Suppression Of Autofluorescence Research Articles

Quantitative investigation of photobleaching-based autofluorescence suppression in formalin-fixed paraffin-embedded human tissue

Immunofluorescence (IF) is a crucial technique in histopathology, enabling the visualization of multiple antibody distributions within single tissue specimens. However, autofluorescence (AF), which originates from endogenous molecules in formalin-fixed paraffin-embedded (FFPE) tissue, poses a persistent challenge by interfering with the fluorescence signal of interest in IF analysis. While photo-irradiative bleaching has emerged as a protocol to suppress the AF signal, there have been minimal quantitative investigations into this method across various experimental conditions. In this study, we investigated the efficacy of a bleaching-based method for reducing AF in FFPE human tissues. This research analyzed AF intensity as a function of exposure time, deparaffinization, emission range, and tissue types. Furthermore, by employing fluorescence lifetime imaging microscopy, we isolated the IF signal from AF to facilitate accurate quantification. This study provides valuable insights into AF reduction methodology to improve the reliability of IF-based histopathological analyses and advance our understanding of tissue biology and pathology.

FRET-Based Nanoprobe with Adaptive Background Suppression for Reliable Detection of ONOO-/ClO- in Whole Blood: Facilitating Monitoring of Sepsis Progression and Hemolytic Disorders.

Abnormal fluctuations in blood biomarker levels serve as critical indicators of the disease. However, detecting endogenous substances in whole blood using fluorescent probes is challenging due to its complex composition. This challenge primarily arises from two factors: the high autofluorescence of whole blood and the intrinsic fluorescence of the probe, both contributing to significant background fluorescence in the detection system. To overcome these obstacles, we introduced a donor-acceptor "one-to-many" FRET-based sensing strategy integrated with blood autofluorescence suppression to design a multifunctional fluorescent nanoprobe. The donor effectively suppresses blood autofluorescence through the inner filter effect and efficiently quenches donor fluorescence by adjusting the acceptor-to-donor ratio, achieving a "zero" background in whole blood detection. Leveraging this excellent background fluorescence quenching effect, we successfully detected endogenous ONOO- and ClO- levels in whole blood samples from mice with sepsis or hemolytic diseases. Furthermore, we monitored the changes in the ONOO- and ClO- levels throughout the disease course, revealing a positive correlation between the ONOO- and ClO- concentrations and disease severity. This innovative sensing strategy for achieving a "zero" background in whole blood detection provides valuable insights for designing fluorescent probes to directly detect endogenous substances in whole blood.

Management of autofluorescence in formaldehyde-fixed myocardium: choosing the right treatment.

Autofluorescence (AF) poses challenges for detecting proteins of interest in situ when employing immunofluorescence (IF) microscopy. This interference is particularly pronounced in strongly autofluorescent tissues such as myocardium, where tissue AF can be comparable to IF. Although various histochemical methods have been developed to achieve effective AF suppression in different types of tissue, their applications on myocardial samples have not been well validated. Due to inconsistency across different autofluorescent structures in sometypes of tissue, it is unclear if these methods can effectively suppress AF across all autofluorescent structures within the myocardium. Here, we quantitatively evaluated the performance of several commonly used quenching treatments on formaldehyde-fixed myocardial samples, including 0.3 M glycine, 0.3% Sudan Black B (SBB), 0.1% and 1% sodium borohydride (NaBH4), TrueVIEW® and TrueBlack®. We further assessed their quenching performance by employing the pre-treatment and post-treatment protocols, designed to cover two common IF staining scenarios where buffers contained detergents or not. The results suggest that SBB and TrueBlack® outperform other reagents in AF suppression on formaldehyde-fixed myocardial samples in both protocols. Furthermore, we inspected the quenching performance of SBB and TrueBlack® on major autofluorescent myocardial structures and evaluated their influence on IF imaging. The results suggest that SBB outperforms TrueBlack® in quenching major autofluorescent structures, while TrueBlack® excels in preserving IF labeling signal. Surprisingly, we found the treatment of NaBH4 increased AF signal and enhanced the AF contrast of major autofluorescent structures. This finding suggests that NaBH4 has the potential to act as an AF enhancer and may facilitate the interpretation of myocardial structures without the need for counterstaining.

Suppression of auto-fluorescence from high-resolution 3D polymeric architectures fabricated via two-photon polymerization for cell biology applications

Suppression of auto-fluorescence from high-resolution 3D polymeric architectures fabricated via two-photon polymerization for cell biology applications

Sudan Black B Pretreatment to Suppress Autofluorescence in Silk Fibroin Scaffolds.

Natural polymers are extensively utilized as scaffold materials in tissue engineering and 3D disease modeling due to their general features of cytocompatibility, biodegradability, and ability to mimic the architecture and mechanical properties of the native tissue. A major limitation of many polymeric scaffolds is their autofluorescence under common imaging methods. This autofluorescence, a particular challenge with silk fibroin materials, can interfere with the visualization of fluorescently labeled cells and proteins grown on or in these scaffolds, limiting the assessment of outcomes. Here, Sudan Black B (SBB) was successfully used prefixation prior to cell seeding, in various silk matrices and 3D model systems to quench silk autofluorescence for live cell imaging. SBB was also trialed postfixation in silk hydrogels. We validated that multiple silk scaffolds pretreated with SBB (hexafluoro-2-propanol-silk scaffolds, salt-leached sponges, gel-spun catheters, and sponge-gel composite scaffolds) cultured with fibroblasts, adipose tissue, neural cells, and myoblasts demonstrated improved image resolution when compared to the nonpretreated scaffolds, while also maintaining normal cell behavior (attachment, growth, proliferation, differentiation). SBB pretreatment of silk scaffolds is an option for scaffold systems that require autofluorescence suppression.

Raman Microspectroscopy Goes Viral: Infection Dynamics in the Cosmopolitan Microalga, Emiliania huxleyi.

Emiliania huxleyi is a cosmopolitan member of the marine phytoplankton. This species’ capacities for carbon sequestration and sulfur mobilization make it a key player in oceanic biogeochemical cycles that influence climate on a planetary scale. Seasonal E. huxleyi blooms are abruptly terminated by viral epidemics caused by a clade of large DNA viruses collectively known as coccolithoviruses (EhVs). EhVs thereby mediate a significant part of material and energy fluxes associated with E. huxleyi population dynamics. In this study, we use spontaneous Raman microspectroscopy to perform label-free and non-invasive measurements of the macromolecular composition of individual virions and E. huxleyi host cells. Our novel autofluorescence suppression protocol enabled spectroscopic visualization of evolving macromolecular redistributions in individual E. huxleyi cells at different stages of EhV infection. Material transfer from E. huxleyi hosts to single EhV-163 virions was confirmed by combining stable isotope probing (SIP) experiments with Raman microspectroscopy. Inheritance of the host cells’ 13C-enriched isotopic signature was quantified based on red shifts of Raman peaks characteristic of phenylalanine’s phenyl ring. Two-dimensional Raman mapping of EhV-infected E. huxleyi cells revealed that the compact region producing an intense Raman DNA signal (i.e., the nucleus) in healthy E. huxleyi cells becomes diffuse during the first hours of infection. Raman DNA emissions integrated throughout individual cells decreased during the infection cycle. Our observations are consistent with EhV-163 degrading the host’s nuclear DNA, scavenging released nucleotides for its own genome replication, and shedding newly-produced virions prior to host lysis via budding.

Time-gated interferometric detection increases Raman scattering to fluorescence signal ratio in biological samples.

Attainable levels of signal-to-background ratio (SBR) in Raman spectroscopy of biological samples is limited by the presence of endogenous fluorophores. It is customary to remove the ubiquitous fluorescence background using postacquisition data processing. However, new approaches are needed to reduce background contributions and maximize the fraction of the sensor dynamical range occupied by Raman photons. Time-resolved detection using pulsed lasers and time-gated measurements can be used to address the signal-to-background problem in biological samples by limiting light detection to nonresonant interaction phenomena with relaxation time scales occurring on sub-nanosecond time scales, thereby excluding contributions from resonant phenomena such as fluorescence. A time-gated Fourier-transform spectrometer was assembled using a commercially available interferometer, a single channel single-photon avalanche diode and time tagging electronics. A time gate of 300 ps increased the signal-to-background-ratio of the 1440 cm-1 Raman band from 36% to 69% in an olive oil sample hereby demonstrating the potential of this approach for autofluorescence suppression.

Applying fluorescence in situ hybridization to aquatic systems with cyanobacteria blooms: Autofluorescence suppression and high‐throughput image analysis

AbstractCyanobacterial Harmful Algal Blooms (CyanoHABs) are expanding geographically in both fresh and marine water bodies due to coastal eutrophication and global climate change and are restructuring the microbial ecology of these systems. Cyanobacterial autofluorescence can pose a significant impediment to accurately identifying prokaryotic taxonomic groups in environmental samples using fluorescence in situ hybridization (FISH). This can hinder our ability to accurately quantify, and therefore fully understand ecological changes. As abundances of FISH target cells and autofluorescent cells can often be of the same the order of magnitude, simply subtracting average autofluorescent cell concentrations—determined from enumerating unhybridized samples—yields apparent concentrations of target cells with unacceptably large analytical uncertainty. Here we present a CuSO4/EtOH chemical pretreatment protocol that significantly reduces undesirable autofluorescence in hybridized environmental samples. We apply a novel data filtration routine to FISH images that efficiently removes residual autofluorescent cells from final cell counts. We then subject images to an automated image analysis routine that accurately enumerates probe‐positive cells. This method is inexpensive and easy to implement as part of a routine FISH workflow. By applying this method to cyanobacteria rich samples, we can better understand how microbial community changes are contributing to globally changing biogeochemical cycles.

Photochromic Fluorophores Enable Imaging of Lowly Expressed Proteins in the Autofluorescent Fungus Candida albicans.

ABSTRACTFluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence.IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes.

Wide Field Spectral Imaging with Shifted Excitation Raman Difference Spectroscopy Using the Nod and Shuffle Technique.

Wide field Raman imaging using the integral field spectroscopy approach was used as a fast, one shot imaging method for the simultaneous collection of all spectra composing a Raman image. For the suppression of autofluorescence and background signals such as room light, shifted excitation Raman difference spectroscopy (SERDS) was applied to remove background artifacts in Raman spectra. To reduce acquisition times in wide field SERDS imaging, we adapted the nod and shuffle technique from astrophysics and implemented it into a wide field SERDS imaging setup. In our adapted version, the nod corresponds to the change in excitation wavelength, whereas the shuffle corresponds to the shifting of charges up and down on a Charge-Coupled Device (CCD) chip synchronous to the change in excitation wavelength. We coupled this improved wide field SERDS imaging setup to diode lasers with 784.4/785.5 and 457.7/458.9 nm excitation and applied it to samples such as paracetamol and aspirin tablets, polystyrene and polymethyl methacrylate beads, as well as pork meat using multiple accumulations with acquisition times in the range of 50 to 200 ms. The results tackle two main challenges of SERDS imaging: gradual photobleaching changes the autofluorescence background, and multiple readouts of CCD detector prolong the acquisition time.

Autofluorescence Suppression by Optically Controlling Dark States of Photoswitchable Fluorescent Proteins on Commercial Microscopes

Autofluorescence Suppression by Optically Controlling Dark States of Photoswitchable Fluorescent Proteins on Commercial Microscopes

Lanthanide ions doped in vanadium oxide for sensitive optical glucose detection

Blood glucose monitoring is essential to avoid the unwanted consequences of glucose level fluctuations. Optical monitors are of special interest because they can be non-invasive. Among optical glucose sensors, fluorescent upconversion nanoparticles (UCNPs) have the advantage of good photostability, low toxicity, and exceptional autofluorescence suppression. However, to sense glucose, UCNPs normally need surface functionalization, and this can be easily affected by other factors in biological systems, and may also affect their ability for real-time sensing of both increasing and decreasing glucose levels. Here, we report YVO4 : Yb3+, Er3+@Nd3+ core/shell UCNPs with Nd and Yb shell and GdVO4 : Yb3+, Er3+@Nd3+ core/shell UCNPs with Nd and Yb shell that show sensitive, reversible, and selective optical glucose detection without the need for any surface functionalization or modifications.

Dual-Excitation Nanocellulose Plasmonic Membranes for Molecular and Cellular SERS Detection.

We demonstrate that cellulose nanofiber (CNF) biomaterials with high transparency and mechanical robustness can be combined with gold nanorods to form a multifunctional porous membrane for dual-mode surface-enhanced Raman scattering (SERS) detection of both small molecules and cells. The nanoporous nature of the nanofiber membranes allows for effective molecular filtration and preconcentration of the analytes, further boosting the SERS performance. Specifically, because of the low fluorescence and Raman background of the CNF matrix, extremely low loading density of gold nanorods can be used. The nanorod assemblies within the CNF network can be resonantly driven by a 532 nm laser (transverse plasmonic mode) and near resonantly driven at by a 785 nm laser (longitudinal mode), facilitating dual operational modes at two excitation wavelengths. The shorter wavelength excitation mode yields better Raman scattering efficiency and has been demonstrated to be capable of detecting rhodamine 6G (R6G) dyes down to picomolar concentrations. On the other hand, the longer wavelength excitation mode provides autofluorescence suppression for the better detection of microorganisms such as Escherichia coli, shortening the required integration time from hours to minutes. Upon drastically lowering the spectral background noise and utilizing nanofiltration, the plasmonic CNF membranes reported here show significantly improved SERS sensitivity and detection fidelity as compared to traditional metal, metal oxide, synthetic polymer, and paper SERS substrates.

Phase confinement of self-migrated plasmonic silver in triphasic system: Offering 3D hot spots on hydrophobic paper for SERS detection

Abstract Hydrophobic paper decorated Ag nanoparticles (NPs) with a uniform distribution was fabricated in an oil-paper-aqueous triphasic system via a facile in situ interfacial reduction approach. Interestingly, the Ag NPs formed at the paper/aqueous interface could migrate to the oil/paper interface. Moreover, these Ag NPs would not continue to migrate away the paper surface into the oil phase but was completely confined in the solid paper matrix. This phase confinement of migrated Ag NPs in the paper matrix creates abundant 3D hot spots for plasmonic enhancement. The obtained Ag-loaded paper exhibits an excellent SERS activity, reliable reproducibility and autofluorescence suppression effect, which enabling it as a promising platform for qualitative and quantitative SERS study. Due to its condensation effect, the hydrophobic paper surface can be used as a concentrator to enrich water-soluble analytes over the SERS active domains for highly sensitive detection (10−9 mol/L−1). Furthermore, the Ag-loaded paper combined with the feature of chromatography and plamon-enhanced optical signals possesses ability to perform synchronous separation and surface-enhanced Raman scattering (SERS) detection of complex samples.

Engineering water-tolerant core/shell upconversion nanoparticles for optical temperature sensing.

Luminescence thermometry is a promising approach using upconversion nanoparticles (UCNPs) with a nanoscale regime in biological tissues. UCNPs are superior to conventional fluorescent markers, benefiting from their autofluorescence suppression and deep imaging in tissues. However, they are still limited by poor water solubility and weak upconversion luminescence intensity, especially at a small particle size. Recently, YVO4:Er+3,Yb+3 nanoparticles have shown high efficiency upconversion (UC) luminescence in water at single-particle level and high contrast imaging in biological models. Typically, a 980-nm laser triggers the UC process in the UCNPs, which overlaps with maximum absorption of water molecules that are dominant in biological samples, resulting in biological tissues overheating and possible damaging. Interestingly, neodymium (Nd+3) possesses a large absorption cross section at the water low absorption band (808nm), which can overcome overheating issues. In this Letter, we introduce Nd+3 as a new near-infrared absorber and UC sensitizer into YVO4:Er+3,Yb+3 nanoparticles in a core/shell structure to ensure successive energy transfer between the new UC sensitizer (Nd+3) to the upconverting activator (Er+3). Finally, we synthesized water-tolerant YVO4:Er+3,Yb+3@Nd+3 core/shell nanoparticles (average size 20nm) with strong UC luminescence at a biocompatible excitation wavelength for optical temperature sensing where overheating in water is minimized.

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.

Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC)

Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer.

Classification of Raman spectra of single cells with autofluorescence suppression by wavelength modulated excitation

Wavelength modulated Raman spectroscopy has recently been shown to suppress the fluorescence background generated by the sample and the substrate. Here we apply this technique to collect wavelength modulated Raman spectra from 697 individual cells for a model system of circulating tumour cells that consists of leukocytes from patient's blood, acute myeloid leukaemia cells (OCI-AML3), and breast tumour cells BT-20 and MCF-7. We study the classification behaviour of wavelength modulated Raman spectra in comparison to a common background correction method in chemometrics. Classifications using a support vector machine with a radial based kernel function were compared for classical Raman spectra, average Raman spectra of each cell and wavelength modulated Raman spectra. The dataset was divided into 80% training spectra and 20% independent validation spectra. The stability of the classification was tested by performing training and validation 200 times with randomly selected datasets. The results are displayed in box whisker plots. Cell identification based on wavelength modulated Raman spectra gives similar classification rates than classical and averaged Raman spectra with a tendency of reduced accuracies and increased modelling variations. Possible explanations and strategies to further improve the wavelength modulated Raman spectroscopy are discussed.

Demonstration of true‐color high‐contrast microorganism imaging for terbium bioprobes

Lanthanide bioprobes offer a number of novel advantages for advanced cytometry, including the microsecond luminescence lifetime, sharp spectral emission, and large stokes shift. However, to date, only the europium-based bioprobes have been broadly studied for time-gated luminescence cell imaging, though a wide range of efficient terbium bioprobes have been synthesized and some of them are commercially available. We analyze that the bottleneck problem was due to the lack of an efficient microscope with pulsed excitation at wavelengths of 300-330 nm. We investigate a recently available 315 nm ultraviolet (UV) light emitting diode to excite an epifluorescence microscope. Substituting a commercial UV objective (40×), the 315 nm light efficiently delivered the excitation light onto the uncovered specimen. A novel pinhole-assisted optical chopper unit was attached behind the eyepiece for direct lifetime-gating to permit visual inspection of background-free images. We demonstrate the use of a commercial terbium complex for high-contrast imaging of an environmental pathogenic microorganism, Cryptosporidium parvum. As a result of effective autofluorescence suppression by a factor of 61.85 in the time domain, we achieved an enhanced signal-to-background ratio of 14.43. This type of time-gating optics is easily adaptable to the use of routine epifluorescence microscopes, which provides an opportunity for high-contrast imaging using multiplexed lanthanide bioprobes.

Time‐resolved microscopy for imaging lanthanide luminescence in living cells

Time-resolved luminescence (TRL) microscopy can image signals from lanthanide coordination complexes or other probes with long emission lifetimes, thereby eliminating short-lifetime (<100 ns) autofluorescence background from biological specimens. However, lanthanide complexes emit far fewer photons per unit time than conventional fluorescent probes, making it difficult to rapidly acquire high quality images at probe concentrations that are relevant to live cell experiments. This article describes the development and characterization of a TRL microscope that employs a light-emitting diode (LED, λ(em) = 365 nm) for pulsed epi-illumination and an intensified charge-coupled device (ICCD) camera for gated, widefield detection. Europium chelate-impregnated microspheres were used to evaluate instrument performance in terms of short-lifetime fluorescence background rejection, photon collection efficiency, image contrast, and signal-to-noise ratio (SNR). About 200 nm microspheres were imaged within the time resolution limit of the ICCD (66.7 ms) with complete autofluorescence suppression. About 40 nm microspheres containing ~400 chelate molecules were detected within ~1-s acquisition times. A luminescent terbium complex, Lumi4-Tb®, was introduced into the cytoplasm of cultured cells at an estimated concentration of 300 nM by the method of osmotic lysis of pinocytic vesicles. Time-resolved images of the living, terbium complex-loaded cells were acquired within acquisition times as short as 333 ms, and the effects of increased exposure time and frame summing on image contrast and SNR were evaluated. The performance analyses show that TRL microscopy is sufficiently sensitive and precise to allow high-resolution, quantitative imaging of lanthanide luminescence in living cells under physiologically relevant experimental conditions.