Confocal pinhole

Fig. 5. Scanning confocal optical microscopy for single-molecule detection. Sample scanning configuration. The fiber exit and the active area of the SPAD serve as confocal pinholes. S sample, O high-NA microscope objective, DM dichroic mirror, L1,L2 lenses, F Filters.

Laser scanning confocal microscope A fluorescence microscope achieving improved depth discrimination and contrast by blocking fluorescence that originates outside the plane of focus by use of a confocal pinhole. 

Fig. 3 Confocal fluorescence detection on a microchip. A laser was used as the excitation source. The laser light passed through a biconvex lens, was focused into an illumination pinhole, and was subsequently reflected by a dichroic mirror and focused in the channel on the capillary electrophoresis (CE) chip by a microscope objective. The fluorescence signal from the sample, collected by the microscope objective, was passed through the dichroic mirror, focused by the tube lens into a confocal pinhole, and then detected by a photomultiplier tube (PMT). To improve the signal-to-noise ratio, band-pass filter and notch filter were inserted above PMT for spectral filtering...

Laser-scanning microscopes can be classified by the way they excite and detect fluorescence in the sample. One-photon microscopes use a NUV or visible CW laser to excite the sample. Two-photon, or Multiphoton , microscopes use a femtosecond laser of high repetition rate. The fluorescence light can be detected by feeding it back through the scanner and through a confocal pinhole. The principle is termed confocal or descanned detection. A second detection method is to divert the fluorescence directly behind the microscope objective. The principle is termed direct or nondescaimed detection. 

An FCS system with two-photon excitation is shown in Fig. 5.108, right [51, 457]. A femtosecond Ti Sapphire laser of high repetition rate is used to excite the sample. Because there is no appreciable excitation outside the focal plane of the microscope lens a small sample volume is achieved without a confocal pinhole. This makes the optical setup very simple. In terms of signal recording there is no difference between one-photon and two-photon FCS. 

Adjust the confocal pinhole to a value between 2 and 4 optical units for an optimal signal-to-noise ratio and spatial resolution. 

In confocal microscopy the PSF is a function of a number of physical components, namely the laser beam, the microscope objective, the confocal pinhole, and the detector. The PSF is a convolution of the effect of all of these components. Despite this apparent complexity it is perhaps surprising that a simple description of the PSF for these microscopes has been widely applied in which the sample volume is described by a three-dimensional Gaussian with a 1/e beam waist diameter 2coq and a length 2zq along the optic axis [6,20,21 ] and is given by,... 

Mechanically registering two scanning systems would be difficult, so commercial LCSMs are reflection microscopes, where (as shown in Fig. 6.1) the beam passes through the same scanner twice. The beam is scanned On the specimen, then de-scanned onto a fixed confocal pinhole. Con-focal microscopes may have a transmission mode, but currently this will not be confocal. It may use a separate regular illumination system, or the scanned illumination. In the latter case the... 

A much better achievement of local studies is possible by using the confocal arrangement, shown in Fig. 23, in which the illuminated region, the focal point of the microscope lens, is optically conjugated to a confocal pinhole. Parasitic light emitted by neighboring volumes is thus eliminated and the locality of the data is highly increased. 

In LSCM, light emitted from the object is focused on a confocal pinhole which acts as a spatial filter light from the focal plane passes the pinhole, while light from other planes is effectively suppressed. The pinhole thus ensures that the information only arrives from a particular level of the specimen with a very high resolution (AF) along the... 

Fig. 2 Optics of confocal microscopy. The incident light emitted from laser (t) is reflected by a dichroic mirror (2). The reflected light is focused on the specimen (4) through objective lens (3). At the focus the light may be reflected by the interior structure or the incident laser excites fluorescent molecules attached to the particular site (position) of the specimen. The reflected light or fluorescence from the focal plane refocuses at the confocal pinhole (5) and thus it passes the pinhole to reach detector (6). The out-of-focus light (fluorescence) is blocked by the confocal pinhole as shown by the dashed line...

The phase-separated structures of the DPB/PB mixtures were observed by LSCM with an incident laser beam wavelength A of 364 nm. A band pass filter (395-440 nm) installed in front of the detector (photomultiplier) was used to detect only fluorescence from the anthracene molecules that were labeled only to the PB. The PB phase was recognized as a bright phase under the fluorescent LSCM (see Fig. 10). The intensity of fluorescence from a particular point in a focal plane (x-y or lateral plane) at a given depth z, I x,y,z), was recorded by the detector behind a pinhole ( confocal pinhole ) which efficiently excludes out-of-focus light, thus achieving an excellent depth resolution. Here, the z -axis denotes the optical axis of the micro-... 

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