Leica Microsystems’ 4Tune detector, the key component of the SP8 DIVE Deep In Vivo Explorer, provides spectrally tunable image recording with non-descanning detection. An innovative solution for multiparameter multiphoton microscopy.
Multiphoton Microscopy is one of the current hot topics in life science research. The new Leica TCS SP8 DIVE from Leica Microsystems presents a series of beneficial new innovations, including a free tunable non-descanning detector and an ingenious beam manipulating device VBE. The variable beam expander offers free tuning of both beam diameter and axial IR-correction for up to four IR beams simultaneously.
When operating a confocal microscope, or when discussing features and parameters of such a device, we inescapably mention the pinhole and its diameter. This short introductory document is meant to explain the significance of the pinhole for those, who did not want to spend too much time to dig into theory and details of confocal microscopy but wanted to have an idea about the effect of the pinhole.
Super-resolution refers to any device or method that can resolve better than the classical Abbe limit. Apart from infinite super-resolution techniques such as STED (stimulated emission depletion) and SMLM (single-molecule localization methods) that can theoretically resolve to any detail, there are also methods for limited super-resolution. Here we present HyVolution by Leica, which merges optical super-resolution and computational super-resolution. The optical part is provided by confocal microscopy, and the computational part by deconvolution. Lateral resolution of 140 nm is demonstrated. HyVolution offers multiple fluorescence recording in truly simultaneous mode.
Super-sensitive detection with low noise plus Huygens deconvolution is the key to resolving structures down to 140 nm with a confocal microscope. The result is crisp images full of contrast revealing details, which cannot be seen with confocal microscopy.
In recent years, light sheet microscopy has emerged as the biological imaging modality of choice to achieve high performance in imaging speed, resolution, and penetration depth – all with minimal photo-induced damage.
Audrey Salles is a specialist for confocal and super-resolution microscopy at Pasteur Institute, Imagopole, PFID, Paris, France. Her research interests are cytokine signaling and skeleton organization of human TCD4-cells.
This article summarizes the development and differences in design and functionality of confocal technology as far as spectral properties are concerned, from classical filter-based excitation and emission color selection to fully flexible spectral excitation and emission tuning. All three major components: light source with excitation color selection, beam splitting for incident illumination and detector emission filtering have been completely transformed.
Modern biomedical research is currently dominated by imaging and measuring with optical microscopes. One branch of the microscopy technology is confocal microscopy. For correlation purposes, multiparameter fluorescence imaging is particularly of unique interest. This article is concerned with the spectral performance of the various modules in a confocal point-scanning microscope ("True Confocal System"), and how these modules have evolved to allow for tunability and flexibility in excitation and emission collection in multiple bands (channels).
The perfect light source for confocal microscopes in biomedical applications has sufficient intensity, tunable color and is pulsed for use in lifetime fluorescence. Furthermore, it should offer means to avoid reflection of excitation light, and the coupling into the beam path must be efficient and homogeneous throughout the full visible spectrum. Such a source has been invented and implemented: the white light laser in combination with acousto-optical beam splitting.