Internal scanning

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Internal scanning

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October Learn how and when to remove this template message Confocal point sensor principle from Minsky's patent The principle of confocal imaging was patented in by Marvin Minsky [2] and aims to overcome some limitations of traditional wide-field fluorescence microscopes.

All parts of the specimen in the optical path are 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 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. As only light produced by fluorescence very close to the focal plane can be detected, the image's optical resolutionparticularly in the sample depth direction, is much better than that of wide-field microscopes.

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.

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 photodiodetransforming the light signal into an electrical one that is recorded by a computer.

The beam is scanned across the sample in the horizontal plane by using one or more servo controlled oscillating mirrors.

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This scanning method usually has a low reaction latency and the scan speed can be varied. Slower scans provide a better signal-to-noise ratioresulting in better contrast and higher resolution.

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 lensbut also by the optical properties of the specimen.

The thin optical sectioning possible makes these types of microscopes particularly good at 3D imaging and surface profiling of samples. Successive slices make up a 'z-stack' which can either be processed by certain software to create a 3D image, or it is merged into a 2D stack predominately the maximum pixel intensity is taken, other common methods include using the standard deviation or summing the pixels.

The fluorescence is visible by confocal microscopy. Confocal microscopy provides the capacity for direct, noninvasive, serial optical sectioning of intact, thick, living specimens with a minimum of sample preparation as well as a marginal improvement in lateral resolution.

However, the actual dye concentration can be low to minimize the disturbance of biological systems: Also, transgenic techniques can create organisms that produce their own fluorescent chimeric molecules such as a fusion of GFP, green fluorescent protein with the protein of interest.

Confocal microscopes work on the principle of point excitation in the specimen diffraction limited spot and point detection of the resulting fluorescent signal. A pinhole at the detector provides a physical barrier that blocks out-of-focus fluorescence.

Only the in-focus, or central spot of the airy disk, is recorded. Raster scanning the specimen one point at a time permits thin optical sections to be collected by simply changing the z-focus.

Internal scanning

The resulting images can be stacked to produce a 3D image of the specimen. Techniques used for horizontal scanning[ edit ] Four types of confocal microscopes are commercially available: Confocal laser scanning microscopes use multiple mirrors typically 2 or 3 scanning linearly along the x- and the y- axes to scan the laser across the sample and "descan" the image across a fixed pinhole and detector.

Internal scanning

Spinning-disk Nipkow disk confocal microscopes use a series of moving pinholes on a disc to scan spots of light.

Since a series of pinholes scans an area in parallel, each pinhole is allowed to hover over a specific area for a longer amount of time thereby reducing the excitation energy needed to illuminate a sample when compared to laser scanning microscopes.

Decreased excitation energy reduces phototoxicity and photobleaching of a sample often making it the preferred system for imaging live cells or organisms.Internal Scanning Buried pipe inspection.

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