Images From a Single Excitation

Because on CCD setups excitation for D, S, and A images is usually filter-selected from a single white light source the relative intensity of excitation is approximately fixed. Confocal microscopes use separate laser lines, often from distinct lasers, that can (and for optimal imaging should) be independently adjusted. Thus, on CCD setups y (Eq. (7.6)) is constant for a given set of filters whereas on the confocal, it varies from image to image (also, see Sect. 7.4.2). 

Figure 10.11 Near-field imaging of SHG signals from a single Au nanoparticle.250 (a) Schematic of experimental setup (b) AFM image of an elliptical nanoparticle (c, d) near-field SHG signal mapping with emission detection polarized parallel and perpendicular to incident excitation. (Reprinted with permission from M. Zavelani-Rossi et al., Appl. Phys. Lett. 2008,92, 093119. Copyright 2008 American Institute of Physics.) (See color insert.)...

Figure S.3. Example of a first-order measurement of combinatorial materials. Polymer branching from measurements of fluorescence spectra from each polymerized material in a 96-element microreactor array at a single excitation wavelength. (A) Reflected-hght image of the microreactor array (B) representative fluorescence spectrum from a single microreactor in the array.

Fluorescence lifetime imaging microscopy (FLIM) is a technique to determine the spatial distribution of excited state lifetimes in microscopic samples. This can mean everything from a single decay time, to an entire decay profile, in two or three dimensions. Typically, FLIM instruments are designed to measure hfe-times in the nanosecond range, since the lifetimes of most fluorochromes used in modern fluorescence microscopy fall within this range. In this chapter, an overview is presented of the various techniques used in FLIM instruments today and of application areas in biology and biomedicine. 

---