Fluorescent probes and fluorescence (microscopy) techniques--illuminating biological and biomedical research - PubMed (original) (raw)

Review

Fluorescent probes and fluorescence (microscopy) techniques--illuminating biological and biomedical research

Gregor P C Drummen. Molecules. 2012.

Abstract

Fluorescence, the absorption and re-emission of photons with longer wavelengths, is one of those amazing phenomena of Nature. Its discovery and utilization had, and still has, a major impact on biological and biomedical research, since it enables researchers not just to visualize normal physiological processes with high temporal and spatial resolution, to detect multiple signals concomitantly, to track single molecules in vivo, to replace radioactive assays when possible, but also to shed light on many pathobiological processes underpinning disease states, which would otherwise not be possible. Compounds that exhibit fluorescence are commonly called fluorochromes or fluorophores and one of these fluorescent molecules in particular has significantly enabled life science research to gain new insights in virtually all its sub-disciplines: Green Fluorescent Protein. Because fluorescent proteins are synthesized in vivo, integration of fluorescent detection methods into the biological system via genetic techniques now became feasible. Currently fluorescent proteins are available that virtually span the whole electromagnetic spectrum. Concomitantly, fluorescence imaging techniques were developed, and often progress in one field fueled innovation in the other. Impressively, the properties of fluorescence were utilized to develop new assays and imaging modalities, ranging from energy transfer to image molecular interactions to imaging beyond the diffraction limit with super-resolution microscopy. Here, an overview is provided of recent developments in both fluorescence imaging and fluorochrome engineering, which together constitute the “fluorescence toolbox” in life science research.

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Figures

Figure 1

Figure 1

Luminescence in Nature. (A) Sepiolidae family of squid; (B) Aurelia aurita, moon jelly fish; please note that the blue glow stems from diffraction and not from bioluminescence; (C) Lampyridae family of fireflies; (D) Phosphorescent zinc sulfide pigment (alkaline earth metal); (E) Fluorescent rocks (top is illuminated with UV-light; bottom white light recording): left = Willemite/Calcite; right = Hardystonite (courtesy Ron Teunissen ©2012); (F) Luminescent water at the Gippsland Lakes, Australia (courtesy Phil Hart ©2012), which is created by Noctiluca scintillans, a non-parasitic marine-dwelling species of dinoflagellate that exhibits bioluminescence; the bioluminescent reaction is instantaneous, as observed in the left picture when the water is mechanically disturbed; (G) Mycena Chlorophos, a large genus of small saprotrophic mushrooms.

Figure 2

Figure 2

Fluorescence principle. (A) Schematic representation of the fluorescence phenomenon in the classical Bohr model. From the ground state _GS_0❶, absorption of a light quantum (blue) causes an electron to move to a higher energy level ❷→❸. After residing in this “excited state” ❸ for a particular time, the fluorescence lifetime, the electron falls back to its original level ❸→❹ and the fluorochrome dissipates the excess energy by emitting a photon (green) ❹. (B) Jabłoński diagram: Upon photon absorption, a ground state _GS_0 electron (electronic singlet) is promoted to a higher and excited state, relaxes quickly to a lower vibrational excited state (white line) and thereby looses energy. When returning to the ground state, it dissipates the remaining energy by emitting a photon with a longer wavelength, i.e., fluorescence emission. The spins of electrons in the singlet states (paired or unpaired anti-parallel spins) compared to the triplet state (unpaired, parallel spin) are depicted. Notice that intersystem crossing from _ES_1→ _ET_1 requires spin conversion and phosphorescence occurs through relaxation from the triplet excited state.

Figure 3

Figure 3

Twisted Intramolecular Charge Transfer (TICT) dynamics. Upon excitation from the ground state (GS), the locally excited state (LES) equilibrates rapidly with the TICT state after fast electron transfer. The TICT state is energetically lower and relaxation from the TICT state occurs either radiatively or non-radiatively to a thermally hot ground state (GS’) which after dissipation of heat becomes the GS. Alternatively, excess energy is dissipated via direct radiative relaxation to the GS. The exact pathway for energy dissipation depends strongly on the polarity of the environment.

Figure 4

Figure 4

Pyrene’s excimer formation in lipid vesicles. (A) Molecular structure of pyrene (Benzo[d,e,f]phenanthrene; C16H10). (B) Emission spectra of pyrene in egg-PC. Spectra are normalized to the 372 nm peak of the monomer and excimer formation can increasingly be observed at ~460 nm with increasing pyrene concentration.

Figure 5

Figure 5

General chemical structure of metal-phthalocyanine dyes. Me denotes the metal ion; R1-4 are peripheral functional groups and X and Y are optional functional groups above and below the molecule’s plane (axial).

Figure 6

Figure 6

Bio-imaging with hyaluronan analogs modified with the near-infrared IR-783 dye.(A) Optical images of SKH mice injected with fluorescent HA-NIRdye (1% dye load). Based on Reference [76]. Note that the probe predominantly accumulates in the upper abdominal and thoracic organs, i.e., liver, heart, lungs, kidneys, stomach, and lymph nodes. After 24−48 h, the probe is exclusively found in the liver. (B) Progression of the fluorescence intensity in the boxes indicated in A.

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