LCS SPIM for high-throughput cleared sample imaging

The Bruker Luxendo LCS SPIM is a compact light-sheet microscope designed for imaging large optically cleared samples. Designed based on lab-user feedback, it is easy to maintain and clean, ensuring safe handling of clearing liquids. It is compatible with all clearing solutions currently on the market, making it ideal for experiments across different clearing methods and multi-user environments.

The system can support both large and small samples by using flexible embedding options. It allows for fast-imaging options, minimal sample distortion, and extended field of view by using three objective lenses, single-side detection, an inverted optical design, and dual-side illumination. LCS SPIM is suitable for use in multi-user environments and high-throughput studies.

Compact, benchtop design

• Easy and safe handling
  • The LCS SPIM is compatible with all clearing solutions with an optical setup to support safe handling of solutions and samples. The benchtop design also has a Laser Safety Class 1 system.

High-throughput capability

• Cutting-edge quality and speed
  • It has an inverted design with dual-side illumination to ensure high-quality imaging with a fast imaging option, minimal sample distortion, and an extended field of view.

Multi-size cuvettes with sample holders

• Bespoke embedding solutions
  • Samples are embedded in an exchangeable quartz-crystal cuvette available in three sizes. Custom models were also designed with users to allow for stable imaging of complex-shaped tissues and objects.

New advanced features

Dual destriping: Impressive elimination of striping artifacts

During imaging, cell nuclei or pigments can interfere with light, leading to striping artifacts and shadows in the images. The new SPIM enables dual destriping by using beam swiping and pivot scanning that allows for creation of a homogeneous illumination profile without changing the acquisition speed.

Image Credit: Bruker Nano Surfaces and Metrology

TAG-lens: Light-sheet length control with axial scanning

Ultra-fast varifocal lenses can be used to adjust the system’s optics. Luxendo SPIMs use tunable acoustic gradient (TAG) lenses. The lenses operate using acoustic waves and help reduce axial stretching while ensuring uniformity and improved light-sheet control across the field of view.

Compact, benchtop design

The LCS SPIM's vibration-free, compact design offers improved stability even during long-term, high-throughput experiments.

The system's basic configuration is cost-effective and suitable for cleared sample imaging. With a highly versatile layout, the flexible upgrade options expand the system’s performance to enhance optical performance and speed. It also ensures controlled thermal conditions and maximal stability of focus.

The class 1 laser system can be customized to meet the user’s lab bench requirements and does not require any vibration-compensation mechanism or air table.

The system's individual parts are lightweight and balanced. Its piezo-crawler stages provide precision and longevity for permanently accurate specimen positioning. Its gentle image acquisition technology does not affect the quality of the images or the sample.

Next-generation imaging

Large and closed cuvette

Image Credit: Bruker Nano Surfaces and Metrology

The system simplifies the process of mounting samples and cleaning and maintenance of the microscope by using exchangeable quartz-crystal cuvettes. The cuvettes are available in three sizes, from [20 mm × 20 mm × 20 mm] to [100 mm × 30 mm × 30 mm] (l × w × h), to accommodate imaging of different samples, from organs to organisms.

Illumination and detection

The cuvette moves in a Z pattern. Then, the focus shift is corrected by making changes in the detection optics to ensure the best illumination. The sCMOS camera is well-suited for experiments requiring high speed, quantification, and detection efficiency.

The ideal setup for cleared samples

High flexibility of sample size

The system is flexible for imaging cleared organs, such as a mouse brain, and whole organisms, such as a whole mouse. By placing samples in closed cuvettes, users can ensure safe handling. It is also compatible with all clearing solutions, covering a refractive index (RI) range of 1.33 to 1.58.

Compared with other microscope systems that face challenges in operating with multiple clearing techniques, LCS SPIM is compatible with and flexible across all currently available clearing solutions.

Innovative and easy sample handling

During the imaging process, reproducible and safe sample handling is critical. The LCS SPIM cuvettes come in three different sizes from [20 mm × 20 mm × 20 mm] up to [100 mm × 30 mm × 30 mm] (l × w × h). This allows imaging of different samples from small organs to medium-sized organisms. The different sizes mean that only a small number of expensive samples are required.

The cuvettes cover the commonly used refractive index (RI) range from 1.33 to 1.58 and more, and hence are compatible with all currently available clearing solutions. Further, custom, easy-to-use sample holders are also available, designed in association with application specialists and end-users. The system also has a 3D printing option for users to design customized sample holders.

Image Credit: Bruker Nano Surfaces and Metrology

Advanced fast-imaging acquisition

The field of view of the illumination beams can be extended by varying the axial position of the light sheet. This reduces phototoxicity and the number of images required, while enhancing the imaging rates. It also reduces shadowing artifacts and improves image quality.

LCS SPIM and advanced fast-imaging acquisition also improve imaging speed and thus find use for imaging large cleared samples. The LuxBundle processing software also enables accurate and fast image processing.

Example case: Speed scanning a whole mouse brain with two labels/channels:

• Magnification: 4.4×
• Tiles: five × four tiles (20 total)
• Resolution: 1.4 × 1.4 × 10 µm (x,y,z)
• Exposure time: 50 ms
• Scan time: 30 minutes
• Processing: 30 minutes

High-throughput capability

One quartz crystal cuvette simplifies quick loading of bulky, fragile specimens. Each item is simply placed inside this container holding the clearing liquid.

The motorized stage moves the sample into position and collects image layers. At the same time, adjustable optics quickly sweep a sheet of light through the sample. Together, they capture high-speed images without disturbing or damaging the sample.

Image Credit: Bruker Nano Surfaces and Metrology

Illumination and detection

Illumination optics is the process of generating a light sheet by passing a beam-shaping unit to adjust the diameter and length and scanning a beam. Dual Nikon 4× (0.2 NA) air objectives project two perfectly aligned light sheets onto the sample from opposite sides. The illumination setup can be expanded and upgraded, making it flexible for selecting different lasers.

The detection optics has a single Olympus 4× 0.28NA air/dry lens. A secondary magnification changer adjusts the total system magnification between 2.2× and 8.8×. The sCMOS camera is ideal for experiments requiring high quantification, detection efficiency, and speed. The spatial detection arm has a 10-position high-speed filter wheel.

Image Credit: Bruker Nano Surfaces and Metrology

LSC SPIM Design

Image Credit: Bruker Nano Surfaces and Metrology

Image Credit: Bruker Nano Surfaces and Metrology

Image Credit: Bruker Nano Surfaces and Metrology

Image Credit: Bruker Nano Surfaces and Metrology

Image Credit: Bruker Nano Surfaces and Metrology

Applications

Whole organ imaging: Geometric patterning of tissues

Integrating light-sheet imaging and tissue clearing is the most suitable option for generating detailed three-dimensional insights of whole organs and tissues. The combination provides novel insights into the in toto geometric patterning of tissues, organs, and organisms.

Intricate branching patterns of mouse lungs and blood vessels. Samples courtesy of: Ayelen Melina Santamans Recchini and Gaudalupe Sabio Buzo, Stress kinases in Diabetes, Cancer and Cardiovascular Disease Laboratory and Unit of Microscopy and Dynamic Imaging, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid. Image Credit: Bruker Nano Surfaces and Metrology

Minimal solution requirements

Using the correctly sized cuvettes for embedding samples ensures minimal use of clearing solution, reduces exposure to chemicals, and saves costs.

Rendering of sample cuvette in the LCS SPIM with a cleared mouse sample (illumination path: blue; detection path: green). Photos show LCS sample cuvette from the top, holding cleared mouse samples, including lung, heart, bone, and head. Image Credit: Bruker Nano Surfaces and Metrology

Whole mouse imaging

Sample imaged on the LCS SPIM. Image Credit: Dr. Chenchen Pan, DKFZ, Heidelberg, Germany. 

Neurogenesis

Elucidating the mechanisms of nervous system development and maintenance is fundamental to understanding its degeneration in aging and disease. Combining optical tissue clearing with LCS SPIM light-sheet microscopy enables advanced 3D visualization of biological structures within intact organisms. Thus, it is possible to study intricate nerves in whole organisms, a capability previously restricted to medical imaging modalities.

Developing nerves in a whole mouse embryo. Sample cleared with DBE. Tiled image (3 × 4) acquisition. Scalebars: 1 mm. Imaged on the LCS SPIM. Sample courtesy of: James Muller, MSKCC, New York, USA. Image Credit: Bruker Nano Surfaces and Metrology

Morphogenesis and health

Imaging with the LCS SPIM is not restricted to already formed tissue. The system helps users understand the formation and maintenance of tissues. The following example shows a visualization of the muscle-specific precursors in a fruit fly, providing key insights into how muscle tissue develops and stays healthy throughout life.

Transgenic Drosophila Line Expressing UAS-mCD8-GFP with the Mef2-GAL4 Driver Line. The fruit fly was optically cleared, and refractive index adjusted using alcohol dilutions and ethyl cinnamate (ECi). Length head-to-tail: 1.7 mm. Imaged on the LCS SPIM. Image Credit: Susanne Önel, Philipps University Marburg, Marburg, Germany.

Cleared-sample/cleared-tissue applications

LCS SPIM can be used by researchers in various applications, including research studies.

Specifications

Source: Bruker Nano Surfaces and Metrology

System comparison

Source: Bruker Nano Surfaces and Metrology

^* compatible module

Software

All-in-one LuxBundle software

Intuitive design

Bruker's LuxBundle software reduces time while improving productivity by offering:

• Completely scriptable microscope control and post-processing through an open interface (e.g., Python or any other language), ready for custom "smart" microscopy
• All-in-one, easy-to-use interface for collection, viewing, and post-processing
• Data formats (.hdf5, .tiff, .ims) compatible with common image processing software: Imaris, BigDataViewer, Aivia, Fiji, Arivis, Matlab, Python, and Napari
• High reproducibility of experiments: All parameters are saved in the metadata and configurations can be saved for future experiments

Image Credit: Bruker Nano Surfaces and Metrology

Impressive image post processor

Bruker’s dual-view light-sheet microscopes can image a sample from different views/angles and generate images with multiple tiles. The LuxBundle software enables crisp 360° images with improved quality that compensate for scattering and absorption. Features include:

• Tile stitching of hundreds of tiles for large samples
• Multi-color alignment
• Multi-view image fusion and deconvolution

Cleared mouse embryo labeled with methylene blue (cyan) and showing autofluorescence (magenta). Image composed of 12 tiles and 920 planes, all processed and stitched with LuxBundle. Image Credit: Montserrat Coll Lladó, European Molecular Biology Laboratory, Barcelona, Spain

3D data viewer

LuxBundle's integrated 3D data viewer enables researchers to analyze the complete dataset directly after acquisition. This gives users control over their data with key capabilities, including:

• Both raw and post-processed images
• The ability to turn tile stitching on or off
• Flexible options to draw and annotate regions and landmarks
• Fast viewing of multi-terabyte data sets