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The project-start architecture contains: An environment model that maps the field of interest, which includes the IT solution to be developed. This indicates the business context in which the IT solution is supposed to function. The demarcation of the IT solution, in both logical and technical terms, including any interfaces with other systems. The design choices that transcend the project. Only those design choices that have an impact outside the project are outlined in the project-start architecture. Standards and guidelines that apply to the project. A good project-start architecture functions as a service-level agreement (SLA), both toward the organization itself and toward an external party, if the project is contracted out. The project-start architecture can be used to test whether or not the project delivers what was agreed upon. The project-start architecture remains valid as a framework, even after the IT solutions have been taken over by the organization. This guarantees that any subsequent adjustments are also tested against the enterprise architecture. At the end of this chapter, we have included the project-start architecture for WWW-TeleBel as an illustration. (More about project-start architectures is discussed in 7.)

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Like the CLSM, the TPLSM uses a laser, a raster scanning mechanism, and an objective lens for point scanning of the specimen But TPLSM has unique characteristics and offers several distinct advantages: Excitation wavelengths in the near IR are provided by a pulsed IR laser such as a titanium:sapphire laser Excitation occurs in the near UV, causing fluorescence emission in the blue portion of the visual spectrum Fluorochromes in the focal plane are differentially excited because with 2P excitation the efficiency of fluorescence excitation depends on the square of the light intensity, not directly on intensity as is the case for 1P excitation Thus, by properly adjusting the amplitude of laser excitation, it is possible to differentially excite fluorochromes within the focal plane The laser power, pulse duration, pulse frequency, and wavelength are critical variables for determining 2P excitation.

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where [0; T ] is the signal observation interval. Here we assume coherent processing for purposes of simplicity. This estimator is a MVUE, and it can be shown that the error variance of tML (which equals the Cramer Rao bound) is N s2ML T 0 2 : t 2 0 cHr t dt 3:23

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Figure 12-13 shows the difference between 2P- and 1P-induced fluorescence in a transparent cuvette containing a fluorescent dye Differential fluorescence excitation in the focal plane of the specimen is the hallmark feature of TPLSM and contrasts with CLSM, where fluorescence emission occurs across the entire thickness of a specimen excited by the scanning laser beam Thus, in TPLSM, where there is no photon signal from out-of-focal-plane sources, the S/N in the image is improved Because the fluorescence emission occurs only in the focal plane, the rate of photobleaching is also considerably reduced for the specimen as a whole The use of near-IR wavelengths for excitation permits examination of relatively thick specimens This is because cells and tissues absorb near-IR light poorly, so cellular photodamage is minimized, and because light scattering within the specimen is reduced at longer wavelengths The depth of penetration can be up to 0.

MLEs of the Incomplete Data Averaged Over 10 Simulations Distribution Inverse Gaussian Gamma Inverse Gaussian p21 MLE (Unrecognized Incomplete Data)a 1 = 7.94 (0.011), 2 = 0.17 (0.003) = 2.28 (0.139), = 12.99 (1.138) 1 = 7.36 (0.082), 2 = 0.06 (0.003) p21 = 0.26 (0.003)

5 mm for some tissues, 10 times the penetration depth of a CSLM A confocal detector pinhole is not required, because there is no out-of-focal-plane fluorescence that must be excluded Nevertheless, emitted fluorescent wavelengths can be collected in the usual way and delivered by the galvanometer mirrors to the pinhole and detector as in CSLM, but the efficiency of detection is significantly reduced This method is called descanned detection A more efficient method of detection involves placing the detector near the specimen and outside the normal fluorescence pathway (external detection) Several other detection methods are also possible Despite the potential advantages, there are still practical limitations and problems that remain to be solved Since the titanium:sapphire laser presently used for TPSLM.

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