Fluorophores for Confocal Microscopy: Photophysics and Photochemistry (original) (raw)

Photophysics and Spectroscopy of Fluorophores in the Green Fluorescent Protein Family

Abstract Proteins homologous to the green fluorescent protein (GFPs) form a large family of unconventional, genetically encoded fluorophores with widely diverse colors and applications, which have profoundly renewed the fields of biological imaging and drug screening. Their detailed spectroscopy stems from a complex interplay between the electronic properties of a relatively simple, yet flexible and multiprotonable chromophore formed after specific biosynthesis, and the spatial and dynamic organization of its protein carrier. Early experimental and theoretical studies of GFP from the Aequorea victoria jellyfish and of model synthetic compounds have revealed that chromophore twisting, cis-trans isomerization, proton transfer, and electron transfer are major excited state reactions that determine its photophysics and photochemistry. It has been found later that quite similar mechanisms are at work in several distant members of the GFP family, suggesting a unified picture that may guide the future development of new GFP-based biosensors. Graphical Abstract

An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy

The Journal of Cell Biology, 1987

Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.

Basic Principles of Fluorescence Microscopy

World Journal of Young Researchers, 2013

Fluorescence microscopy is a basic requirement in cell biology, molecular biology and biotechnology. Advancements over the years has helped scientist to trace molecules in live cells and understand the basis of cell metabolism, exchange, mutation and toxicity. In this short communication we seek to explain in simple terms the basic principles of how a fluorescence microscope works. The principles of excitation and emission focuses on the ability of fluorophores to absorb energy from photons and to emit such absorbed energy. The difference between the chemical structures of these fluorephores determines how much energy that is required to excite them and how long a fluorescence signal from a fluorophore will last. The principles of epi-illumination on the other hand describe the arrangement and function of the various components of a fluorescence microscope.

Quantification and calibration of images in fluorescence microscopy

Analytical Biochemistry, 2010

Fluorescence microscopy is a method widely used in life sciences to image biological processes in living and fixed cells or in fixed tissues. Quantification and calibration of images in fluorescence microscopy is notoriously difficult. We have developed a new methodology to prepare tissue ''phantoms" that contain known amounts of (i) fluorophore, (ii) DNA, (iii) proteins, and (iv) DNA oligonucleotide standards. The basis of the phantoms is the ability of gelatin to act as a matrix for the conjugation of fluorophores as either a free-flowing liquid or a gelatinous solid depending on temperature (P40 and 64°C).

Fluorescence performance standards for confocal microscopy

Proceedings of SPIE, 2010

State of the art confocal microscopes offer diffraction limited (or even better) spatial resolution, highest (single molecule) sensitivity and ps-fluorescence lifetime measurement accuracy. For developers, manufacturers, as well as users of confocal microscopes it is mandatory to assign values to these qualities. In particular for users, it is often not easy to ascertain that the instrument is properly aligned as a large number of factors influence resolution or sensitivity. Therefore, we aspire to design a set of performance standards to be deployed on a day-to-day fashion in order to check the instruments characteristics. The main quantities such performance standard must address are: Spatial resolution, Sensitivity, Fluorescence lifetime To facilitate the deployment and thus promote wide range adoption in day-to-day performance testing the corresponding standards have to be ready made, easy to handle and to store. The measurement procedures necessary should be available on as many different setups as possible and the procedures involved in their deployment should be as easy as possible. To this end, we developed two performance standards to accomplish the mentioned goals: Resolution reference, Combined molecular brightness and fluorescence lifetime reference. The first one is based on sub-resolution sized Tetra-Speck (TM) fluorescent beads or alternatively on single molecules on a glass surface to image and to determine quantitatively the confocal volume, while the latter is a liquid sample containing fluorescent dyes of different concentrations and spectral properties. Both samples are sealed in order to ease their use and prolong their storage life. Currently long-term tests are performed to ascertain durability and road capabilities.

Advanced Methods in Fluorescence Microscopy

Analytical Cellular Pathology, 2013

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbé limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres.