Chapter 5 Visualizing cells and the csk visible Light microscopy




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Figure 5.4 Ca Wave




To see the effect of a sudden rise in Ca++ into a cell, it can be introduced into the cell while trapped in a cage compound loaded with Ca++, and then release it all of a sudden have been devised to see its instantaneous effects on cells. One such is the ‘caged compound’ technique, where an organic compound is structured to hold Ca++ in a cage until it is needed. This procedure is analogous to placing a Trojan horse into the cell. The cage releases upon deliverance of a pulse of high-energy light. An example of a caged release of Ca++ in a nerve cell is shown below, where green is the color of the Ca dye.




Figure 5.5 Caged Ca release




Physical Basis of Immunofluorescence

(Adapted From http://www.probes.com/handbook/sections/0001.html)

Fluorescence is the result of a three-stage process that occurs in certain molecules (generally polyaromatic hydrocarbons or heterocycles) called fluorophores or fluorescent dyes. A fluorescent probe is a fluorophore designed to localize within a specific region of a biological specimen or to respond to a specific stimulus. The process responsible for the fluorescence of fluorescent probes and other fluorophores is illustrated by the simple electronic-state diagram (Jablonski diagram) shown in Figure 1.

Stage 1: Excitation


A photon of energy hEX is supplied by an external source such as an incandescent lamp or a laser and absorbed by the fluorophore, creating an excited electronic singlet state (S1'). This process distinguishes fluorescence from chemiluminescence, in which the excited state is populated by a chemical reaction.

Stage 2: Excited-State Lifetime


The excited state exists for a finite time (typically 1–10 nanoseconds). During this time, the fluorophore undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment. These processes have two important consequences. First, the energy of S1' is partially dissipated, yielding a relaxed singlet excited state (S1) from which fluorescence emission originates. Second, not all the molecules initially excited by absorption (Stage 1) return to the ground state (S0) by fluorescence emission. Other processes such as collisional quenching, fluorescence resonance energy transfer (FRET) and intersystem crossing (see below) may also depopulate S1. The fluorescence quantum yield, which is the ratio of the number of fluorescence photons emitted (Stage 3) to the number of photons absorbed (Stage 1), is a measure of the relative extent to which these processes occur.

Stage 3: Fluorescence Emission


A photon of energy hEM is emitted, returning the fluorophore to its ground state S0. Due to energy dissipation during the excited-state lifetime, the energy of this photon is lower, and therefore of longer wavelength, than the excitation photon hEX. The difference in energy or wavelength represented by (hEX – hEM) is called the Stokes shift. The Stokes shift is fundamental to the sensitivity of fluorescence techniques because it allows emission photons to be detected against a low background, isolated from excitation photons. In contrast, absorption spectrophotometry requires measurement of transmitted light relative to high incident light levels at the same wavelength.

Fluorescence Spectra


The entire fluorescence process is cyclical. Unless the fluorophore is irreversibly destroyed in the excited state (an important phenomenon known as photobleaching, see below), the same fluorophore can be repeatedly excited and detected. The fact that a single fluorophore can generate many thousands of detectable photons is fundamental to the high sensitivity of fluorescence detection techniques. For polyatomic molecules in solution, the discrete electronic transitions represented by hEX and hEM in Figure 1 are replaced by rather broad energy spectra called the fluorescence excitation spectrum and fluorescence emission spectrum, respectively. The bandwidths of these spectra are parameters of particular importance for applications in which two or more different fluorophores are simultaneously detected (see below). With few exceptions, the fluorescence excitation spectrum of a single fluorophore species in dilute solution is identical to its absorption spectrum. Under the same conditions, the fluorescence emission spectrum is independent of the excitation wavelength, due to the partial dissipation of excitation energy during the excited-state lifetime, as illustrated in Figure 1. The emission intensity is proportional to the amplitude of the fluorescence excitation spectrum at the excitation wavelength (Figure 2).



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