Background Autofluorescence Research Articles

Thermally Activated Delayed Fluorescence Organic Dots (TADF Odots) for Time‐Resolved and Confocal Fluorescence Imaging in Living Cells and In Vivo

The fluorophores with long‐lived fluorescent emission are highly desirable for time‐resolved fluorescence imaging (TRFI) in monitoring target fluorescence. By embedding the aggregates of a thermally activated delayed fluorescence (TADF) dye, 2,3,5,6‐tetracarbazole‐4‐cyano‐pyridine (CPy), in distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐poly(ethylene glycol) (DSPE‐PEG2000) matrix, CPy‐based organic dots (CPy‐Odots) with a long fluorescence lifetime of 9.3 μs (in water at ambient condition) and high brightness (with an absolute fluorescence quantum efficiency of 38.3%) are fabricated. CPy‐Odots are employed in time‐resolved and confocal fluorescence imaging in living Hela cells and in vivo. The green emission from the CPy‐Odots is readily differentiated from the cellular autofluorescence background because of their stronger emission intensities and longer lifetimes. Unlike other widely studied DSPE‐PEG2000 encapsulated Odots which are always distributed in cytoplasm, CPy‐Odots are located mainly in plasma membrane. In addition, the application of CPy‐Odots as a bright microangiography agent for TRFI in zebrafish is also demonstrated. Much broader application of CPy‐Odots is also prospected after further surface functionalization. Given its simplicity, high fluorescence intensity, and wide availability of TADF materials, the method can be extended to develop more excellent TADF Odots for accomplishing the challenges in future bioimaging applications.

Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine.

The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM) for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

Accurate Quantitative Sensing of Intracellular pH based on Self-ratiometric Upconversion Luminescent Nanoprobe

Accurate quantitation of intracellular pH (pHi) is of great importance in revealing the cellular activities and early warning of diseases. A series of fluorescence-based nano-bioprobes composed of different nanoparticles or/and dye pairs have already been developed for pHi sensing. Till now, biological auto-fluorescence background upon UV-Vis excitation and severe photo-bleaching of dyes are the two main factors impeding the accurate quantitative detection of pHi. Herein, we have developed a self-ratiometric luminescence nanoprobe based on förster resonant energy transfer (FRET) for probing pHi, in which pH-sensitive fluorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively. Under 980 nm excitation, upconversion emission bands at 475 nm and 645 nm of NaYF4:Yb3+, Tm3+ UCNPs were used as pHi response and self-ratiometric reference signal, respectively. This direct quantitative sensing approach has circumvented the traditional software-based subsequent processing of images which may lead to relatively large uncertainty of the results. Due to efficient FRET and fluorescence background free, a highly-sensitive and accurate sensing has been achieved, featured by 3.56 per unit change in pHi value 3.0–7.0 with deviation less than 0.43. This approach shall facilitate the researches in pHi related areas and development of the intracellular drug delivery systems.

Self‐Assembled Dual Dye‐Doped Nanosized Micelles for High‐Contrast Up‐Conversion Bioimaging

Sensitized triplet–triplet annihilation based photon up‐conversion (TTA‐UC) greatly improves the scope and applicability of fluorescence bioimaging by enabling anti‐Stokes detection at low powers, thus eliminating the background autofluorescence and limiting the potential damage of the living tissues. Here the authors present a facile, one‐step protocol to prepare dual dye‐doped, TTA‐UC active nanomicelles starting from the commercially available surfactant Kolliphor EL, a component of several FDA approved preparations. These nanosized micelles show an unprecedented up‐conversion yield of 6.5% under 0.1 W cm−2 excitation intensity in an aqueous, nondeaerated dispersion. The supramolecular architecture obtained preserves the embedded dyes from oxygen quenching, allowing satisfactory anti‐Stokes fluorescence imaging of 3T3 cells. This is the first example of efficient multicomponent up‐converters prepared using highly biocompatible materials approved by the international authority, paving the way for the development of new complex and multifunctional materials for advanced theranostics.

Near-infrared (808 and 980 nm) excited photoluminescence study in Nd-doped Y2O3 phosphor for bio-imaging

The upconversion (UC) process in lanthanide-doped nanophosphors has attracted great research interest for its extensive application potential in biological in vitro and in vivo imaging due to the high tissue penetration depth of near-infrared excitation and low autofluorescence background. In this article, the authors report the synthesis of oxide nanophosphor of size ⩽50 nm, which forms stable aqueous dispersion. The photoluminescence study is made on the nanophosphor upon 808 and 980 nm diode laser excitations. The 808 nm excitation resulted in strong emission at 795 nm due to the 4K13/2 → 4I13/2 transition along with other emissions from the Nd3+ ion. The 980 nm excitation has resulted in it turning green in the 525–560 nm range and is assigned to the 4S3/2 → 4I15/2 transition of the Er3+ ion, which is supposed to be present in a trace amount in the sample. The observation of strong UC emission indicates that the sample can be used for UC-based bio-imaging applications.

Monitoring of nanodiamonds in human urine using artificial neural networks

This paper presents the results of application of the elaborated methods for monitoring of nanodiamonds in human urine using fluorescence spectroscopy. High efficiency of artificial neural networks applied for recognition and estimation of the concentration of nanodiamonds in urine with a strong autofluorescence background is demonstrated. It was found that minimal concentration of nanodiamonds with strong fluorescence containing nitrogen‐vacancy (NV) centers can be detected in urine by fluorescence spectroscopy at a level of 3.06 × 10−4 g L−1; while use of artificial neural networks for detection of weakly fluorescent detonation nanodiamonds provides reasonably high accuracy of detection, not worse than 6.8 × 10−3 g L−1.

Sensitive Time-Gated Immunoluminescence Detection of Prostate Cancer Cells Using a TEGylated Europium Ligand

We describe the application of a synthetically developed tetradentate β-diketonate-europium chelate with high quantum yield (39%), for sensitive immunodetection of prostate cancer cells (DU145). MIL38 antibody, a mouse monoclonal antibody against Glypican 1, conjugated directly to the chelate via lysine residues, resulted in soluble (hydrophilic) and stable immunoconjugates. Indirect labeling of the antibody by a europium chelated secondary polyclonal antibody and a streptavidin/biotin pair was also performed. All of these bright luminescent conjugates were used to stain DU145 cells, a prostate cancer cell line, using time gated luminescence microscopy for imaging, and their performances were compared to conventional FITC labeling. For all prepared conjugates, the europium chelate in conjunction with a gated autosynchronous luminescence detector (GALD) completely suppressed the cellular autofluorescence background to allow capture of vivid, high contrast images of immune-stained cancer cells.

Direct Fluorescent Detection of Blood Potassium by Ion-Selective Formation of Intermolecular G-Quadruplex and Ligand Binding.

G-quadruplex analogues have been widely used as molecular tools for detection of potassium ion (K(+)). However, interference from a higher concentration of sodium ion (Na(+)), enzymatic degradation of the oligonucleotide, and background absorption and fluorescence of blood samples have all limited the use of G-quadruplex for direct detection of K(+) in blood samples. Here, we reported, for the first time, an intermolecular G-quadruplex-based assay capable of direct fluorescent detection of blood K(+). Increased stringency of intermolecular G-quadruplex formation based on our screened G-rich oligonucleotide (5'-TGAGGGA GGGG-3') provided the necessary selectivity for K(+) against Na(+) at physiological ion level. To increase long-term stability of oligonucleotide in blood, the screened oligonucleotide was modified with an inverted thymine nucleotide whose 3'-terminus was connected to the 3'-terminus of the upstream nucleotide, acting as a blocking group to greatly improve antinuclease stability. Lastly, to avoid interference from background absorption and autofluorescence of blood, a G-quadruplex-binding, two-photon-excited ligand, EBMVC-B, was synthesized and chosen as the fluorescence reporter. Thus, based on selective K(+) ion-induced formation of intermolecular G-quadruplex and EBMVC-B binding, this approach could linearly respond to K(+) from 0.5 to 10 mM, which matches quite well with the physiologically relevant concentration of blood K(+). Moreover, the system was highly selective for K(+) against other metal ions, including Na(+), Ca(2+), Mg(2+), Zn(2+) common in blood. The practical application was demonstrated by direct detection of K(+) from real blood samples by two-photon fluorescence technology. To the best of our knowledge, this is the first attempt to exploit molecular G-quadruplex-based fluorescent sensing for direct assay of blood target. As such, we expect that it will promote the design and practical application of similar DNA-based sensors in complex real systems.

Bifunctional Luminomagnetic Rare-Earth Nanorods for High-Contrast Bioimaging Nanoprobes.

Nanoparticles exhibiting both magnetic and luminescent properties are need of the hour for many biological applications. A single compound exhibiting this combination of properties is uncommon. Herein, we report a strategy to synthesize a bifunctional luminomagnetic Gd2−xEuxO3 (x = 0.05 to 0.5) nanorod, with a diameter of ~20 nm and length in ~0.6 μm, using hydrothermal method. Gd2O3:Eu3+ nanorods have been characterized by studying its structural, optical and magnetic properties. The advantage offered by photoluminescent imaging with Gd2O3:Eu3+ nanorods is that this ultrafine nanorod material exhibits hypersensitive intense red emission (610 nm) with good brightness (quantum yield more than 90%), which is an essential parameter for high-contrast bioimaging, especially for overcoming auto fluorescent background. The utility of luminomagnetic nanorods for biological applications in high-contrast cell imaging capability and cell toxicity to image two human breast cancer cell lines T47D and MDA-MB-231 are also evaluated. Additionally, to understand the significance of shape of the nanostructure, the photoluminescence and paramagnetic characteristic of Gd2O3:Eu3+ nanorods were compared with the spherical nanoparticles of Gd2O3:Eu3+.

Sulfone-Rhodamines: A New Class of Near-Infrared Fluorescent Dyes for Bioimaging.

Given the wavelength dependence of tissue transparency and the requirement for sufficiently low background autofluorescence, the development of fluorescent dyes with excitation and emission maxima beyond 700 nm is highly desired, but it is a challenging task. Herein, a new class of fluorescent dyes, named sulfone-rhodamines (SO2Rs), was developed on the basis of the one-atom replacement of the rhodamine 10-position O atom by a sulfone group. Such a modification makes their absorption and emission maxima surprisingly reach up to 700-710 and 728-752 nm, respectively, much longer than their O-, C-, and Si-rhodamine analogs, due to the unusual d*-π* conjugation. Among these dyes, SO2R4 and SO2R5, bearing disubstituted meso-phenyl groups, show the greatest potentials for bioimaging applications in view of their wide pH range of application, high photostability, and big extinction coefficients and fluorescence quantum yields. They could quickly penetrate cells to give stable NIR fluorescence, even after continuous irradiation by a semiconductor laser, making them suitable candidates for time-lapse and long-term bioimaging applications. Moreover, they could specifically localize in lysosomes independent of alkylmorpholine targeted group, thus avoiding the problematic alkalization effect suffered by most LysoTrackers. Further imaging assays of frozen slices of rat kidney reveal that their tissue imaging depth is suprior to the widely used NIR labeling agent Cy5.5.

Super-resolution spectroscopic microscopy via photon localization.

Traditional photon localization microscopy analyses only the spatial distributions of photons emitted by individual molecules to reconstruct super-resolution optical images. Unfortunately, however, the highly valuable spectroscopic information from these photons have been overlooked. Here we report a spectroscopic photon localization microscopy that is capable of capturing the inherent spectroscopic signatures of photons from individual stochastic radiation events. Spectroscopic photon localization microscopy achieved higher spatial resolution than traditional photon localization microscopy through spectral discrimination to identify the photons emitted from individual molecules. As a result, we resolved two fluorescent molecules, which were 15 nm apart, with the corresponding spatial resolution of 10 nm—a four-fold improvement over photon localization microscopy. Using spectroscopic photon localization microscopy, we further demonstrated simultaneous multi-colour super-resolution imaging of microtubules and mitochondria in COS-7 cells and showed that background autofluorescence can be identified through its distinct emission spectra.

A near-infrared fluorescent probe for monitoring fluvastatin-stimulated endogenous H2S production

Most reported fluorescent probes have limitations in practical applications in living systems due to the strong autofluorescence background, construction of probes with near-infrared (NIR) fluorescence emission is an accessible approach for addressing this challenge. We here designed a NIR fluorescent probe for monitoring the endogenous production of H2S in living cells. The designed probe showed significant NIR fluorescence turn-on response to H2S with high selectivity, enabling the sensitive detection H2S. Importantly, the probe could be applied in monitoring the endogenous production of H2S in raw264.7 macrophages. This study showed that fluvastatin can promote the activity of cystathionine γ-lyase (CSE) for generation H2S.

Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.

Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas – from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH.

Three-Dimensional Segmentation and Quantitative Measurement of the Aqueous Outflow System of Intact Mouse Eyes Based on Spectral Two-Photon Microscopy Techniques

PurposeTo visualize and quantify the three-dimensional (3D) spatial relationships of the structures of the aqueous outflow system (AOS) within intact enucleated mouse eyes using spectral two-photon microscopy (TPM) techniques.MethodsSpectral TPM, including two-photon autofluorescence (TPAF) and second-harmonic generation (SHG), were used to image the small structures of the AOS within the limbal region of freshly enucleated mouse eyes. Long infrared excitation wavelengths (930 nm) were used to reduce optical scattering and autofluorescent background. Image stacks were collected for 3D image rendering and structural segmentation. For anatomical reference, vascular perfusion with fluorescent-conjugated dextran (150 KDa) and trans-corneal perfusion with 0.1 μm fluorescent polystyrene beads were separately performed to identify the episcleral veins (EV) and the trabecular meshwork (TM) and Schlemm's canal (SC), respectively.ResultsThree-dimensional rendering and segmentation of spectral two-photon images revealed detailed structures of the AOS, including SC, collector channels (CC), and aqueous veins (AV). The collagen of the TM was detected proximal to SC. The long and short axes of the SC were 82.2 ± 22.2 μm and 6.7 ± 1.4 μm. The diameters of the CC averaged 25.6 ± 7.9 μm where they originated from the SC (ostia), enlarged to 34.1 ± 13.1 μm at the midpoint, and narrowed to 18.3 ± 4.8 μm at the junction of the AV. The diameter of the AV averaged 12.5 ± 3.4 μm.ConclusionsSpectral TPM can be used to reconstruct and measure the spatial relationships of both large and small AOS structures, which will allow for better understanding of distal aqueous outflow dynamics.

Wide-field time-gated photoluminescence microscopy for fast ultrahigh-sensitivity imaging of photoluminescent probes.

Fluorescence microscopy is a fundamental technique for the life sciences, where biocompatible and photostable photoluminescence probes in combination with fast and sensitive imaging systems are continually transforming this field. A wide-field time-gated photoluminescence microscopy system customised for ultrasensitive imaging of unique nanoruby probes with long photoluminescence lifetime is described. The detection sensitivity derived from the long photoluminescence lifetime of the nanoruby makes it possible to discriminate signals from unwanted autofluorescence background and laser backscatter by employing a time-gated image acquisition mode. This mode enabled several-fold improvement of the photoluminescence imaging contrast of discrete nanorubies dispersed on a coverslip. It enabled recovery of the photoluminescence signal emanating from discrete nanorubies when covered by a layer of an organic fluorescent dye, which were otherwise invisible without the use of spectral filtering approaches. Time-gated imaging also facilitated high sensitivity detection of nanorubies in a biological environment of cultured cells. Finally, we monitor the binding kinetics of nanorubies to a functionalised substrate, which exemplified a real-time assay in biological fluids. 3D-pseudo colour images of nanorubies immersed in a highly fluorescent dye solution. Nanoruby photoluminescence is subdued by that of the dye in continuous excitation/imaging (left), however it can be recovered by time-gated imaging (right). At the bottom is schematic diagram of nanoruby assay in a biological fluid.

What to do with high autofluorescence background in pancreatic tissues – an efficient Sudan black B quenching method for specific immunofluorescence labelling

High levels of autofluorescence in tissue samples can entirely mask specific labellings with fluorophores and thus impair immunofluorescence histochemistry. In pancreatic tissue samples we observed autofluorescence as a common problem often mediated by the fixation and processing procedure. Using epifluorescence microscopy, we analysed the intensity and spatial distribution of autofluorescence in formalin-fixed, paraffin-embedded human pancreatic tissues and developed an efficient quenching method to reduce the unwanted light emission. The optimized quenching protocol using Sudan black B reduced the unequally distributed tissue autofluorescence to a low and intensity-equalized background level. Quantitative image analysis demonstrated autofluorescence suppression by 65-95%, depending on the selected fluorescence filter setups. The procedure did not affect specific immunofluorescence labelling or tissue integrity. As a clear result of Sudan black B treatment, a tremendous improvement of the signal-to-noise ratio was achieved, allowing a reliable detection and quantification of specific fluorescent labels. Other tissue treatment methods, such as cupric sulphate, toluidine blue and ultraviolet irradiation, or combinations of these with Sudan black B, were not as efficient. The easy-to-perform Sudan black B technique improves dramatically qualitative and quantitative fluorescence analysis of critical pancreatic tissue sections and rescues even overfixed tissues for immunofluorescence application.

Phosphorescent Polymeric Thermometers for In Vitro and In Vivo Temperature Sensing with Minimized Background Interference

Temperature plays a crucial role in many biological processes. Accurate temperature determination is important for diagnosis and treatment of diseases. Autofluorescence is an unavoidable interference in luminescent bioimaging. Hence, a large amount of research works has been devoted to reducing background autofluorescence and improving signal‐to‐noise ratio (SNR) in biodetection. Herein, a dual‐emissive phosphorescent polymeric thermometer has been developed by incorporating two long‐lived phosphorescent iridium(III) complexes into an acrylamide‐based thermosensitive polymer. Upon increasing temperature, this polymer undergoes coil‐globule transition, which leads to a decrease in polarity of the microenvironment surrounding the iridium(III) complexes and hence brings about emission enhancement of both complexes. Owing to their different sensitivity to surrounding environment, the emission intensity ratio of the two complexes is correlated to the temperature. Thus, the polymer has been used for temperature determination in vitro and in vivo via ratiometric luminescence imaging. More importantly, by using the long‐lived phosphorescence of the polymer, temperature mapping in zebrafish has been demonstrated successfully with minimized autofluorescence interference and improved SNR via time‐resolved luminescence imaging. To the best of our knowledge, this is the first example to use photoluminescent thermometer for in vivo temperature sensing.

Target/Background Classification Regularized Nonnegative Matrix Factorization for Fluorescence Unmixing

Nonnegative matrix factorization (NMF) is usually applied to multispectral fluorescence imaging for fluorescence unmixing. Unfortunately, most NMF-based fluorescence unmixing methods fail to take advantage of spatial information in data. Besides, NMF is an inherently ill-posed problem, which gets worse in the sparse acquisition of multispectral data (from a small number of spectral bands) due to its insufficient measurements and severe discontinuities in spectral emissions. To overcome these limitations by exploiting the spatial difference between multiple-target fluorophores and background autofluorescence (AF), we propose improved normalized cut to automatically classify all multispectral pixels into target fluorophores and background AF groups. We then initialize NMF by extracting the endmember spectra of target/background fluorescent components in the two groups, and impose a $L_{1/2}$ -norm partial sparseness constraint on merely the abundances of target fluorophores within hierarchical alternating least squares framework of NMF. Experimental results based on synthetic and in vivo fluorescence data show the superiority of the proposed algorithm with respect to other state-of-the-art approaches.

Penetrating Peptide-Bioconjugated Persistent Nanophosphors for Long-Term Tracking of Adipose-Derived Stem Cells with Superior Signal-to-Noise Ratio.

Reliable long-term in vivo tracking of stem cells is of great importance in stem cell-based therapy and research. Fluorescence imaging with in situ excitation has significant autofluorescence background, which results in poor signal-to-noise ratio (SNR). Here we report TAT penetrating peptide-bioconjugated long persistent luminescence nanoparticles (LPLNP-TAT) for long-term tracking of adipose-derived stem cells (ASC) without constant external excitation. LPLNP-TAT exhibits near-infrared emitting, red light renewable capability, and superior in vivo imaging depth and SNR compared with conventional organic dye and quantum dots. Our findings show that LPLNP-TAT can successfully label ASC without impairing their proliferation and differentiation and can effectively track ASC in skin-regeneration and tumor-homing models. We believe that LPLNP-TAT represents a new generation of cell tracking probes and will have broad application in diagnosis and therapy.

A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices.

Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation.