Confocal microscopy is an optical imaging technique for increasing optical resolution and contrast of an image by using a spatial pinhole to block out-of-focus light in image generation. A distinct feature of confocal microscopy is the generation of sharp images of the focused plane, without any disturbing fluorescent light from the background or other regions of the specimen. 3D objects can be visualized by staking images from different optical planes.
Laser Scanning Confocal Microscopy (LSCM)
Laser scanning confocal microscopy create images by scanning laser point-by-point over sample and recording intensity at each spot. Coherent light from laser illuminates the specimen onto a defined focal plane. This leads to the emission of fluorescent light at exactly this point. The pinhole is placed in front of the photomultiplier (PMT) detector. It cuts off out-of-focus light into PMT, allowing the production of images with higher resolution, higher contrast and reduced noise. The PMT detects photons from emitted fluorescent light and convert a detectable signal. This technique is capable of very thin optical sectioning of samples, enabling 3D structures. One major drawback of single-point laser scanning confocal microscope is generally limited by the relatively slow speed and the higher potential for phototoxicity since it scans every point in the sample. And low light efficiency of the PMT (generally less than 35%) is another drawback. Recently, PMT employing gallium arsenide phosphide (GaAsP) on its photoelectric surface offers superior photon efficiency (high up to 45%) compared to conventional alkali-metal-based PMTs. The high sensitivity of GaAsP PMT can be made of dark samples that were difficult to observe and of samples easy to make photobleaching.
Equipment and Software:
Olympus FV1000 with Prior motorized stage
FV10-ASW
Spinning Disk Confocal Microscopy (SDCM)
Spinning disk confocal microscopes is a powerful tool for rapid spatial and temporal imaging of living cells. Spinning disk confocal microscopy is an alternative to LSCM utilizing full of pinholes arranged in the disk, rather than a single pinhole. As the disk spun around its axis, the pinholes arranged in a spiral scan across the sample in rows, building up an image. SDCM scans multiple points at once and can spin at very high speeds, thus requiring high-quality camera. High-performance camera systems, e.g. EMCCD, are capable of providing high acquisition speeds with acceptable contrast and minimal photobleaching at the low light levels available with this technique. SDCM is highly useful for high-speed imaging of living cells and for the imaging of samples sensitive to photobleaching and phototoxicity. Drawbacks of SDCM include the lack of a scan zoom function to alter the digital image sampling rate and the inability to adjust the pin-hole size to alter the optical sectioning strength and imaging resolution.
Equipment and Software:
Leica DMi8 inverted microscope with Prior motorized stage
Yokogawa CSU-X1 spinning disk scanner
Hamamatsu 9100-02 CCD and Hamamatsu 9100-23B CCD
Volocity