Multi-user equipment approved in grant 15/50040-4: microscopy confocal TCS SP8 DLS Hyvolution

Abstract

Conventional epifluorescence microscopy, often utilized to screen the distribution of cellular components by use of immunolabelling, is a classical technique extensively present in different areas of research. However, it suffers from an important disadvantage, namely out-of-focus blur. This effect is due to the excitation of an entire field of view, which activates emission in the whole depth of the specimen, rather than just at the focal plane. The nature of the illumination in this microscopy technique hinders the analysis of thick structures, as the blur greatly affects contrast of the samples. Confocal fluorescence microscopy emerged to circumvent this imaging artifact by excluding the detection of out-of-focus light by means of a pinhole. The images acquired in this manner display sharp contrast throughout the whole thickness of the sample, with lateral and axial resolution improvement. This enhanced way of visualizing the samples provide a gain of information, as it can display structures reliably in 3 dimensions, even in thicker samples. Other improvements in signal detection are also very relevant, as confocal light path allow spectral detection of multicolor-labelled samples, which is crucial for live imaging. This mode of detection can be used to its full potential when implemented with individual detection devices for each label, and the spectral separation made by means of highly transparent devices, as prisms. This features permits image acquisition without signal crosstalk, which are mandatory for colocalization experiments, with minimum photo damage to the sample. The usage of laser scanning in confocal microscopy also provides new approaches to biological imaging. Dynamic experiments can be performed to investigate the behavior of live cells in time lapse or the mobility of subcellular components by FRAP (Fluorescence Recovery After Photobleaching), and even experiments of fluorescence correlation spectroscopy within the confocal volume. The use of the laser also allowed the development of new ways to image the sample. Selective Plane Illumination Microscope (SPIM), described in the early 1900s, is getting more attention nowadays. This technique, also known as lighsheet microscopy, decouples the illumination lightpath from the detection of light, consequently drastically decreasing photo bleaching effect. Here, a thin sheet of light excites only the volume that will be imaged, preserving the sample. With this approach, big volumes can be imaged quickly, with optical sectioning, and with minimal disturbance. These imaging characteristics make SPIM an optimal method for imaging samples in areas as developmental biology, 3D cell cultures and long-term live cell experiments. Furthermore, the combination of these two imaging modalities - confocal and lightsheet - bring certain advantages, as speed, a more intuitive specimen preparation and the possibility of using the confocal lasers to photo manipulate the sample imaged by SPIM. By using deflector mirror on the focus plane of the detection objective, one can integrate these two approaches in one multimodal instrument that can fulfill different imaging needs from different research groups, with different biological questions, making this microscope a well-suited candidate for a multiuser imaging facility. (AU)