Use Of Fluorophores Research Articles

Multispectral imaging for characterizing autofluorescent tissues

Selective Plane Illumination Microscopy (SPIM) has become an emerging technology since its first application for 3D in-vivo imaging of the development of a living organism. An extensive number of works have been published, improving both the speed of acquisition and the resolution of the systems. Furthermore, multispectral imaging allows the effective separation of overlapping signals associated with different fluorophores from the spectrum over the whole field-of-view of the analyzed sample. To eliminate the need of using fluorescent dyes, this technique can also be applied to autofluorescence imaging. However, the effective separation of the overlapped spectra in autofluorescence imaging necessitates the use of mathematical tools. In this work, we explore the application of a method based on Principal Component Analysis (PCA) that enables tissue characterization upon spectral autofluorescence data without the use of fluorophores. Thus, enabling the separation of different tissue types in fixed and living samples with no need of staining techniques. Two procedures are described for acquiring spectral data, including a single excitation based method and a multi-excitation scanning approach. In both cases, we demonstrate the effective separation of various tissue types based on their unique autofluorescence spectra.

Validation of Calcein Violet as a New Marker of Semen Membrane Integrity in Domestic Animals.

Many fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones (Apis mellifera). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR®14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry.

Blue Fluorescence of Cyano-tryptophan Predicts Local Electrostatics and Hydrogen Bonding in Biomolecules.

Cyano-tryptophan is an unnatural fluorescent amino acid that emits in the visible region. Along with the structural similarity with tryptophan, the unique photophysical properties of this fluorophore make it an ideal probe for biophysical research. Herein, combining fluorescence spectroscopy, infrared spectroscopy, and molecular dynamics simulations, we show that the cyano-tryptophan's emission energy quantifies the underlying bond-specific noncovalent interactions in terms of the electric field. We further report the use of fluorophore's emission energy to predict its hydrogen bond characteristics. We demonstrate that combining experiments with molecular dynamics simulations can provide the hydrogen bonding status of the nitrile moiety. In addition, we report a method to differentiate between aqueous and nonaqueous hydrogen-bonding partners. Using a phenomenological approach, we demonstrate that the presence of the cyano-indole moiety is responsible for the distinct correlations between the fluorophore's emission and the electrostatic forces on the nitrile bond. As indole is a privileged scaffold for both native amino acids and nucleobases, cyano-indoles will have many multifaceted applications.

Narrative review on applications of fluorescence-guided surgery in adult and paediatric urology

: More recently, there has been a substantial increase in the use of fluorophores to guide open and laparoscopic procedures with the ultimate aim to optimize both oncological and functional outcomes and to reduce operative time and blood loss. Fluorescent dyes are currently adopted to allow safer and more accurate dissections during both oncological and reconstructive procedures thanks to their ability of highlighting tumour margins and tissue vascularization. Urology is one of the fields in which fluorescence-guided surgery (FGS) has proved to be most useful. In particular, it has assumed a pivotal role in the surgical treatment of oncological patients affected by kidney, bladder, prostate and penis cancers and in the management of paediatric urological conditions. This review aims to provide an update on the use of FGS in adult and paediatric urology, drawing attention to its most recent and interesting applications in this very innovative field of research. Although FGS has been only recently introduced in the clinical scenario, it can be already considered a powerful tool to improve oncological, anatomical and functional outcomes in both adult and paediatric urology. An increased identification of lymph nodes (LNs), a more accurate visualization of tumour margins and a better definition of blood supply and lymphatic drainage have proved to be greatly beneficial for patients undergoing urological procedures, whether they are adults or children. Longitudinal studies with larger sample sizes are still needed to draw firm conclusions and to confirm its benefits in urology.

Fluorescence-Guided Surgery: A Review on Timing and Use in Brain Tumor Surgery.

Fluorescence-guided surgery (FGS) allows surgeons to have improved visualization of tumor tissue in the operating room, enabling maximal safe resection of malignant brain tumors. Over the past two decades, multiple fluorescent agents have been studied for FGS, including 5-aminolevulinic acid (5-ALA), fluorescein sodium, and indocyanine green (ICG). Both non-targeted and targeted fluorescent agents are currently being used in clinical practice, as well as under investigation, for glioma visualization and resection. While the efficacy of intraoperative fluorescence in studied fluorophores has been well established in the literature, the effect of timing on fluorophore administration in glioma surgery has not been as well depicted. In the past year, recent studies of 5-ALA use have shown that intraoperative fluorescence may persist beyond the previously studied window used in prior multicenter trials. Additionally, the use of fluorophores for different brain tumor types is discussed in detail, including a discussion of choosing the right fluorophore based on tumor etiology. In the following review, the authors will describe the temporal nature of the various fluorophores used in glioma surgery, what remains uncertain in FGS, and provide a guide for using fluorescence as a surgical adjunct in brain tumor surgery.

Choice of fluorophore affects dynamic DNA nanostructures.

The ability to dynamically remodel DNA origami structures or functional nanodevices is highly desired in the field of DNA nanotechnology. Concomitantly, the use of fluorophores to track and validate the dynamics of such DNA-based architectures is commonplace and often unavoidable. It is therefore crucial to be aware of the side effects of popular fluorophores, which are often exchanged without considering the potential impact on the system. Here, we show that the choice of fluorophore can strongly affect the reconfiguration of DNA nanostructures. To this end, we encapsulate a triple-stranded DNA (tsDNA) into water-in-oil compartments and functionalize their periphery with a single-stranded DNA handle (ssDNA). Thus, the tsDNA can bind and unbind from the periphery by reversible opening of the triplex and subsequent strand displacement. Using a combination of experiments, molecular dynamics (MD) simulations, and reaction-diffusion modelling, we demonstrate for 12 different fluorophore combinations that it is possible to alter or even inhibit the DNA nanostructure formation—without changing the DNA sequence. Besides its immediate importance for the design of pH-responsive switches and fluorophore labelling, our work presents a strategy to precisely tune the energy landscape of dynamic DNA nanodevices.

Probing the Role of Actin in Membrane Organization

The actin cortex has long been hypothesized to organize membrane components, especially ordered membrane domains thought to play important functional roles at the plasma membrane. Recent developments in super-resolution fluorescence localization microscopy have enabled imaging of the actin cortex in chemically fixed cells through the use of fluorophores that spatially sample filaments at densities required to trace actin meshworks. This poster describes our ongoing efforts to quantify how actin impacts the domain structure of cell plasma membranes, utilizing these actin binding probes in conjunction with well characterized peptide markers of membrane domains. We aim to employ super-resolution imaging to validate past studies, which have characterized meshwork compartment sizes in several cell types based on diffusion data. The majority of our studies are conducted in B cells, where past work indicates that actin structure plays important roles in both antigen mediated B cell receptor signaling and in the tonic signaling that occurs in the absence of ligands. Through this approach, we explore the extent to which these signaling functions are impacted by coupling between actin and membrane domains.

Thermal Stability of Thiolated DNA SAMs in Buffer: Revealing the Influence of Surface Crystallography and DNA Coverage via In Situ Combinatorial Surface Analysis.

The thermal stability of thiol based DNA SAMs prepared on gold surfaces is an important parameter that is correlated to sensor lifetime. The thermal stability of DNA SAMs was evaluated in aqueous buffer through the use of fluorophore labeled DNA, a single crystal gold bead electrode, and microscopy. The stability of different crystallographic regions on the electrode was studied for thermal treatments up to 95 °C for 90 min. Using a in situ combinatorial surface analytical measurement showed that the crystallography of the underlying gold surface played a significant role, with the square or rectangular lattices (e.g., 110, 100, 210) having the highest stability. Surfaces with hexagonal lattices (e.g., 111, 311, 211) were less stable toward thermal treatments. These crystallographic trends were observed for both high and low coverage DNA SAMs. High coverage DNA SAMs were the most stable, with stability decreasing with decreasing coverage on average. Increasing DNA SAM coverage appears to slow the kinetics of thermal desorption, but the coordination to the underlying surface determined their relative stability. Preparing the DNA SAMs under nominally similar conditions were found to create surfaces that were similar at room temperature, but had significantly different thermal stability. Optimal DNA sensing with these surfaces most often requires low coverage DNA SAMs which results in poor thermal stability, which is predictive of a poor shelf life, making optimization of both parameters challenging. Furthermore, the crystallographically specific results should be taken into account when studying the typically used polycrystalline substrates since the underlying surface crystallography maybe different for different samples. It appears that preparing DNA SAMs with low coverage and significant thermal stability will be challenging using the current SAM preparation procedures.

Surgery-Guided Removal of Ovarian Cancer Using Up-Converting Nanoparticles.

Ovarian cancer survival and the recurrence rate are drastically affected by the amount of tumor that can be surgically removed prior to chemotherapy. Surgeons are currently limited to visual inspection, making smaller tumors difficult to be removed surgically. Enhancing the surgeon's ability to selectively remove cancerous tissue would have a positive effect on a patient's prognosis. One approach to aid in surgical tumor removal involves using targeted fluorescent probes to selectively label cancerous tissue. To date, there has been a trade-off in balancing two requirements for the surgeon: the ability to see maximal tumors and the ability to identify these tumors by eye while performing the surgery. The ability to see maximal tumors has been prioritized and this has led to the use of fluorophores activated by near-infrared (NIR) light as NIR penetrates most deeply in this surgical setting, but the light emitted by traditional NIR fluorophores is invisible to the naked eye. This has necessitated the use of specialty detectors and monitors that the surgeon must consult while performing the surgery. In this study, we develop nanoparticles that selectively label ovarian tumors and are activated by NIR light but emit visible light. This potentially allows for maximal tumor observation and real-time detection by eye during surgery. We designed two generations of up-converting nanoparticles that emit green light when illuminated with NIR light. These particles specifically label ovarian tumors most likely via tumor-associated macrophages, which are prominent in the tumor microenvironment. Our results demonstrate that this approach is a viable means of visualizing tumors during surgery without the need for complicated, expensive, and bulky detection equipment. Continued improvement and experimentation could expand our approach into a much needed surgical technique to aid ovarian tumor removal.

Study of a Miniaturizable System for Optical Sensing Application to Human Cells

Conventional approaches to human intracellular optical sensing, generally, require dedicated laboratories with bulky detection systems. They are performed by cell labeling procedures based on the use of fluorophores that are, mostly, phototoxic, invasive, bleached in case of prolonged light exposures, which require carriers and/or structural modifications for the cellular uptake. These issues, together with the sensitivity of the eukaryotic cell model, could be problematic towards the development of a robust sensing system suitable for biomedical screening. In this work, we studied a sensing system resulting from the combination of the commercial tris(2,2’bipyridyl)ruthenium(II) fluorophore, for cell labeling, with a potentially miniaturizable optical system composed by a laser source and a photomultiplier tube, for the fluorescence analysis.

Oral Biofilm Formation on Different Materials for Dental Implants.

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Bioinspired water-soluble two-photon fluorophores

The structural modification of plant pigments allows the development of new functional dyes with tailored photophysical properties. Two-photon excitation fluorescence microscopy has emerged as an important technique, but requires the use of fluorophores having large two-photon absorption cross-sections in water. This feature is rarely found in natural or artificial dyes, but betalains, the main pigments of beetroots and some species of fluorescent flowers, can be modified to produce water-soluble two-photon fluorophores. Herein we describe the semisynthesis and photophysical characterization of two water-soluble artificial coumarinic betalains. The excitation of these compounds in the near-infrared results in yellow fluorescence with two-photon cross-sections in water of 323 GM and 179 GM.

Quantifying the Selective Modification of Au(111) Facets via Electrochemical and Electroless Treatments for Manipulating Gold Nanorod Surface Composition.

Manipulating the composition of a mixed alkylthiol self-assembled monolayer (SAM) modified gold surface using both electrochemical and electroless methods is demonstrated. Through the use of fluorophore labeled thiolated DNA and in situ fluorescence microscopy with a gold single crystal bead electrode, a procedure was developed to study and quantify the selective desorption of an alkylthiolate SAM. This method enabled a self-consistent measurement of the removal of the SAM from the 111 surface compared to the 100 surface region at various potentials. A 20-fold increase in the electrochemical removal and replacement of the SAM from the 111 surface over the 100 surface was realized at -0.8 V/AgAgCl. A related procedure was developed for the solution-based electroless removal of the SAM using NaBH4 achieving a similar selectivity at the same potential. Unfortunately, in the electroless process fine control over the reducing potential was difficult to achieve. In addition, working in the presence of O2 complicates the solution potential measurement due to depolarization by the reduction of O2, resulting in a less clear relationship between selectivity and measured solution potential. Interestingly, the electrochemical method was not disturbed by the presence of O2. In preparation for work with Au nanorods, electrochemical measurements were performed in electrolyte that included 1 mM CTAB and was found to not interfere with this method. Preliminary results are promising for using this methodology for treatment of acid-terminated alkylthiol modified Au nanorods.

Using Biotechnology to Solve Engineering Problems: Non-Destructive Testing of Microfabrication Components.

In an increasingly miniaturised technological world, non-destructive testing (NDT) methodologies able to detect defects at the micro scale are necessary to prevent failures. Although several existing methods allow the detection of defects at that scale, their application may be hindered by the small size of the samples to examine. In this study, the application of bacterial cells to help the detection of fissures, cracks, and voids on the surface of metals is proposed. The application of magnetic and electric fields after deposition of the cells ensured the distribution of the cells over the entire surfaces and helped the penetration of the cells inside the defects. The use of fluorophores to stain the cells allowed their visualisation and the identification of the defects. Furthermore, the size and zeta potential of the cells and their production of siderophores and biosurfactants could be influenced to detect smaller defects. Micro and nano surface defects made in aluminium, steel, and copper alloys could be readily identified by two Staphylococcus strains and Rhodococcus erythropolis cells.

Tunable Probes with Direct Fluorescence Signals for the Constitutive and Immunoproteasome.

Electrophiles are commonly used for the inhibition of proteases. Notably, inhibitors of the proteasome, a central determinant of cellular survival and a target of several FDA-approved drugs, are mainly characterized by the reactivity of their electrophilic head groups. We aimed to tune the inhibitory strength of peptidic sulfonate esters by varying the leaving groups. Indeed, proteasome inhibition correlated well with the pKa of the leaving group. The use of fluorophores as leaving groups enabled us to design probes that release a stoichiometric fluorescence signal upon reaction, thereby directly linking proteasome inactivation to the readout. This principle could be applicable to other sulfonyl fluoride based inhibitors and allows the design of sensitive probes for enzymatic studies.

Increased Light Absorption in Dye-Sensitized Solar Cells with Light Harvesting Dye

In plants, photosynthetic protein, light harvesting complexes (LHC), holds reaction center called photosystem I or II (PSI or PSII). PSI and PSII receive light energy and convert it to electric flow. This reaction is done in high quantum efficiency. This light-harvesting system enables the plants to use the sun-light efficiently. The converted electron is spatially transferred in multiple chemical steps, and this is called Z scheme. The Z scheme is known to be highly efficient which have nearly no loss of electric energy. Dye-sensitized solar cells (DSSCs) have attracted increasing attention because of its multiple advantages such as low-cost, lightweight, and flexibility. In DSSCs, photogenerated charge carriers in dye are separated and the electrons are injected to conduction band of semiconductor. The charge separating process of DSSCs is similar to that of photosynthesis. Here, we report DSSCs further imitated photosynthesis. The DSSCs we reported have applied the light-harvesting system in photosynthesis. Perylene-based fluorophore was dispersed in electrolyte in order to imitate the roll of chlorophyll, the light harvesting dyes, in nature. This material was chosen due to the high stability against photo-oxidation. Phthalocyanine, the sensitizing dye, was adsorbed on the surface of photoanode to convert photoenergy into an electric flow. The two synthetic dyes are selected to imitate the light-harvesting complex and reaction center, respectively. The energy absorbed at the fluorophore undergoes Förster resonant energy transfer (FRET) to the sensitizing dye. This allows broader spectral absorption, which is beneficial as a characteristic of a solar cell. We have fabricated the above-mentioned imitated system using synthetic dyes, and evaluated its performances on FRET and following electron transfer. Experiments were done as follows. Perylene-based fluorophore was dispersed in iodine/triiodide electrolytes. Three types of fluorophores with different emission wavelengths were used, and electrolytes with different fluorophore concentrations were prepared at the purpose of comparison. Two phthalocyanine-based sensitizing dyes, PcS2 and PcS20 (structures shown in Figure1 a and b), were chosen to investigate the impact of modifications around the main structure. Absorption characteristics of the two materials are nearly the same [1]. The TiO2electrode consisted of a 10 µm nanoporous layer with 3 µm scattering layer was prepared by screen printing. Solar cells were assembled using Surlyn film with platinized counter electrode. The electrolyte solution was introduced through two holes predrilled in the counter electrode. Evaluation of the samples was done using incident-photon-to-current conversion efficiency (IPCE). Figure 2 shows IPCE result of the solar cell using one of the three fluorophore and PcS2. As a result, the DSSCs fabricated using fluorophore with emission well matching the absorption of the sensitizing dye had shown a notable photo-electric conversion around 400-600 nm. The overall photovoltaic performance was greater than the case of no use of fluorophore at all. The solar cells with other two types of fluorophores had not shown this characteristic. Since the emission band of these two fluorophores does not match the adsorption band of the sensitizing dye, it is suggested that FRET reaction is involved in the trend seen in Fig. 2. In conclusions, DSSCs with plant-inspired structure was fabricated and their photovoltaic performance was investigated. Occurrence of FRET among the fluorophore introduced in the electrolyte and the sensitizing dye was suggested from the comparison between the evaluation using fluorophores with different emission band. Synthetizing dyes with different modification had shown the difference, in which suggests that the bulkiness of the dye affects the reaction in the photoanode surface. The overall photovoltaic performance was shown to improve by creating a structure that induces FRET reaction. Ref.1 S. Mori, M. Nagata, Y. Nakahata, K. Yasuta, R. Goto, M. Kimura, and M. Taya J. Am. Chem. Soc. 2010, 132, 4054-4055 Figure 1

Multichannel Hyperspectral Imaging and Characterization of Far-Red Fluorophores using a Thin-Film Tunable Filter

Multicolour fluorescence imaging has traditionally been limited to the use of fluorophores with non-overlapping emission spectra. The advent of hyperspectral imaging has made it possible to distinguish signals with partial overlap by acquiring fluorescence over narrow emission bands. We report here the implementation of one such approach, using the Semrock Versachrome™ thin film tunable filter integrated via a custom-built insert into an Olympus IX-83 dual-deck inverted TIRF microscope. We demonstrate the ability to distinguish and resolve the emission spectra of two far-red fluorophores, Alexa 647 and ATTO 680, by varying the angle of incidence of the emitted light on the Versachrome. We have implemented a galvanometer based solution that provides fast and precise control over the transmission band. The high transmission coefficient of the thin film filter allowed us to reduce the exposure time and acquire a full-spectrum sequence in less than 100ms. Deconvolution of the fluorescence signals allowed determination of the relative fluorophore population maximizing the recovery of information. We demonstrate the application of the technique to imaging multiple sub-cellular targets with well-characterized supra-molecular morphology, including cytoskeletal, mitochondrial, plasma membrane and nuclear proteins. This technique for high-speed spectral imaging will bring new applications into focus and improve performance in already established approaches.

Steps Towards in-Situ Studies of the Mechanical Degradation of Li-Ion Batteries Using Fluorescence Confocal Microscopy

The study of various degradation mechanisms in lithium ion batteries (LIB) that lead to capacity fade during cycling is of primary interest to the future progress of consumer electronic products, including the desire for advancements in hybrid electric vehicles. Considering that significant improvements to performance properties such as cycle life and energy density are crucial; it is necessary to advance the understanding of electrode and electrolyte materials, and the physical and chemical processes that ultimately lead to capacity fade in commercial LIB. By quantifying the degradation processes that occur within LIB, the predictive capability in modeling battery performance will improve. This work introduces a novel approach to studying, in-situ, the degradation mechanisms of the cell as a whole with focus on the LiCoO2 electrode. The research involves utilizing fluorescence confocal microscopy to observe direct changes on the mesoscopic structural level as well as changes in the electrochemical cell performance in order to quantify the mechanical stresses that develop due to various processes (SEI formation, electrode expansion/ contraction, external loading, etc.). It is expected that this work will realize, for the first time, the effect of stress on particle configuration over time, loss of electronic contact to particles, and the role of binder in controlling and affecting stress evolution. In this report, we report validation testing of the use of fluorophores in LIB for studying the interactions among particles and binder during cycling. Cyclic voltammetry (CV) is used to determine the electrochemical stability of fluorescents in electrolyte of 1.2M LiPF6 EC:EMC (3:7 by wt). CV testing was performed within the potential window of an operating LiCoO2 cell; 0 to 4.2 V. Fluorescents that were tested include acridine, acriflavine, boron-dipyrromethene (BODIPY FL), fluorescein, naphthofluorescein, quinine, rhodamine 6G, and rhodamine B. Figure 1 shows the CV results for all fluorescents. Many of these dyes are not suitable for use in LIB due to strong reduction and/or oxidation peaks within the potential window. The most stable dyes that are best suited for use in LIB are fluorescein and naphthofluorescein. Meanwhile, coin cell testing is performed to ensure that the addition of fluorescents to the electrode and electrolyte materials does not adversely affect the long-term performance of the cell. Coin cells were made with and without naphthofluorescein and fluorescein added to the electrolyte and binder, respectively. Cells are cycled for a minimum of 100 charge-discharge cycles at a C/10 charge and discharge rate. In addition to performing validation testing and ensuring that dyes do not leach from their tagged component, preliminary imaging of the dyed cell using the confocal microscope is done in preparations for in-situ imaging and testing. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000. Figure 1

Now you see it, now you don't: A biochemist's primer for advances in high-resolution microscopy: Comment on “Biomolecular dynamics and binding studies in the living cell” by Stephan Diekmann and Christian Hoischen

For many scientists, the first glimpse of the inner workings of the world around us comes from the use of a simple light microscope. Indeed, ground glass was used to make lenses for magnifying glasses as early as the time of the Romans (Seneca, Pliny). In the mid-15th century, the Janssens put together a series of lenses in a tube, discovering the ability of compound microscopes to magnify objects greatly. This advance allowed Hooke and Leeuewenhoek to conclude that life was composed of cells, and resulted in Schleiden, Schwann, Virchows’ observations that cells regenerated new cells. Warp speed to 400 years later, with a hop in between for Coon’s tagging of antibodies, and for the development of GFP-tags by Shimomura, Chalfie, Tsien, and the humble light microscope has transformed our understanding of the natural and physical world. In recent years, revolutionary changes to microscopes and the application of powerful computational algorithms to analyze data have yielded information far surpassing the physical limits set by the diffraction of light. In this comprehensive review, Diekmann and Hoeschien discuss the application of new technologies such as FRET, FRAP, FCS, STORM/PALM, FLIM-FRET, now commonly used to illuminate the inner mysteries of the sanctum sanctorum of the cell – the nucleus. They demonstrate that with careful and rigorous measurements, an almost quantitative knowledge of the nucleus is well within our grasp. For the biochemist, geneticist, or molecular biologist, confounded by the bewildering array of acronyms denoting different techniques in light microscopy, this review is both timely, and a time saver. It describes in great detail how each advance named above can be applied to the quantitative and qualitative measurement of protein–protein interactions, positional information, and dynamics of multi-protein complexes in living cells, down to 10 nm resolution. These technological advances, as Diekmann and Hoeschien explain, are powerful in their ability to gather every pixel of information available, and when carefully analyzed, able to yield tremendous insights on the behavior of proteins in a biologically meaningful setting. The review is particularly useful in systematically and objectively comparing the benefits and the limitations of each approach, as well as highlighting the potential artifacts associated with the use of fluorophores or covalently-attached

Metal-enhanced fluorescence of nano-core–shell structure used for sensitive detection of prion protein with a dual-aptamer strategy

Metal-enhanced fluorescence (MEF) as a newly recognized technology is widespread throughout biological research. The use of fluorophore–metal interactions is recognized to be able to alleviate some of fluorophore photophysical constraints, favorably increase both the fluorophore emission intensity and photostability. In this contribution, we developed a novel metal-enhanced fluorescence (MEF) and dual-aptamer-based strategy to achieve the prion detection in solution and intracellular protein imaging simultaneously, which shows high promise for nanostructure-based biosensing. In the presence of prion protein, core–shell Ag@SiO2, which are functionalized covalently by single stranded aptamer (Apt1) of prions and Cyanine 3 (Cy3) decorated the other aptamer (Apt2) were coupled together by the specific interaction between prions and the anti-prion aptamers in solution. By adjusting shell thickness of the pariticles, a dual-aptamer strategy combined MEF can be realized by the excitation and/or emission rates of Cy3. It was found that the enhanced fluorescence intensities followed a linear relationship in the range of 0.05–0.30nM, which is successfully applied to the detection of PrP in mice brain homogenates.