SHG Microscopy

Fluorescence has proven to be a versatile tool for a wide range of applications in physical and life sciences. It's a powerful technique for studying immediate molecular environment, inter and intramolecular interactions. The technique is nearly background-free and offers sensitivity down to single molecule detection. Fluorescence methodology, however offers much more than just excellent signal/noise ratio. Continuing new developments in fluorescent probes, instrumentation, methodology, non-linear excitation, detectos, imaging technology, applications and software have resulted in its booming rennaissance and popularity.

Fluorescence is the phenomenon in which absorption of light of a given wavelength by a fluorescent molecule is followed by the emission of light at longer wavelengths. The distribution of wavelength-dependent intensity that causes fluorescence is known as the fluorescence excitation spectrum, and the distribution of wavelength-dependent intensity of emitted energy is known as the fluorescence emission spectrum.

Fluorescence detection has three major advantages over other light-based investigation methods: high sensitivity, high speed, and safety. The point of safety refers to the fact that samples are not affected or destroyed in the process, and no hazardous byproducts are generated.

Sensitivity is an important issue because the fluorescence signal is proportional to the concentration of the substance being investigated. Relatively small changes in ion concentration in living cells can have significant physiological effects. Whereas absorbance measurements can reliably determine concentrations only as low as several tenths of a micromolar, fluorescence techniques can accurately measure concentrations one million times smaller -- pico- and even femtomolar. Quantities less than an attomole (<10-18 mole) may be detected.

Using fluorescence, one can monitor very rapid changes in concentration. Changes in fluorescence intensity on the order of picoseconds can be detected if necessary.

Because it is a non-invasive technique, fluorescence does not interfere with a sample. The excitation light levels required to generate a fluorescence signal are low, reducing the effects of photo-bleaching, and living tissue can be investigated with no adverse effects on its natural physiological behavior.

The ratio fluorescence technique was developed to identify the location of intracellular free calcium in living cells, to measure the concentration of intracellular ions and pH, and to monitor the changes in intracellular ions.

The advent of ion-sensitive fluorescent probes, or dyes, has unleashed the power of fluorescence to identify and measure biologically-significant non-fluorescent substances. Until the introduction of such dyes, it was not possible to detect calcium, sodium, potassium and magnesium ions by means of fluorescence because these substances do not naturally fluoresce. In 1980 the first fluorescent dye specific to calcium was reported. This was followed by dyes for pH, sodium, magnesium, potassium and more, and new dyes and applications are being announced regularly.

Fluorescent dyes are designed to have a high affinity to specific ions; for example, Fura-2 is specific to calcium ions. When they bind to an ion, their fluorescent properties are altered. By quantitatively interpreting the changes in fluorescent properties, the concentration of the ion being investigated can be measured.

Fluorescence microscopy is perhaps the most powerful functional imaging modality available to study biological function. The development of genetically expressed fluorophore labels such as green fluorescence protein (GFP) offers an unparalleled opportunity to follow the location of specific proteins inside cells by imaging fluorescence intensity distributions. To obtain quantitative images, however, and to study cell (and protein) function, it is often necessary to do more than just image fluorescence intensity. One established way to gain quantitative information concerning fluorophore distributions is ratiometric fluorescence imaging, applying spectral or temporal resolution. This can make the measurements more robust against changes in quantum efficiency or scattering losses.

In principle, straightforward fluorescence intensity imaging should report the distribution of a fluorophore, with the intensity being a function of the local fluorophore concentration. In practice the observed fluorescence intensity may originate from multiple fluorophores (with different quantum efficiencies), the quantum efficiency of an individual fluorophore population may change across a sample and the detected intensity may be modified by optical scattering and absorption in the sample. Thus it is often necessary to eliminate these uncertainties using ratiometric techniques or to address them using more sophisticated measurements to obtain more information.

Further information can be gained from the quantum efficiency, which is a function of the radiative (g) and non-radiative (k) decay rates and so is associated the state or local environment of a fluorophore molecule. The radiative decay rate is essentially a function of the electronic energy level structure of the fluorophore molecule (although it does depend weakly on the local refractive index) while the non-radiative decay rate can be a sensitive function of the local fluorophore environment. Thus by observing changes in quantum efficiency, one can, in principle, probe local variations in e.g. viscosity, temperature, refractive index, pH, [Ca], [O2], electric field, etc, depending on the nature of the particular fluorophore molecule. Unfortunately determination of the quantum efficiency from intensity measurements requires knowledge of the absorbed and emitted radiation fluxes and of the fluorophore concentration. This is difficult or impossible to achieve in heterogeneous media such as biological tissues, which are also strongly scattering, and is not possible with autofluorescence.

Spectral resolution of fluorescence can provide functional information when the spectral profile changes as a function of the molecular state or environment but it is not straightforward to engineer fluorescent labels whose spectral profile changes in a predictable manner to permit quantitative imaging and nature is not always so kind as to provide them. Spectral discrimination is important, however, when contrasting different fluorophores – both in excitation and emission. This is particularly important when imaging in heterogeneous biological tissue where multiple fluorophores can present a significant background to measurements on a specific fluorophore.