnest details, have a larger ﬁeld of view, or adapt to the refractive index of your imaging medium, you can pick the optimal detection objective from a growing number of options.
|Objective||Live cell imaging||Sugar based clearing reagents||BAAB clearing compatible|
|FLUOTAR L 25x/0.95 W||Yes||No||No|
|APO L 20x/0.5 W||Yes||No||No|
|Fluotar L 16x/0.6 IMM||Yes||Yes||Yes|
|APO L 10x/0.3||Yes||Yes||No|
Convenient data handling
Observing processes in 3D and over long periods of time generates a lot of data. Several tools implemented into the LAS X software help you to manage your data conveniently. The online fusion tool gives you the choice to keep raw data or to gain disc space by saving only the merged image. Your data is automatically stored during acquisition. The smart loading facilitates data review giving you direct access to the time points or z stacks of interest in large time-lapse data sets. You can compile your desired post-processing steps in a pipeline which is automatically processed.
Optimal environmental conditions
A software controlled climate chamber keeps the environmental conditions that your specimens like best. Users gain full control of the experimental conditions with the LAS X Environmental Control module. The logged environmental data can be monitored during the experiment. All environmental conditions can be set within one interface, so for example, you can run temperature profiles for heat shock experiments.
Choose between an environmental chamber or stage top incubation solutions.
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