The LSFM setup maintained the essential components from the first modern reports. Thus, the possible resolution enhancements due to STED might be transferred to higher imaging penetration depths and also multitude higher imaging fields of view. 16 – 18 Incorporation of STED into a light sheet microscope does not use the excitation objective as the imaging objective. 15 Many configurations of STED microscopes exist, which allow for the implementation of fluorophore multiplexing, high-resolution imaging of live samples and even with multiphoton excitation. Configurations of STED microscopy implemented into confocal microscopes have achieved imaging resolutions of < 7 nm. The implementation consists of creating the STED beam around the confocal scanning beam to make the effective fluorescence excitation area smaller, and thus, improve the resolution from the confocal microscope beyond the predicted diffraction limit of light. STED microscopy in its present form takes advantage of STED with the implementation of it in scanning confocal fluorescence microscopy. STED is a photophysical process by which the excited state electrons are induced and forced to emit a de-exciting photon by a separate incident photon. Additionally, the incorporation of STED into a light sheet microscope could also be advantageous over other types of STED microscopy. 14 The successful incorporation of STED with light sheet excitation could, in principle, be added to any type of planar or light sheet microscopy to further enhance resolution. Our initiative is the incorporation of stimulated emission depletion (STED) light sheets to reduce the effective excitation sheet waist in an LSFM configuration. In practice, a numerical aperture of 0.8 is the highest usable where the beam waist is equivalent to the depth of focus of the objective to achieve penetration of 1 mm and more. To obtain the necessary penetration depth and field of view for the specimen, the resulting excitation field compromises the Gaussian light sheet width. 4 This is due at least in part to the excitation field, which limits both the lateral and axial resolutions. 10 – 12 However, with the recent exception of structured illumination and line scanning sheet illumination microscopy, resolution enhancements have been limited to postprocessing of multiview imaging, which is a technique that requires much more time and effort for both acquisition and analysis and reduces the benefits of the high-speed imaging.Ī disadvantage of LSFM is the low image resolution despite the deep tissue penetration offered. 5 – 9 Recently, as the wealth of information increases from LSFM, advancements to LSFM, such as multiple beam and multiple view imaging, introduce improvements to distortions and artifacts inherent in LSFM.
1, 2, 4 Imaging with LSFM continuously reveals novel scientific findings in immunology, neurology, pathology, cardiology, and development. 1 The advantages of LSFM are high optical sectioning, imaging rates, and penetration depths with low photobleaching and scattering. 1 – 3 Light sheet based fluorescence microscopy (LSFM) uses a separate optical excitation path orthogonal to the detection objective that limits the excitation to the focal imaging plane. For the imaging of whole model organisms and tissues, planar (or sheet) illumination microscopy remains a top choice for subcellular resolution of complete, large specimen three-dimensional (3-D) reconstructions.
0 kommentar(er)
