By Melbourne Centre for Nanofabrication
Laser Scanning Confocal Microscopy (LSCM) uses solid-state lasers to irradiate and excite specimen at specific wavelengths, scanning a fluorescent subject point-by-point, line-by-line and layer-by-layer and collecting light from these small spots to give excellent resolution. It is then able to image the subject by reconstructing the scanned information. It enables the 3D reconstruction of topologically complex structures in high resolution (approximately 250nm) as well as the sharp imaging of thick specimens at varying depths. LSCM is useful for the topographical study of materials as well as life science applications such as the imaging of live and fixed cells. It is able to image both opaque and transparent fluorescent materials at different depths, providing an understanding of surface structure for opaque materials and internal structures for transparent materials. A unique feature of LSCM is the ability to capture both very fast and very slow biological reactions. With fast resonant scanning, the LSCM can capture up to 400 frames per second enabling the imaging of processes such as calcium reactions. By providing time-lapse capabilities, the LSCM can capture long-term processes such as live cell cleaving and drug delivery into cells. MCN has two LSCM. The Nikon A1Rsi which is attached to an inverted microscope where a laser is combined with a photomultiplier tube detector. Images are then collected and displayed on a monitor. The LSCM is able to image specimens at an axial and lateral resolution of 250nm to create high resolution X,Y and Z images of 4000 2 pixels. It features four solid state lasers to cover almost all types of fluorescent dyes of 405nm, 488nm, 561nm and 635nm, as well as hybrid high resolution galvano scanner and fast resonant scanner. The Leica Stellaris 5, equipped with 5 Continuous Wave Lasers: 405 nm, 448 nm, 514 nm, 561 nm, 638 nm and Pulsed Lasers: 485–685 nm, can image samples at an axial resolution of 200nm and a lateral resolution of 120nm. The POWER HyD S detectors have a spectral sensitivity range of 410 to 850nm. The TauSense technology enhances functional imaging through lifetime based information.
