Overlay image showing the eye of an intact unlabeled zebrafish. Green: Stimulated Raman Scattering (SRS) image of lipid components (at 2850 cm⁻¹). Red: SRS image of protein components (at 2935 cm⁻¹). Blue: second-harmonic signals, mainly from the sclera and cornea. Sample provided by Elena Remacha Motta and Julien Vermot, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Strasbourg, France.

Gain the power to image targets inaccessible with traditional methods

While traditional fluorescence microscopy methods are highly successful research tools, the type and number of targets that can be visualized are limited. STELLARIS 8 CRS helps you overcome these limitations:

  • Direct visualization of chemical bonds to target events and structures which are largely inaccessible with traditional methods; 
  • Three-dimensional image information that enables observation of fine details even inside complex 3D specimens; 
  • Minimally perturbative excitation for dynamic studies with the specimen maintained as close to physiological conditions as possible, from video-rate imaging to long-term observations of sensitive specimens.

Image structures and events without a need for fluorescent dyes

The STELLARIS 8 CRS microscope allows users to image and differentiate structures and events using their chemical properties. This way, it can provide access to a vast amount of biochemical, metabolic, and pharmacokinetic information that is inaccessible via traditional methods. 

Image contrast in CRS is provided by the characteristic, intrinsic vibrational states of the different molecules in the specimen. Thus, no staining of the specimen is needed eliminating the drawbacks of dye-based imaging methods, such as photobleaching and staining artifacts.

Multi-color SRS imaging reveals the subcellular distribution of a Raman-tagged pharmacological compound (yellow, SRS imaging at 2230 cm⁻¹), in the context of the endogenous lipids and proteins inside an otherwise unlabeled cellular sample. Sample courtesy of Dr. Matthäus Mittasch, Dewpoint Therapeutics GmbH.

Three-dimensional imaging in brain tissues: Z-stack of a 200 µm thick mouse brain slice, showing simultaneous SRS imaging of myelinated axons (glow) and two-photon fluorescence from Thy1-YFP labeled neurons (cyan). Sample courtesy of Dr. Monika Leischner-Brill, Institute of Neuronal Cell Biology, TU München, Germany.

Built-in three-dimensional imaging for 3D samples 

STELLARIS 8 CRS is perfectly suited to image 3D samples, such as tissues, organoids, or intact small model organisms, at subcellular resolution by directly using their chemical properties. 3D imaging without a need for post-processing is a built-in property of CRS, thanks to a combination of two features: 

  • CRS signals are generated through a nonlinear optical effect that takes place exclusively inside the focal volume of the excitation lasers, providing intrinsically three-dimensional image information.
  • The near-infrared laser beams used for CRS excitation propagate through the sample with minimal perturbation, allowing for efficient imaging even inside intact 3D specimens.

Image live specimens as close to physiological conditions as possible

The highly efficient excitation of molecular bonds by CRS provides chemically specific image contrast at unprecedented speeds. It enables the imaging of live specimens at video rates. 

STELLARIS 8 CRS is equipped with a Tandem Scanner that allows both conventional and high-speed imaging of many specimen morphologies. 

In addition to speed, gentle imaging is essential to preserve live samples during long-term observation. The stain-free approach combined with the use of near-infrared lasers keep phototoxicity and photodamage at minimal levels. 

Label-free investigation of subcellular dynamics in a live small-intestinal organoid. The time lapse movie of SRS signals (2940 cm⁻¹) shows endogenous proteins and lipids, providing insights in epithelial cell organization and lipid droplet dynamics in this model system. Sample courtesy of Dr. Ruslan Dmitriev, Ghent University, Belgium.

Amyloid-β plaques and associated pathological lipid deposits visualized in unlabeled brain tissues. Spectroscopic analysis shows an enrichment of membrane lipids and a depletion of cholesterol compared to nearby healthy brain structures, providing a new window to study connections between lipid metabolism and Alzheimer’s pathology. Sample courtesy of Dr. Martin Fuhrmann, Andrea Baral, German Center for Neurodegenerative Diseases, Bonn.

Explore the potential of morpho-chemical and functional information in your imaging experiment

To tackle challenging problems in the life sciences and fundamental medical research, it is frequently necessary to maximize the information gained from your samples. This often includes the need for imaging of non-traditional targets, such as changes in lipid metabolism.

STELLARIS 8 CRS provides you with a fully integrated system that allows you to acquire and correlate a wide range of biochemical and biophysical contrasts – in addition to confocal fluorescence intensity and lifetime information – to get the most out of your experiment.
 

Gain information on the biochemical composition of your sample

The combination of morphological and biochemical information can be crucial for the understanding of healthy biological functions and any changes caused by disease.

STELLARIS 8 CRS provides label-free imaging with chemical contrast at an unprecedented spatial resolution. With CRS, biological functions can be probed on many spatial scales, ranging from subcellular organelles to groups of cells in a tissue or even pathological structures that perturb tissue function. 

Visualizing the endogenous biochemical composition of a fresh, untreated apple slice. (A) Representative frames of an SRS spectroscopic image stack. (B) SRS spectra of the regions of interest shown in (A). Yellow: outermost peel consisting of a waxy phase of long-chain saturated fatty acids. Green, red: inner cuticular layers made of short-chain unsaturated fatty acids. Blue, magenta: polyphenolic compounds. Cyan: cell walls made of polysaccharides. Orange: carotenoid pigments. (C) 8-color spectral unmixing result showing the biochemically distinct structures.
Label-free SRS imaging reveals the core-shell architecture of a multi-cellular skin cancer spheroid model and uncovers the appearance of an unexpected, lipid-rich cell phenotype (isolated, bright yellow cells). Sample courtesy of Dr. Julia Klicks, Prof. Rüdiger Rudolf, Hochschule Mannheim, Germany.

Reveal new dimensions relevant to development and disease

Direct visualization of cellular phenotypes and metabolic states is key to understand biological processes in health and disease. Sample processing may alter these properties, so a label-free approach can be a preferred alternative.

CRS imaging provides the spectroscopic capabilities that enable a detailed study of your sample under conditions as close to real life as possible. 

Combine confocal fluorescence imaging with chemical imaging

To get an unparalleled view of the multiple biological dimensions of your sample, STELLARIS 8 CRS provides several imaging methods tightly integrated into the confocal system. They enable multi-modal optical imaging with biochemical, biophysical, and molecular contrast. 

  • Stimulated Raman Scattering (SRS)
  • Coherent Anti-Stokes Raman Scattering (CARS)
  • Single-photon or multi-photon fluorescence
  • Second Harmonic Generation (SHG)
  • Imaging with infrared (IR), visible (VIS), and ultraviolet (UV) lasers in simultaneous or sequential mode
     
Multimodal optical imaging of osteogenesis in a mouse skullcap explant using a combination of visible confocal fluorescence microscopy with multi-color chemical imaging via SRS and added physical contrast via SHG. In a single sample, the localization of osteoblasts, the deposition of extracellular collagen fibers, and the formation of bone mineral are visualized. In addition, lipid-rich structures are observed predominantly inside isolated osteoblasts scattered throughout the developing bone structures. Sample courtesy of Jacqueline Tabler and Sebastian Bundschuh, MPI-CBG Dresden, Germany.
Upper left: CARS microscope image of lipids in a brain tissue, showing lipid-rich white matter and gray matter regions. Upper right: The average photon arrival time image reveals shorter arrival times from the lipid-rich white matter and longer arrival times from gray matter. This result indicates that the instantaneous CARS signals are accompanied by 2-photon autofluorescence signals with a finite lifetime. Bottom row: Lifetime-based separation of the instantaneous CARS signals and autofluorescence signals with an average arrival time of 1.9 ns. Right: Overlay image.

Explore new possibilities with vibrational and lifetime imaging  

Many biological specimens exhibit fluorescence emission, arising either from endogenous fluorophores or from intentional fluorescent labeling. Whereas SRS signals are not affected by fluorescence, CARS signals may experience some degree of fluorescent crosstalk.

The TauSense tools in the STELLARIS platform can help solve this issue. By using fluorescence lifetime-based information, you can separate instantaneous CARS signals and fluorescence signals. 

Boost your productivity with inherently quantifiable data

STELLARIS 8 CRS offers all the versatility and ease-of-use available with the STELLARIS platform. This integration allows you to handle a wide range of challenging samples and helps you maximize the benefits of CRS imaging, including obtaining inherently quantifiable data from ratiometric and spectroscopic imaging approaches. 

SRS images and spectra of dodecane (a fully saturated hydrocarbon, cyan) and linoleic acid (a poly-unsaturated fatty acid, magenta) droplets immersed in water. The ratio of intensities at 1660 cm⁻¹ to 1440 cm⁻¹ allows for quantification of lipid unsaturation.

Getting to a CRS image in a few clicks using the intuitive ImageCompass user interface.

Easy setup of experiments with a fully integrated system

Every aspect of your experiment is fully controlled through the ImageCompass user interface, offering a convenient and intuitive approach to CRS microscopy for both experts and beginners.

Additionally, the integration of the CRS laser control into ImageCompass allows users to go from single-chemical-bond imaging to spectroscopic imaging or multi-modal imaging in just a few clicks. 

Navigate large and complex samples with ease

The LAS X Navigator is a powerful tool that allows you to quickly switch from searching image by image to seeing a full overview of your sample. The full integration of CRS multi-position experiments with Navigator allows you to perform full tile scans of large samples, providing all the information needed to choose regions of interest for subsequent, more detailed investigations.

Automated imaging of large-area samples: Shown here is a high-resolution tile scan of an entire mouse brain slice. A comparison of corresponding cortical tissue regions from mice grown on a high-fat diet vs a regular diet reveals the occurrence of pathological, lipid-rich arterial plaques on a high-fat diet, but not on a regular diet. Sample courtesy of Judith Leyh and Prof. Ingo Bechmann, Universität Leipzig, Germany.
SRS spectroscopic imaging provides detailed information on the chemical composition of brain structures. Left: SRS image showing healthy, lipid-rich white matter structures (top) and pathological lipid deposits surrounding an Amyloid-β plaque (bottom left). Right: SRS spectra show that pathological deposits are enriched with membrane lipids (sphingomyelin, phosphatidylcholine) compared to the more cholesterol-rich white matter.

Quantifiable information from hyperspectral or ratiometric imaging

Inspired by approaches developed by the Raman spectroscopy community, CRS enables ratiometric and spectroscopic imaging that provide reproducible and quantifiable information about the sample's chemical composition. These basic quantification tools are integrated in the LAS X software.

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