Methods and Applications in Fluorescence最新文献

Using computational chemistry, we have scanned a set of four-membered N-heterocyclic carbenes with bulky substituents for their ability to form carbene metal amides (CMAs) with excellent thermally activated delayed fluorescence (TADF) properties. In comparison to the properties of their well-known five- and six-membered analogs, the transition dipole moments of the first excited singlet states of the corresponding Cu^(I)carbazolide (Cz) complexes increase. For CMAs of the most promising four-membered carbene, a lactam-based carbene (4LAC), detailed investigations of the TADF properties have been performed using advanced quantum chemical methods. Due to the small energy separation between its singlet and triplet ligand-to-ligand charge-transfer (LLCT) states, 4LAC-Ag^(I)-Cz exhibits the best ratio between reverse intersystem crossing (rISC) and intersystem crossing in the coinage metal triad for a coplanar orientation of the ligands. The TADF properties of the corresponding Cu^(I)and Au^(I)complexes benefit from twisted ligand-ligand alignments, achieved by using tetrafluorocarbazolide (4FCz) as donor ligand. The moderate reduction of the fluorescence rate constant upon twisting by about 45-50° is overcompensated by a decrease of the singlet-triplet energy gap, thus improving the TADF performance. Overall, with fluorescence rate constants of the order of 10⁷s^-1and rISC rate constants between 10⁹and 10¹⁰s^-1, TADF should have competitive advantage over common triplet deactivation processes such as triplet-triplet annihilation. Like in other CMAs, full excited-state geometry relaxation in liquid solution is detrimental for the emission properties. In the solid state, where the formation of a perpendicular ligand-ligand alignment is sterically hindered by the environment, 4LAC-M-Cz and 4LAC-M-4FCz are predicted to be efficient TADF compounds with red to orange emission.

2-Aminopurine (2AP) is the most widely used fluorescent nucleobase analog in DNA and RNA research. While quenching of 2AP by DNA bases has been extensively characterized, the effect of extrinsic quenchers has received far less attention. This study examines the fluorescence quenching mechanisms of 2AP by commonly used buffers in biochemical research. We systematically investigated four Good's buffers-MES, MOPS, HEPES, and PIPES-along with their parent compounds morpholine and piperazine across a range of pH conditions and concentrations. For morpholine-containing buffers (MES and MOPS), quenching occurs predominantly at pH values at or above their respective pKa values and is negligible at more than two pH units below the pKa. In contrast, piperazine-containing buffers (HEPES and PIPES) exhibit substantial quenching even below their pKa values due to the presence of two basic nitrogen atoms in the piperazine ring, one of which remains unprotonated and reactive across the investigated pH range. Time-resolved fluorescence measurements demonstrate that quenching is primarily dynamic for MES, MOPS, and HEPES, while PIPES shows significant static quenching contributions. Results are consistent with a mechanism involving photoinduced electron transfer from unprotonated tertiary amines to excited-state 2AP. The thermodynamic feasibility of this mechanism is supported by the low oxidation potentials of these tertiary amines compared to primary amine-containing buffers such as TRIS, which does not quench 2AP fluorescence. These results have significant practical implications for fluorescence-based studies using 2AP as a structural or dynamic probe in nucleic acid research. Buffer selection can substantially alter both quantum yields and fluorescence lifetimes of 2AP, potentially leading to misinterpretation of experimental data if these effects are not properly accounted for.

The fluorescent on-chip imaging system differs from a conventional fluorescent microscope in terms of the imaging method because the sample is directly placed on the imaging sensor (i.e., charge-coupled device (CCD)). While this imaging modality presents several advantages, including a wide field of view and rapid scanning speed, it can be difficult to detect certain particles in dense and scattering environments, such as whole blood and tissue. These difficulties lead to a decreased signal-to-noise ratio (SNR) in the captured images, influenced by both the medium's light-transmitting capability and the excitation techniques used. In this paper, we quantitatively examine the effect of beam shaping techniques on a fluorescent on-chip imaging system from the SNR perspective. An experimental comparison is conducted between a Gaussian beam and plane-wave illumination generated by a novel phase modulation schema using our developed imaging platform. The results indicate that the Gaussian beam produces higher SNR images than plane waves when detecting fluorescent particles in a microchannel. Gaussian beam's higher energy confinement ability enhances the image quality of on-chip fluorescent imaging systems, particularly involving scattering-like medium limitations.

Uranium(VI) speciation in aqueous carbonate solutions was systematically investigated using time-resolved laser-induced fluorescence spectroscopy (TRLFS) across a broad pH range (4.3-13.0) at room temperature. Distinct uranyl complexes were identified based on their luminescence lifetimes and emission spectra, and their formation was correlated with the theoretical speciation models. Particular emphasis was placed on alkaline conditions, where uranium speciation is less understood due to weak luminescence signals. This study revealed the presence of multiple hydroxo and carbonato complexes, including non-luminescent species at high pH. These findings provide new insights into uranium(VI) behaviour in cementitious environments relevant to deep geological repositories. Moreover the dynamics of complex formation were investigated, and the quantity of precipitates were quantified using the methods based on the luminescent properties of uranium and the presence of a complexing agent. The luminescence intensity was shown to be independent of pH and linearly correlated with uranium concentration, confirming TRLFS as a robust tool for uranium quantification in variable geochemical settings. This work contributes to a more accurate understanding of uranium mobility and stability in nuclear waste management scenarios and in locations contaminated with elevated uranium levels as a resulting of ore extraction or processing.

Fluorescence-based optical techniques are developing rapidly, giving access to high spatiotemporal information on live biological systems with single molecule sensitivity. However, these techniques are typically restricted to expert labs and are not easilyaccessibleto the general user. While the development of customized systems and their wider distribution is difficult, as it requires expert manpower, software developments are easy to distribute. However, in reality only few users outside an expert community are exploring and using these tools. This is due to theusabilityof the software which often requires expert skills to operate and is neither intuitive nor easy to use. These issues of accessibility and usability limit the spread of state-of-the-art techniques. And while accessibility of custom instrumentation is difficult to solve, the accessibility and usability of software is an easier target. In this perspective, therefore, we concentrate on the software issue and examine the major translational barriers that prevent biologists from adopting the available fluorescence microscopy techniques. We discuss key developments in the field such as open-source tools, standardized file formats and AI-driven analysis platforms, and suggest a roadmap to bring advanced tools to a wider community.

Super-resolution microscopy (SRM) has revolutionized fluorescence imaging enabling insights into the molecular organization of cells that were previously unconceivable. Latest developments now allow the visualization of individual molecules with nanometer precision and imaging with molecular resolution. However, translating these achievements to imaging under physiological conditions in cells remains challenging. The higher the spatial resolution is pushed by the development of improved SRM methods the more challenging the problems we are confronted when aiming to use them for sub-10 nm fluorescence imaging in cells. It turns out that most developed SRM methods that demonstrate nanometer resolution cannot be directly implemented for molecular resolution imaging in cells. Particularly, fluorescence labeling, i.e. high-density covalent labeling of the molecules of interest with fluorophores with minimal linkage error represents currently a nearly insurmountable obstacle. In addition, even if high labeling densities can be realized it has to be considered that fluorophores can interact via different energy pathways and thus impede super-resolution imaging in the sub-10 nm range. Here, we describe the boundaries, discuss the challenges we must accept and show strategies to circumvent them and achieve true molecular resolution fluorescence imaging under physiological conditions in cells.

Accurate and efficient autofocusing is essential for the automation of fluorescence microscopy, but background noise and shallow depth of field at high magnifications make autofocusing particularly challenging. Here, we present a fast and accurate autofocus algorithm to address these challenges. It is highly effective for high-magnification imaging, while performing equally well for low-magnification imaging tasks. The method is based on the mountain climbing search algorithm and yields improvements on autofocusing precision of up to 200-fold over current methods, whilst offering competitive speed and greatly extended search ranges. Our approach is broadly applicable: it demonstrated good stability and reproducibility across magnifications ranging from 20X to 100X, excels in both live cell imaging and high-resolution fixed sample imaging, and it is compatible with various microscopy techniques without the need for fiducial markers or hardware modifications on existing microscopes. To maximise its accessibility, we constructed a user-friendly interface compatible with the widely used Micromanager software. It generalises well across various imaging modalities and hardware platforms, making it particularly suitable for use in high-resolution screening of candidate drugs.

Ultraviolet (UV) microscopy is a powerful imaging modality that harnesses the shorter wavelengths of UV light to achieve high-resolution imaging and probe molecular-level chemical and structural properties of biological and biomedical specimens, often without the need for extrinsic labelling. Innovations in technologies such as low-cost illuminators, detectors, and new ways of preparing specimens for imaging have led to a better understanding of complex biological systems. Here we review the latest advances and trends in UV microscopy for applications in the life sciences, including histology, cell biology and haemotology. By examining these developments, we highlight the evolving potential of UV and we conclude by considering the future of this longstanding technique.

Multifocal Structured Illumination Microscopy (MSIM) was initially introduced as a parallel version of image scanning microscopy, aiming to enhance the temporal resolution of the imaging process. Beyond its capacity in super-resolution imaging, MSIM exhibits optical sectioning capabilities akin to confocal microscopy, making it well-suited for imaging thick tissue samples. Traditional MSIM reconstruction algorithms rely on digital pinholes to eliminate out-of-focus signals, demanding precise illumination information. However, controlling and accurately reconstructing illumination patterns can be challenging or impractical in certain experimental settings. To address this, our paper proposes a blind reconstruction method for MSIM that circumvents the need for exact illumination information. Leveraging the stability of the standard deviation for each pixel in illumination, this method achieves optical sectioning effectively and provides approximately 1.76 times better resolution than widefield imaging. The efficacy of our proposed blind reconstruction method for both super-resolution imaging and optical sectioning is validated through both simulations and experimental results.