Description
Employs a pinhole aperture and focused laser beams to collect serial optical sections, allowing for high-contrast 3D reconstruction.
How this lab uses this technology
Laser Scanning Confocal Microscopy (LSCM) is an advanced fluorescence imaging technique that provides high-resolution optical sections of biological samples. The system uses a focused laser beam to scan the specimen point by point, while a spatial pinhole located in front of the detector removes most of the out-of-focus fluorescence. This produces images with improved contrast and spatial resolution compared with conventional widefield fluorescence microscopy.
By acquiring consecutive optical sections at different depths, LSCM enables the visualization of internal structures without physically sectioning the sample. These optical sections can be combined to generate three-dimensional reconstructions of cells, tissues and microorganisms. The technique also supports multichannel imaging, allowing several fluorescent markers to be detected within the same sample and facilitating the study of their spatial distribution and potential colocalization.
The Leica STELLARIS platform is a point-scanning confocal system designed for flexible and sensitive fluorescence imaging. Depending on the installed configuration, the platform may combine tunable laser excitation, spectral detection and highly sensitive photon-counting detectors. These features enable precise selection of excitation and emission ranges, efficient detection of weak fluorescence signals and improved separation of fluorophores with partially overlapping emission spectra.
Advanced tools available on STELLARIS platforms may include spectral imaging, fluorescence-lifetime-based contrast through TauSense, and LIGHTNING confocal super-resolution processing. These technologies can provide additional contrast, improve image quality and help distinguish fluorophores or biological structures that are difficult to separate using fluorescence intensity alone. Availability of these functions depends on the specific configuration and software modules installed in the instrument.
LSCM can be applied to both fixed and living samples, including cultured cells, animal and plant tissues, microorganisms, organoids and other fluorescently labelled biological materials. It is particularly useful for studying cellular and subcellular organization, protein and molecular localization, cytoskeletal structures, organelles, tissue architecture, host–microorganism interactions and dynamic cellular processes.
Typical applications include:
Acquisition of high-resolution single-plane fluorescence images.
Sequential and simultaneous multichannel fluorescence imaging.
Optical sectioning and acquisition of z-stacks.
Two-dimensional and three-dimensional reconstruction of biological structures.
Colocalization and spatial distribution studies.
Spectral separation of fluorophores with overlapping emission profiles.
Time-lapse imaging of living cells and dynamic biological processes.
Quantitative analysis of fluorescence intensity, morphology, area, volume and object distribution.
Imaging of large sample areas through mosaic or tile-scanning acquisition.
Photobleaching-based experiments, such as fluorescence recovery after photobleaching, when supported by the experimental configuration.
The main outputs include high-resolution two-dimensional images, three-dimensional reconstructions, multichannel fluorescence datasets, time-series recordings and quantitative measurements of fluorescent structures. Consequently, LSCM is a powerful and versatile technique for investigating the morphology, organization, localization and dynamics of biological systems at cellular and subcellular levels.