The thin optical sectioning possible makes these types of microscopes particularly good at 3D imaging and surface profiling of samples. The achievable thickness of the focal plane is defined mostly by the wavelength of the used light divided by the numerical aperture of the objective lens, but also by the optical properties of the specimen. Slower scans provide a better signal-to-noise ratio, resulting in better contrast. This scanning method usually has a low reaction latency and the scan speed can be varied. The beam is scanned across the sample in the horizontal plane by using one or more ( servo controlled) oscillating mirrors. a rectangular pattern of parallel scanning lines) in the specimen. Īs only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (i.e. To offset this drop in signal after the pinhole, the light intensity is detected by a sensitive detector, usually a photomultiplier tube (PMT) or avalanche photodiode, transforming the light signal into an electrical one. However, as much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of decreased signal intensity – so long exposures are often required. As only light produced by fluorescence very close to the focal plane can be detected, the image's optical resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes. In contrast, a confocal microscope uses point illumination (see Point Spread Function) and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal – the name 'confocal' stems from this configuration. All parts of the sample can be excited at the same time and the resulting fluorescence is detected by the microscope's photodetector or camera including a large unfocused background part. In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen is flooded evenly in light from a light source. The principle of confocal imaging was patented in 1957 by Marvin Minsky and aims to overcome some limitations of traditional wide-field fluorescence microscopes. The fluorescence is visible by confocal microscopy. The CLSM achieves a controlled and highly limited depth of field.īasic concept Confocal point sensor principle from Minsky's patent GFP fusion protein being expressed in Nicotiana benthamiana. Light travels through the sample under a conventional microscope as far into the specimen as it can penetrate, while a confocal microscope only focuses a smaller beam of light at one narrow depth level at a time. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures (a process known as optical sectioning) within an object. Optical imaging technique Confocal Microscopyįluorescence and confocal microscopes operating principleĬonfocal microscopy, most frequently confocal laser scanning microscopy ( CLSM) or laser scanning confocal microscopy ( LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation.

Z stack confocal