**4. Comparison of fluorescence spectroscopy with other spectroscopic methods as an analytical technique**

Compare to other analysis techniques, it must suffice here to add that fluorescence measurements are rapid, accurate and require only very small quantities of sample (nanomole or less). Fluorescence instrumentation is also relatively inexpensive and easy to use. In general, fluorescence experiments are relatively easy to perform; as in many fields, it is the planning of appropriate experiments, the analysis, and accurate interpretation of the data that require more extensive experience.

Fluorescent methods have three significant advantages over absorption spectroscopy and other typical optical spectroscopy. First, two wavelengths are used in fluorimetry, but only one in absorption spectroscopy. Emitted light from each fluorescent color can be easily separated because each color has unique and narrow excitation spectra. This selectivity can be further enhanced by narrowing the slit width of the emission monochromator so that only emitted light within a narrow spectral range is measured. Multiple fluorescent colors within a single sample can be quantified by sequential measurement of emitted intensity using a set of excitation and emission wavelength pairs specific for each color. The second advantage of fluorescence over absorption spectroscopy is low signal to noise, since emitted light is read at right angles to the exciting light. For absorption spectrophotometry, the excitation source, sample and transmitted light are configured in line, so that the absorption signal is the small difference between the exciting light and the transmitted light, both of which are quite intense. The third advantage is that fluorescent methods have a greater range of linearity. Because of these differences, the sensitivity of fluorescence is approximately 1,000 times greater than absorption spectrophotometric methods (Guilbault, 1990). However, a major disadvantage of fluorescence is the sensitivity of fluorescence intensity to fluctuations in pH and temperature. However, pH effects can be eliminated by using nonaqueous solvents, and normal room temperature fluctuations do not significantly affect the fluorescence intensities of commercial dye solutions.

In addition there are other useful fluorescent techniques that have an advantages over the other typical optical spectroscopy, that not have been mentioned in this chapter, these are: (1) polarization (anisotropy), this technique gives information about the rotation of a fluorophore, and allow to infer protein shape, membrane fluidity (order parameters), and binding analysis; (2) quenching fluorescence, these processes can occur during the excited state lifetime –for example collisional quenching, energy transfer, charge transfer reactions or photochemistry –or they may occur due to formation of complexes in the ground state, thus this technique can give a useful kinetic information of the tested system, and give information about accessibility of fluorophoresis obtained allowing to correlate it with changes in protein or membrane structure; (3) Förster resonance energy transfer (FRET), this technique can be considered as a molecular ruler, which allow to determine distance from 10 to 80 Å; (4) fluorescence microscopy for image analysis, this technique gives magnification (in order to see small parts), resolution (in order to distinguish details of the small parts), and contrast (in order to magnify and resolve details, fluorescence emission provides contrast); (5) multiphoton excitation fluorescence microscopy, as an example for two photon excitation a molecule can be excited by using simultaneous photons and get fluorescence as happens with one photon excitation, this techniques has so many advantages such as sectioning effect without pinholes, low photobleaching and photodamage rate, separation of excitation and emission, no Raman from the solvent, deep penetration in tissues, single excitation wavelength for many dyes, avoid chromatic aberrations, and no expensive UV optics (for UV excited fluorophores) needed. Even though it has some disadvantages such as only is suitable for fluorescence images (reflected light images is not currently available), the technique is not suitable for imaging highly pigmented cells and tissues which absorb near infrared light, and laser source is expensive. It is worth mentioning that all analysis that can be done using fluorescence spectroscopy can be done using a multi-photon excitation fluorescence microscopy. However, multi-photon excitation fluorescence microscopy has a unique analysis applications over the fluorescence spectroscopy such as: analysis of deep tissue imaging (Brain, skin, etc), can prime photochemical reaction within subfemtoliter volumes inside solutions, cells and tissues (photolabile "caged" compounds), can be used for imaging of living specimen for longer period of time, and for live animal imaging (intrinsic fluorophores).
