Georgina A.
Rivera-Ingraham
Life-imaging techniques
Keeping cells, tissues and even complete individuals alive and functioning as normally as possible during our analyses of biological processes is on of the greatest challenges in biological and medical sciences. A good way to study undisturbed living models is through the use of the so-called life-imaging techniques (LIT) or live-imaging, which allows visualization of living cells through images obtained with microscopes. These methods allow researchers to investigate in-vivo or ex-vivo biological functions through the use of fluorescence compounds. There are numerous fluorescent probes which enable us to detect particular components of complex biomolecular assemblies, such as live cells, structures, compounds or processes, with sensitivity and selectivity.
But what is fluorescence?
Fluorescence is the property of some atoms and molecules to absorb light at a particular wavelength and to subsequently emit light of longer wavelength after a brief interval, termed the fluorescence lifetime.
The most striking examples of fluorescence occur when the absorbed radiation is in the ultraviolet region of the spectrum, and thus invisible to the human eye, and the emitted light is in the visible region.
More precisely, 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 respond to a specific stimulus or to localize within a specific region of a biological specimen. The process responsible for the fluorescence of fluorescent probes and other fluorophores is illustrated by the simple electronic-state diagram (Jablonski diagram) shown on figure 1.
Figure 1. Jablonski diagram illustrating the processes involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of fluorescence. The labeled stages 1, 2 and 3 are explained in the adjoining text (from The Molecular Probes® Handbook)
Stage 1: Excitation.
A photon of energy hνEX 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) (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 hνEM 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 hνEX. The difference in energy or wavelength represented by (hνEX – hνEM) 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.
How do we detect fluorescence?
Four essential elements of fluorescence detection systems can be identified from the preceding discussion:
1) an excitation light source,
2) a fluorophore,
3) wavelength filters to isolate emission photons from excitation photons,
4) a detector that registers emission photons and produces a recordable output, usually as an electrical signal.
Fluorescence instruments are primarily of four types, each providing distinctly different information:
a) Spectrofluorometers and microplate readers measure the average properties of bulk (μL to mL) samples.
b) Fluorescence microscopes resolve fluorescence as a function of spatial coordinates in two or three dimensions for microscopic objects (less than ~0.1 mm diameter).
c) Fluorescence scanners, including microarray readers, resolve fluorescence as a function of spatial coordinates in two dimensions for macroscopic objects such as electrophoresis gels, blots and chromatograms.
d) Flow cytometers measure fluorescence per cell in a flowing stream, allowing subpopulations within a large sample to be identified and quantitated.
But no method is flawless. What problems can we encounter?
One of the main problems which we encounter when conducting life-imaging, and specially when we are using fluorescent microscopy, is photobleaching. This is irreversible loss of fluorescence signal due to long exposure to excitation light. This becomes a major problem when we conduct time-lapse microscopy, that is, when we record image sequences over time to later view them at great speed to give an accelerated view of microscopic processes.
What are the applications of live-imaging?
There are a great variety of fluorescent probes which target very different and specific physical conditions (e.g. pH, membrane potentials), structures (nuclei, membranes of different compositions, mitochondria, lysosomes...), molecules (including different species of reactive oxygen and nitrogen species...), etc.
Fluorescent probes can be used either individually or in conjunction with other probes (as long as their fluorescent spectra don't overlap). Therefore, purposes and applications of these techniques become unlimited.
Here we present some of our own examples: