A confocal microscope uses a pinhole to only allow light from a thin slice of objects in focus to reach a photosensor. Out of focus light above and below that slice is filtered out. Images from confocals are therefore cleaner and sharper than from widefields. Point confocals take pictures pixel by pixel and are relatively slow, but very accurate. Speed can be improved using a resonant scanner, which is scanning point by point very fast, but fairly noisily. Noisy samples can be denoised post image acquisition. Another way to go faster is to use a spinning disk confocal, which parallelises the scanning process (i.e. multiple points are scanned at the same time) and sends signals through filters to cameras. All the confocals are capable of creating z-stacks. Expand all Collapse all Leica SP8 4 detectors The Leica SP8 4 detectors is a point confocal equipped with four tuneable detectors – three PMT and two ultrasensitive HyD with a detection range between 410-750nm - and four very stable LED laser lines at 405, 488, 552 and 638nm. This inverted microscope is equipped with 10x, 20x, 40x and 63x lenses as well as a 25x water lens for long distance work on thick samples. The microscope has an incubation chamber for live cell work. Special applications and features: Navigator scan for quick mapping with spiral option. The navigator allows for mark and find, as well as tiling of large and complex objects. 3D viewer to visualise samples in 3 dimensions, as well as save movies. Location: IRR Imaging Hub Contact IRR.Imaging@ed.ac.uk for access Leica SP8 5 detectors The Leica SP8 5 detectors is a point confocal equipped with five tuneable detectors –two PMT and three ultrasensitive HyD with a detection range between 410-750nm - and nine laser lines at 405, 458, 476, 488, 496, 514, 561, 594 and 638nm. This inverted microscope is equipped with 10x, 20x, 40x and 63x lenses as well as a 25x and 40x water lenses for long distance work on thick samples such as tissue slices or organoids. The microscope has an incubation chamber for live cell work. Special applications and features: You can use this microscope in resonant scanner mode and denoise the image using the lightning algorithm. This is ideal for large and thick samples. Navigator scan for quick mapping with spiral option. The navigator allows for mark and find, as well as tiling of large and complex objects. 3D viewer to visualise samples in 3 dimensions, as well as save movies. FRAP (Fluorescence recovery after photobleaching) is used to observe how dynamic trafficking in cell is. FRAP works by bleaching a region of a cell and observing fluorescence recovery thereafter –through dynamic transport or diffusion. FRET (Fluorescence/Förster resonance energy transfer) is used to identify if two fluorophores are within 10nm of each other. FRET works by illuminating a donor fluorophore, the emitted photon of which activates an acceptor, which turns visible. Contact us to ensure you use a correct pair of fluorophores, and that you have the necessary controls! Location: IRR Imaging Hub Contact IRR.Imaging@ed.ac.uk for access Leica Stellaris FALCON The Leica Stellaris FALCON is a point confocal equipped with five tuneable detectors with a detection range between 410-850nm, including one optimised for near infrared detection (Cy5-7 range). This microscope has a white laser that can be tuned in 1nm step between 440 to 790nm, making all fluorophores specifically excitable. This inverted microscope is equipped with 10x, 20x, 40x and 63x lenses as well as a 25x and 40x water lenses for long distance work on thick samples. The microscope has an incubation chamber for live cell work. Special applications and features: Complete flexibility with the combination of tuneable white laser and receptors: any fluorescent molecule of choice should now be detectable. Navigator scan for quick mapping with spiral option. The navigator allows for mark and find, as well as tiling of large and complex objects. 3D viewer to visualise samples in 3 dimensions, as well as save 3D animations. FRAP (Fluorescence recovery after photobleaching) is used to observe how dynamic trafficking in cell is. FRAP works by bleaching a region of a cell and observing fluorescence recovery thereafter –through dynamic transport or diffusion. FRET (Fluorescence/Förster resonance energy transfer) is used to identify if two fluorophores are within 10nm of each other. FRET works by illuminating a donor fluorophore, the emitted photon of which activates an acceptor, which turns visible. Contact us to ensure you use a correct pair of fluorophores, and that you have the necessary controls! FLIM (Fluorescence lifetime imaging microscopy) is used to separate signals excited by the same lasers (either a combination of dyes or dyes and background). FLIM excites fluorophores by pulses instead of continuously. During interval between pulses, it analyses the speed at which fluorophores emit their photons. The difference in emission speed, which needs to be at least 0.2ns, allows for signal separation. Since fluorophore environment also influences fluorescence speed, FLIM can be used to study e.g. metabolism. Location: IRR Imaging Hub Contact IRR.Imaging@ed.ac.uk for access Opera Phenix Plus The Opera Phenix is a high content spinning disk confocal microscope. It is equipped with dual cameras and five laser lines at 375, 425, 488, 561, and 640nm. With the right combination of dyes, you can image up to four channels concomitantly, which makes the Phenix blisteringly fast. The Opera Phenix has 5x, 10x and 20x and 40x air lenses, as well as high quality automated water immersion lenses at 20x, 40x and 63x. These lenses are better than equivalent air lenses in term of sensitivity and resolution along the XY and Z axes. This system has an in-built incubator, for live imaging, and a liquid dispenser allowing delivery of pharmacological compounds and probes, ideal e.g. for Calcium imaging. Opera Phenix Plus is equipped additionally with robotic arm which automatically loads plates and slide holders allowing for 24/7 usage. This system is specifically designed for large pharmaceutical screens in multiple well plates, but is extremely versatile enough to to accommodate various applications such as: Imaging tissue slices (live and fixed) imaging live and fixed organoids, imaging zebrafish embryos . FRET in combination with compound screens fast event imaging such as calcium flux. low magnification prescan to find rare event and high magnification rescan. be used on sample slices, organoids, tissue culture samples, zebrafish embryos… Data from the Opera Phenix are analysed using the Harmony software capable of 3D reconstructions and 3D analysis and additionally we host a server - Image Artist on a virtual machine to facilitate large dataset analysis.Data from the Opera Phenix are analysed using the Harmony software on our custom-built server. Location: IRR Translational Imaging Hub, contact irr-hcs@ed.ac.uk for access. This article was published on 2024-07-08
