Objective magnification

G(t) decays with correlation time because the fluctuation is more and more uncorrelated as the temporal separation increases. The rate and shape of the temporal decay of G(t) depend on the transport and/or kinetic processes that are responsible for fluctuations in fluorescence intensity. Analysis of G(z) thus yields information on translational diffusion, flow, rotational mobility and chemical kinetics. When translational diffusion is the cause of the fluctuations, the phenomenon depends on the excitation volume, which in turn depends on the objective magnification. The larger the volume, the longer the diffusion time, i.e. the residence time of the fluorophore in the excitation volume. On the contrary, the fluctuations are not volume-dependent in the case of chemical processes or rotational diffusion (Figure 11.10). Chemical reactions can be studied only when the involved fluorescent species have different fluorescence quantum yields. 

The condenser must be designed for phase contrast. A different objective magnification often requires a different condenser setting. Condenser turrets are often used for this purpose. If work will be performed at various magnifications, be sure that rotating the turret doesn t require the condenser to be recentered. The loss of time in this situation can be serious when it is desirable to work at various magnifications. The numerical aperture of the condenser must equal or exceed the numerical aperture of the objective. 

Point spread function (PSF) If a tiny population of 100 nm fluorescent beads sandwiched between a coverslip and a microscope slide are examined at high resolution (i.e. at 100x objective magnification, 1.4 NA. and in a correctly matched refractive index of oil), it can actually show a tiny set of rings in the horizontal (XY) view (also called an airy disk (see Fig. below). This airy disk cannot be avoided due to diffraction and the wave nature of light. If a specimen is optically sectioned and projected in a vertical (XZ) view (see Fig. xx), a set of concentric rings will flare from the center. When a three-dimensional image of this specimen is collected, a complete point spread function is said to be recorded for each bead. The (PSF)... 

The experimental design of a DHM for measurements of microscopic flows requires optimal choice of many parameters (i.e., illumination wavelength, microscope objective magnification. 

Micro-holographic PIV/PTV Technique, Table 1 The design parameters for three-dimensional velocity field measurements inside microchannel using digital holographic microscope (DHM) for microscope objective magnifications, M = 5x andM = lOx ... 

M-velocity profile plots along y-diiectirai are shown in (c) and (d) corresponding to microscope objective magnifications, M = 5x and lOx, respectively... 

X plane measurement volume for the microscope objective magnifications ofM = 5x and 10x, respectively... 

Objective magnification NA d = 0.6X/NA Total system magnification Image resolution (jim)... 

In wide-field microscopy, lateral sampling is achieved by the CCD chip and is a function of the CCD pixel size Pj, camera binning b, and objective magnification M, as follows ... 

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