Confocal detection

Furthermore, we assume the more general, but also more complex case in which confocal detection is used. This allows for example flexibility in setting independent detector gains for D, S, and A. When this flexibility is not required the expressions simplify considerably. 

Fig. 12 CNTs act as a vector for drug delivery into living cells. After incubation of HeLa cells with AlexaFluor594-labeled SWNTs for 12 h at 37 °C, living cells were observed under confocal fluorescence microscope for a CNT uptake study, (a) Images show dual confocal detection of AlexaFluor594-SWNT (red) internalized into cells with the membrane stained by AlexaFluor488 (green), (b) Series of images of different z-focal scanning planes down through cells. (Adapted from [61])...

Another area of rapid growth in application of the fluorescence techniques to proteins is in imaging of cells and tissues. Through use of an array detector such as a CCD camera with wide-fleld illumination or beam scanning with confocal detection, the spatial position of fluorophores can be recorded with a precision... 

Single Enzyme Experiments with Confocal Detection Schemes... 

Measurements at low light levels are routinely performed with photon-counting techniques. The development of ultrasensitive optical detectors has made great progress in the last couple of years. Integrated photon-counting modules with cooled avalanche photodiodes (APD) have been available for some years [31]. These detectors can have quantum efficiencies of 50% with less than 10 dark counts per second. The light sensitive area of such a device has a diameter of about 200 (im and can serve directly as a pinhole in a confocal detection channel. 

By using a field stop in front of the detector, light from outside the focus can be suppressed. This can be useful to suppress fluorescence of the cuvette walls, fluorescence of dye moleeules bound to the cuvette walls, or distortions by scattering or reabsorption. Confocal detection can also be used to reduce the daylight-sensitivity of the detection system. 

Ophthalmic imagers often use a scanning technique with the pupil of the eye placed in the exit pupil of the scanning system [461]. This reduces image distortion and blurring due to the poor optical quality of the lens of the eye. Moreover, confocal detection can be used to suppress reflection, scattering, and fluorescence signals from the lens of the eye. 

Optically driven photon correlation experiments normally require confining the detection or the excitation to an extremely small sample volume. This is achieved either by confocal detection or two-photon excitation in a microscope. The optical principles are the same as in confocal and two-photon laser scanning microscopes (see Sect. 5.7, page 129). However, most correlation experiments do not require scanning and can be performed in relatively simple microscopes. 

Similar to capillaries, the most common detection method for high-speed separations on microdevices is fluorescence, yet few methods have matched the limits of detection found in sheath-flow formats routinely used in capillary systems. This is due in part to the microfluidic substrate being in a planar format and therefore difficult to reproduce the three-dimensional Taylor cone found in sheath-flow capillary systems. The most simple and common method for high-sensitivity detection in microdevices is the use of a confocal detection scheme. ... 

Hgure 5 Schematic of data acquisition in microarray technobgy (A) charge coupled device (CCD) cameras (B) laser scanners with confocal detection optics. 

Figure 3.5 Illustration of the principle of confocal detection to limit the collection volume in the direction of the propagation of the excitation beam. Light emerging from near the focal plane (black spot) is collected and collimated by the microscope objective and then focused by a second lens to pass through an aperture and onto the detector. Light that originates from in front or behind the focal plane (grey spot) is out of focus at the aperture and only a small proportion continues to the detector. The aperture is said to be confoca/with the objective. (Optics delivering the excitation light have been omitted for clarity).

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