Saturated Fluorescence Research Articles

Modern Methods of Fluorescence Nanoscopy in Biology

Optical microscopy has undergone significant changes in recent decades due to the breaking of the diffraction limit of optical resolution and the development of high-resolution imaging techniques, which are collectively known as fluorescence nanoscopy. These techniques allow researchers to observe biological structures and processes at a nanoscale level of detail, revealing previously hidden features and aiding in answering fundamental biological questions. Among the advanced methods of fluorescent nanoscopy are: STED (Stimulated Emission Depletion Microscopy), STORM (STochastic Optical Reconstruction Microscopy), PALM (Photo-activated Localization Microscopy), TIRF (Total Internal Reflection Fluorescence), SIM (Structured Illumination Microscopy), MINFLUX (Minimal Photon Fluxes), PAINT (Points Accumulation for Imaging in Nanoscale Topography) и RESOLFT (REversible Saturable Optical Fluorescence Transitions) and others. In addition, most of these methods make it possible to obtain volumetric (3D) images of the objects under study. In this review, we will look at the principles of these methods, their advantages and disadvantages, and their application in biological researches.

Parallel illumination for depletion microscopy through acousto-optic spatial light modulation

Several types of super-resolution microscopy techniques, such as Stimulated Emission Depletion (STED), Reversible Saturable Optical Fluorescence Transitions (RESOLFT) or Switching Laser Mode (SLAM) microscopies, employ Laguerre-Gaussian beams (also called vortex or doughnut beams) to obtain fluorescence information within a sub-wavelength region of the specimen under observation, thus breaking the diffraction limit and producing images of greatly improved quality. However, in general, these techniques operate on a point-by-point basis, so scanning the sample in order to build a full, meaningful image, takes time. Parallelization of the illumination is the only way to make these microscopy techniques more suitable for real-time live cell imaging applications. Here, we demonstrate the parallel production of arbitrary arrays of Gaussian and Laguerre-Gaussian lasers foci suitable for super-resolution microscopy, together with the possibility to fast scan through the sample, by means of acousto-optic spatial light modulation, a technique that we have pioneered in the past in several other fields. Parallelized illumination with both Gaussian and doughnut beams will be then used to acquire super-resolution images.

Fluorescence saturation imaging microscopy: molecular fingerprinting with a standard confocal microscope.

Molecular specificity in fluorescence imaging of cells and tissues can be increased by measuring parameters other than intensity. For instance, fluorescence lifetime imaging became a widespread modality for biomedical optics. Previously, we suggested using the fluorescence saturation effect at pulsed laser excitation to map the absorption cross-section as an additional molecular contrast in two-photon microscopy [Opt. Lett.47(17), 4455 (2022).10.1364/OL.465605]. Here, it is shown that, somewhat counterintuitive, fluorescence saturation can be observed under cw excitation in a standard confocal microscopy setup. Mapping the fluorescence saturation parameter allows obtaining additional information about the fluorophores in the system, as demonstrated by the example of peptide hydrogel, stained cells and unstained thyroid gland. The suggested technique does not require additional equipment and can be implemented on confocal systems as is.

Activatable near-infrared fluorescence and chemical exchange saturation transfer MRI multimodal imaging probe for tumor detection in vitro and in vivo

Multimodal imaging has emerged as a powerful tool in biomedical research and clinical diagnostics. Ideally, it combines multiple imaging techniques to provide complementary anatomical and molecular information in living subjects. Particularly desirable are multimodal imaging probes capable of providing differential diagnostic signals upon interaction with specific molecular targets. Hydrogen peroxide (H2O2) is a key target in this regard, since it is typically overexpressed in cancer cells. In this study, we present a small-molecule probe that not only selectively detects endogenous H2O2 through multimodal imaging, with a significant H2O2-triggered 15-fold fluorescence enhancement but also turns “on” a chemical exchange saturation transfer (CEST) magnetic resonance (MR) response with 60-fold signal enhancement at pH 7.4. Excellent selectivity against various other biologically relevant species is seen. Using this probe, we observed 3.4–4.5-fold and 2.8–5.8-fold higher H2O2 levels in cancerous cell lines and tumor tissues compared to normal cell lines and tissues, respectively. Time-dependent in vivo fluorescence and CEST imaging in a HeLa (Henrietta Lacks) tumor xenograft mouse model revealed probe-dependent tumor detection by fluorescence and CEST MRI contrast in the tumor area. These observations are attributed to the relatively high endogenous H2O2 levels produced during mitosis. This newly developed probe holds promise for advancing our understanding of H2O2-related biology and for cancer detection both in vitro and in vivo.

Numerical study of transient absorption saturation in single-layer graphene for optical nanoscopy applications

Transient absorption, or pump–probe microscopy is an absorption-based technique that can explore samples ultrafast dynamic properties and provide fluorescence-free contrast mechanisms. When applied to graphene and its derivatives, this technique exploits the graphene transient response caused by the ultrafast interband transition as the imaging contrast mechanism. The saturation of this transition is fundamental to allow for super-resolution optical far-field imaging, following the reversible saturable optical fluorescence transitions (RESOLFT) concept, although not involving fluorescence. With this aim, we propose a model to numerically compute the temporal evolution under saturation conditions of the single-layer graphene molecular states, which are involved in the transient absorption. Exploiting an algorithm based on the fourth order Runge–Kutta (RK4) method, and the density matrix approach, we numerically demonstrate that the transient absorption signal of single-layer graphene varies linearly as a function of excitation intensity until it reaches saturation. We experimentally verify this model using a custom pump–probe super-resolution microscope. The results define the intensities necessary to achieve super-resolution in a pump–probe nanoscope while studying graphene-based materials and open the possibility of predicting such a saturation process in other light-matter interactions that undergo the same transition.

Super-sectioning with multi-sheet reversible saturable optical fluorescence transitions (RESOLFT) microscopy

Light-sheet fluorescence microscopy is an invaluable tool for four-dimensional biological imaging of multicellular systems due to the rapid volumetric imaging and minimal illumination dosage. However, it is challenging to retrieve fine subcellular information, especially in living cells, due to the width of the sheet of light (>1 μm). Here, using reversibly switchable fluorescent proteins (RSFPs) and a periodic light pattern for photoswitching, we demonstrate a super-resolution imaging method for rapid volumetric imaging of subcellular structures called multi-sheet RESOLFT. Multiple emission-sheets with a width that is far below the diffraction limit are created in parallel increasing recording speed (1–2 Hz) to provide super-sectioning ability (<100 nm). Our technology is compatible with various RSFPs due to its minimal requirement in the number of switching cycles and can be used to study a plethora of cellular structures. We track cellular processes such as cell division, actin motion and the dynamics of virus-like particles in three dimensions.

Sol-gel combustion synthesis and near-infrared luminescence of Ni2+-doped MgAl2O4 spinel phosphor

Near-infrared phosphor converted light-emitting diode (NIR pc-LED) has many advantages over traditional NIR light sources, and has broad application prospects in night vision surveillance, non-destructive testing, bioimaging, and other fields. Phosphors are typically synthesized by the high-temperature solid-phase method as chunks or large granules, which need to be ground to small particles for practical application. This process introduces surface defects and reduces the luminous efficiency of phosphors. In this work, near-infrared phosphors composed of spinel MgAl2O4:Ni2+ with particle size less than 100 nm was synthesized in high-purity by sol-gel combustion method using nitrate salts with citric acid were used as raw materials. Under 365 nm excitation, MgAl2O4:Ni2+ phosphors emit broadband near-infrared luminescence of 1000–1600 nm with a width at half maximum of 237 nm. The optimal Ni2+ doping concentration was determined to be 1.5 mol% and the optimal sintering temperature was 1200 °C. The thermal activation energy of this phosphor was found to be 0.409 eV and the phosphor had good luminescence thermal stability. An NIR pc-LED device was assembled from the phosphor and commercially available near-ultraviolet LED, which showed a current-dependent increased of NIR luminescence up to 160 mA and an efficiency drop appeared with further increase of the current, suggesting a thermal dependent quenching may have occurred. On the other hand, the output of NIR luminescence increased nearly linearly with the increased of current when a 360 nm laser diode was used as the excitation light source, indicating the NIR phosphor has a very high fluorescence saturation threshold. Our study provides the utility for the sol-gel method in the preparation and the potential application of the nanoscale NIR-II phosphor materials.

Experimental Verification of the Accuracy of Oxygen-Varying Correlated Fluorescence for Determining the Quenching Constant KSV.

The oxygen-varying correlated fluorescence provides an effective strategy to gain the quenching constant KSV for a non-phosphorescent system. Owing to the absence of detectable phosphorescence emission in general optical spectroscopy, the facticity of this method has never been verified. Here, the correct way to determine KSV by oxygen-varying correlated fluorescence was systematically studied in detail. We selected gadolinium protoporphyrin IX (Gd-PpIX) to establish a fluorescence and phosphorescence dual emission system. Then, KSV of Gd-PpIX can be obtained by the change of fluorescence intensity (IF) with oxygen concentration using the method of correlated fluorescence at intense pump power; meanwhile, the value can also be determined from the relationship of the phosphorescence intensity ratio and oxygen content by the Stern-Volmer equation under relatively low power density. It was found that the KSV values obtained by the above two methods were 12.2(1) and 11.9(7) kPa-1, respectively. Our results successfully verified the accuracy of oxygen-varying correlated fluorescence for determining the KSV by the phosphorescence property and fluorescence saturation of Gd-PpIX.

Super-resolution computational saturated absorption microscopy.

Imaging beyond the diffraction limit barrier has attracted wide attention due to the ability to resolve previously hidden image features. Of the various super-resolution microscopy techniques available, a particularly simple method called saturated excitation microscopy (SAX) requires only simple modification of a laser scanning microscope: The illumination beam power is sinusoidally modulated and driven into saturation. SAX images are extracted from the harmonics of the modulation frequency and exhibit improved spatial resolution. Unfortunately, this elegant strategy is hindered by the incursion of shot noise that prevents high-resolution imaging in many realistic scenarios. Here, we demonstrate a technique for super-resolution imaging that we call computational saturated absorption (CSA) in which a joint deconvolution is applied to a set of images with diversity in spatial frequency support among the point spread functions (PSFs) used in the image formation with saturated laser scanning fluorescence microscopy. CSA microscopy allows access to the high spatial frequency diversity in a set of saturated effective PSFs, while avoiding image degradation from shot noise.

Double-stemmed and split structural variants of fluorescent RNA Mango aptamers.

Aptamers with fluorogenic ligands are emerging as useful tools to quantify and track RNA molecules. The RNA Mango family of aptamers have a useful combination of tight ligand binding, bright fluorescence and small size. However, the simple structure of these aptamers, with a single base-paired stem capped by a G-quadruplex, can limit the sequence and structural modifications needed for many use-inspired designs. Here we report new structural variants of RNA Mango that have two base-paired stems attached to the quadruplex. Fluorescence saturation analysis of one of the double-stemmed constructs showed a maximum fluorescence that is ~75% brighter than the original single-stemmed Mango I. A small number of mutations to nucleotides in the tetraloop-like linker of the second stem were subsequently analyzed. The effect of these mutations on the affinity and fluorescence suggested that the nucleobases of the second linker do not directly interact with the fluorogenic ligand (TO1-biotin), but may instead induce higher fluorescence by indirectly altering the ligand properties in the bound state. The effects of the mutations in this second tetraloop-like linker indicate the potential of this second stem for rationale design and reselection experiments. Additionally, we demonstrated that a bimolecular mango designed by splitting the double-stemmed Mango can function when two RNA molecules are co-transcribed from different DNA templates in a single in vitro transcription. This bimolecular Mango has potential application in detecting RNA-RNA interactions. Together, these constructs expand the designability of the Mango aptamers to facilitate future applications of RNA imaging.

Achieving High Temporal Resolution in Single‐Molecule Fluorescence Techniques Using Plasmonic Nanoantennas

AbstractSingle‐molecule fluorescence techniques are essential for investigating the molecular mechanisms in biological processes. However, achieving sub‐millisecond temporal resolution to monitor fast molecular dynamics remains a significant challenge. The fluorescence brightness is the key parameter that generally defines the temporal resolution for these techniques. Conventional microscopes and standard fluorescent emitters fall short in achieving the high brightness required for sub‐millisecond monitoring. Plasmonic nanoantennas are proposed as a solution, but despite huge fluorescence enhancement having been obtained with these structures, the brightness generally remains below 1 million photons/s/molecule. Therefore, the improvement of temporal resolution is overlooked. This article presents a method for achieving high temporal resolution in single‐molecule fluorescence techniques using plasmonic nanoantennas, specifically optical horn antennas. This work demonstrates about 90% collection efficiency of the total emitted light, reaching a high fluorescence brightness of 2 million photons/s/molecule in the saturation regime. This enables observations of single molecules with microsecond binning time and fast fluorescence correlation spectroscopy measurements. This work expands the applications of plasmonic antennas and zero‐mode waveguides in the fluorescence saturation regime toward brighter single‐molecule signal, faster temporal resolutions, and improved detection rates to advance fluorescence sensing, DNA sequencing, and dynamic studies of molecular interactions.

An open-source microscopy framework for simultaneous control of image acquisition, reconstruction, and analysis

We present a computational framework to simultaneously perform image acquisition, reconstruction, and analysis in the context of open-source microscopy automation. The setup features multiple computer units intersecting software with hardware devices and achieves automation using python scripts. In practice, script files are executed in the acquisition computer and can perform any experiment by modifying the state of the hardware devices and accessing experimental data. The presented framework achieves concurrency by using multiple instances of ImSwitch and napari working simultaneously. ImSwitch is a flexible and modular open-source software package for microscope control, and napari is a multidimensional image viewer for scientific image analysis.The presented framework implements a system based on file watching, where multiple units monitor a filesystem that acts as the synchronization primitive. The proposed solution is valid for any microscope setup, supporting various biological applications. The only necessary element is a shared filesystem, common in any standard laboratory, even in resource-constrained settings. The file watcher functionality in Python can be easily integrated into other python-based software.We demonstrate the proposed solution by performing tiling experiments using the molecular nanoscale live imaging with sectioning ability (MoNaLISA) microscope, a high-throughput super-resolution microscope based on reversible saturable optical fluorescence transitions (RESOLFT).

Quantifying protein-protein interactions using confined photoconversion and single-particle tracking.

Single particle tracking (SPT) has become a powerful tool to investigate the dynamics of single molecules in live cells. Typical SPT measurement require sparsely labeled molecules in order to identify and localize the individual molecules. This low density limits the rate at which diffusion-limited reactions can be observed. We are developing a novel technique that increases the rate of observable interactions by two orders of magnitude. We call this method SPT-REversible Saturable OpticaL Fluorescence Transitions (SPT-RESOLFT) because it uses a concept from the RESOLFT super-resolution method. The concept relies on saturated labeling and selectively imaging proteins of interest within roughly 100 nm spot. Fluorophores are activated using diffraction-limited Gaussian beam and deactivated outside of 100 nm by a Laguerre-Gauss donut beam of (p, l) = (0, 2) mode which corresponds to topological charge of 2. We experimentally demonstrate the concept of SPT-RESOLFT using EGFR-mEos4b, which form dimers with ∼1 s lifetimes, and with the gamma subunit of FcεRI, gamma-mEos4b, which forms stable dimers.

Ultra-high efficiency green-emitting LuAG: Ce phosphor-in-ceramic applied for high-power laser lighting

Phosphor-in-ceramic (PiC) has garnered considerable attention in high-power white laser lighting owing to its advantages, such as high efficiency and good thermal stability. In this study, a transparent hydroxyapatite (HA) ceramic was successfully prepared via spark-plasma sintering (SPS) under relatively mild conditions. A series of ultra-high efficiency green-emitting PiCs were obtained by pure HA ceramics with 1–12% Lu3Al5O12: Ce (LuAG) phosphors. Owing to the rapid sintering of SPS and the stability of HA ceramics, HA ceramic matrix has high transparency, and does not damage the crystal structure of the LuAG phosphor. When excited by a blue laser, the HA-LuAG PiC exhibited an excellent luminous efficacy of 300.48 lm·W−1. The fluorescence saturation threshold of the laser power was approximately 7.90 W. These results demonstrate that HA-LuAG PiCs have great potential for applications as efficient green-emitting phosphor conversion materials for high-power laser lighting.

Parallel illumination for depletion microscopy through acousto-optic spatial light modulation

Several types of super-resolution microscopy, such as Stimulated Emission Depletion (STED), Reversible Saturable Optical Fluorescence Transitions (RESOLFT) or Switching Laser Mode (SLAM) microscopies, employ Laguerre-Gaussian beams (also called vortex or doughnut beams) to obtain fluorescence information within a sub-wavelength region of the specimen under observation, thus breaking the diffraction limit and producing images of greatly improved quality. However, in general, these techniques operate on a point-by-point basis, so we need to raster scan the sample in order to build a full, meaningful image, which takes time. Parallelization of the illumination is the only way to make these microscopies more suitable for live cell imaging applications. Here, we demonstrate the parallel production of arbitrary arrays of Gaussian and Laguerre-Gaussian lasers foci suitable for super-resolution microscopy, together with the possibility to fast scan through the sample, by means of acousto-optic spatial light modulation, a technique that we have pioneered in the past in several other fields.

Multi-foci parallelised RESOLFT nanoscopy in an extended field-of-view.

Live-cell imaging of biological structures at high resolution poses challenges in the microscope throughput regarding area and speed. For this reason, different parallelisation strategies have been implemented in coordinate- and stochastic-targeted switching super-resolution microscopy techniques. In this line, the molecular nanoscale live imaging with sectioning ability (MoNaLISA), based on reversible saturable optical fluorescence transitions (RESOLFT), offers resolution of large fields of view in a few seconds. In MoNaLISA, engineered light patterns strategically confine the fluorescence to sub-diffracted volumes in a large area and provide optical sectioning, thus enabling volumetric imaging at high speeds. The optical setup presented in this paper extends the degree of parallelisation of the MoNaLISA microscope by more than four times, reaching a field-of-view of . We set up the periodicity and the optical scheme of the illumination patterns to be power-efficient and homogeneous. In a single recording, this new configuration enables super-resolution imaging of an extended population of the post-synaptic density protein Homer1c in living hippocampal neurons.

Self-aligned patterning technique for fabricating high-performance diamond sensor arrays with nanoscale precision.

Efficient, nanoscale precision alignment of defect center creation in photonics structures in challenges the realization of high-performance photonic devices and quantum technology applications. Here, we propose a facile self-aligned patterning technique based on conventional engineering technology, with doping precision that can reach ~15 nm. We demonstrate this technique by fabricating diamond nanopillar sensor arrays with high consistency and near-optimal photon counts. The sensor array achieves high yield approaching the theoretical limit, and high efficiency for filtering sensors with different numbers of nitrogen vacancy centers. Combined with appropriate crystal orientation, the system achieves a saturated fluorescence rate of 4.34 Mcps and effective fluorescence-dependent detection sensitivity of 1800 cps−1/2 . These sensors also show enhanced spin properties in the isotope-enriched diamond. Our technique is applicable to all similar solid-state systems and could facilitate the development of parallel quantum sensing and scalable information processing.

Fluorescence saturation imaging microscopy: molecular fingerprinting in living cells using two-photon absorption cross section as a contrast mechanism.

Imaging of molecular-specific photophysical parameters such as fluorescence intensity, emission band shape, or fluorescence decay is widely used in biophysics. Here we propose a method for quantitative mapping of another molecular-specific parameter in living cells, two-photon absorption cross section, based on the fluorescence saturation effect. Using model dye solutions and cell culture, we show that the analysis of the fluorescence signal dependencies on the intensity of two-photon excitation within the range typical for routine two-photon microscopy experiments allows one to reconstruct two-photon absorption cross section maps across the sample. We believe that the absorption cross section contrast visualized by the proposed fluorescence saturation imaging microscopy could be a new tool for studying processes in living cells and tissues.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Supramolecular Complex of Photochromic Diarylethene and Cucurbit[7]uril: Fluorescent Photoswitching System for Biolabeling and Imaging.

Photoswitchable fluorophores—proteins and synthetic dyes—whose emission is reversibly switched on and off upon illumination, are powerful probes for bioimaging, protein tracking, and super-resolution microscopy. Compared to proteins, synthetic dyes are smaller and brighter, but their photostability and the number of achievable switching cycles in aqueous solutions are lower. Inspired by the robust photoswitching system of natural proteins, we designed a supramolecular system based on a fluorescent diarylethene (DAE) and cucurbit[7]uril (CB7) (denoted as DAE@CB7). In this assembly, the photoswitchable DAE molecule is encapsulated by CB7 according to the host–guest principle, so that DAE is protected from the environment and its fluorescence brightness and fatigue resistance in pure water improved. The fluorescence quantum yield (Φfl) increased from 0.40 to 0.63 upon CB7 complexation. The photoswitching of the DAE@CB7 complex, upon alternating UV and visible light irradiations, can be repeated 2560 times in aqueous solution before half-bleaching occurs (comparable to fatigue resistance of the reversibly photoswitchable proteins), while free DAE can be switched on and off only 80 times. By incorporation of reactive groups [maleimide and N-hydroxysuccinimidyl (NHS) ester], we prepared bioconjugates of DAE@CB7 with antibodies and demonstrated both specific labeling of intracellular proteins in cells and the reversible on/off switching of the probes in cellular environments under irradiations with 355 nm/485 nm light. The bright emission and robust photoswitching of DAE-Male3@CB7 and DAE-NHS@CB7 complexes (without exclusion of air oxygen and addition of any stabilizing/antifading reagents) enabled confocal and super-resolution RESOLFT (reversible saturable optical fluorescence transitions) imaging with apparent 70–90 nm optical resolution.