Application Of Fluorescence Microscopy Research Articles

A highly photostable and bright green fluorescent protein

The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.

Real-time image denoising of mixed Poisson–Gaussian noise in fluorescence microscopy images using ImageJ

Fluorescence microscopy imaging speed is fundamentally limited by the measurement signal-to-noise ratio (SNR). To improve image SNR for a given image acquisition rate, computational denoising techniques can be used to suppress noise. However, common techniques to estimate a denoised image from a single frame either are computationally expensive or rely on simple noise statistical models. These models assume Poisson or Gaussian noise statistics, which are not appropriate for many fluorescence microscopy applications that contain quantum shot noise and electronic Johnson–Nyquist noise, therefore a mixture of Poisson and Gaussian noise. In this paper, we show convolutional neural networks (CNNs) trained on mixed Poisson and Gaussian noise images to overcome the limitations of existing image denoising methods. The trained CNN is presented as an open-source ImageJ plugin that performs real-time image denoising (within tens of milliseconds) with superior performance (SNR improvement) compared to conventional fluorescence microscopy denoising methods. The method is validated on external datasets with out-of-distribution noise, contrast, structure, and imaging modalities from the training data and consistently achieves high-performance ( > --> 8 d B ) denoising in less time than other fluorescence microscopy denoising methods.

Application of super-resolution fluorescence microscopy in hematologic malignancies

Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells. The examination of bone marrow cells combined with immunodeficiency typing is of great significance to the diagnostic type, treatment and prognosis of hematologic malignancies. Super-resolution fluorescence microscopy (SRM) is a special kind of optical microscopy technology, which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014. With the development of SRM, many related technologies have been applied to the diagnosis and treatment of clinical diseases. It was reported that a major type of SRM technique, single molecule localization microscopy (SMLM), is more sensitive than flow cytometry (FC) in detecting cell membrane antigens’ expression, thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools. Furthermore, SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases. In this paper, we mainly discuss the application of SRM in clone hematological malignancies.

Single-shot mid-infrared incoherent holography using Lucy-Richardson-Rosen algorithm

In recent years, there has been a significant transformation in the field of incoherent imaging with new possibilities of compressing three-dimensional (3D) information into a two-dimensional intensity distribution without two-beam interference (TBI). Most of the incoherent 3D imagers without TBI are based on scattering by a random phase mask exhibiting sharp autocorrelation and low cross-correlation along the depth. Consequently, during reconstruction, high lateral and axial resolutions are obtained. Imaging based on scattering requires an astronomical photon budget and is therefore precluded in many power-sensitive applications. In this study, a proof-of-concept 3D imaging method without TBI using deterministic fields has been demonstrated. A new reconstruction method called the Lucy-Richardson-Rosen algorithm has been developed for this imaging concept. We believe that the proposed approach will cause a paradigm-shift in the current state-of-the-art incoherent imaging, fluorescence microscopy, mid-infrared fingerprinting, astronomical imaging, and fast object recognition applications.

Extreme dependence of Chloroflexus aggregans LOV domain thermo- and photostability on the bound flavin species.

Light-oxygen-voltage (LOV) domains are common photosensory modules that found many applications in fluorescence microscopy and optogenetics. Here, we show that the Chloroflexus aggregans LOV domain can bind different flavin species (lumichrome, LC; riboflavin, RF; flavin mononucleotide, FMN; flavin adenine dinucleotide, FAD) during heterologous expression and that its physicochemical properties depend strongly on the nature of the bound flavin. We show that whereas the dissociation constants for different chromophores are similar, the melting temperature of the protein reconstituted with single flavin species varies from ~ 60°C for LC to ~ 81°C for FMN, and photobleaching half-times vary almost 100-fold. These observations serve as a caution for future studies of LOV domains in non-native conditions yet raise the possibility of fine-tuning various properties of LOV-based fluorescent probes and optogenetic tools by manipulating the chromophore composition.

Recent advances in the standardization of fluorescence microscopy for quantitative image analysis.

Fluorescence microscopy is a broadly used technique within a variety of different fields, including life sciences and medical research, and its quantitative aspects are becoming emphasized. The challenge now lies in improving the accuracy and precision of the measurements obtained from fluorescence microscopy. Improving these will facilitate the comparison of results between different instruments/institutions and therefore ensure the reproducibility of results. In this review, recent standardization procedures, including the benchmarking of the instrument performance and the standardization of the image itself, as well as the reference materials for calibration are summarized, and an overview of the advances in fluorescence microscopy standardization and the current limitations are presented. A procedure for the comparison of the image data obtained using different instruments, by different analysts and/or at different times, should be developed to improve the standardization of this data. The standardization of image data would lead to the development of new applications of fluorescence microscopy not only in academic research but also in regulatory science.

Application of fluorescent microscopy for improved detection of acid fast bacilli in sputum smear preparation

Background: Tuberculosis remains a global health problem with an enormous burden of disease. Ziehl-Neelsen stain is the most commonly used method worldwide to detect tuberculous lymph node aspirates, however, Auramine – O Staining is being preferred owing to its high sensitivity, rapidity, and ease of screening. The aim of the present study was to compare the efficacy of fluorescent microscopy using Auramine O stain with conventional microscopy using Ziehl- Neelsen stain as a screening method to detect clinically suspected cases of pulmonary tuberculosis. Method: A total of 100 clinically suspected cases of pulmonary tuberculosis referred to RNTCP laboratory from chest and TB OPD Medicine and Surgery and all other inpatient and out-patient departments were studied with detailed clinical history and sputum examination using Ziehl–Neelsen and Auramine O staining. Results: Among 100 cases, 28% of cases were detected sputum smear-positive by Ziehl-Neelsen staining, and 30% cases were detected by Auramine O stain using a fluorescence microscope. Maximum sputum smears positive (36.6%) cases were in the age group of 31-40 years with male predominance (75%). Auramine O stain showed higher grading in 4 cases in which one case was scanty on Ziehl-Neelsen smear showed positive 1+ by Auramine O and other 2 cases which were negative by Ziehl-Neelsen stain were showed 1+ result on Auramine O stain. One case which showed 2+ grading on Ziehl-Neelsen stain showed 3+ grading with Auramine O stain. Fluorescence microscopy using Auramine O showed better grading than the Ziehl-Neelsen stain. Conclusion: Fluorescence microscopy is more superior in detecting sputum smear-positive cases over Ziehl-Neelsen stain.

Using optical interference filters to measure autofluorescence in substrates and coatings

In fluorescence microscopy and related applications, fluorescence from optical elements in the system can be a limiting factor in achieving low background levels. Such “autofluorescence” is considerably weaker than the fluorescence produced by the organic dyes used in fluorescence-based analytical techniques. While spectrally detailed information about autofluorescence is desirable, such information comes at a cost of sensitivity. We present a straightforward approach to making sensitive fluorescence measurements using a simple chopped laser and phase-lock detection scheme. By using high-quality optical filters in the excitation and detection paths, the trade-off between spectral information and ultimate sensitivity is controlled by the choice of filter edge locations and bandwidths. An initial implementation of this approach, using a 405 nm diode laser as an excitation source, achieves a sensitivity of ~1 pW optical power of detected fluorescence over a bandwidth of ~125 nm centered at 505 nm. Data from measurements on glass substrates are presented, and challenges for evaluating autofluorescence from coatings on glass are discussed.

Systematic Tuning of Rhodamine Spirocyclization for Super-resolution Microscopy.

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a nonfluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, and an adjustment of the equilibrium poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows one to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell-stimulated emission depletion (STED) microscopy or a spontaneously blinking dye for single-molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

Excitation light-induced phototoxicity during fluorescence imaging

Fluorescence microscopy has revolutionized life science research. It is a powerful technique to visualize molecular structures, physiological functions, and dynamic processes in living cells, tissues, and organisms. Excitation-light and/or excited-fluorescent proteins-mediated dysregulation in cell physiology are widely known but poorly considered. Although there are vast applications of fluorescence microscopy in research, imaging results may suffer from the excitation-light-induced artefacts. Here, we highlight potential excitation light-induced alterations in cell functions thereby misinterpretation of imaging results and strategies to minimize common artefacts that can be produced during imaging experiments.

Emerging solvatochromic push-pull dyes for monitoring the lipid order of biomembranes in live cells.

Solvatochromic dyes have emerged as a new class of fluorescent probes in the field of lipid membranes due to their ability to identify the lipid organization of biomembranes in live cells by changing the colour of their fluorescence. This type of solvatochromic function is useful for studying the heterogeneous features of biomembranes caused by the uneven distribution of lipids and cholesterols in live cells and related cellular processes. Therefore, a variety of advanced solvatochromic dyes have been rapidly developed over the last decade. To provide an overview of the works recently developed solvatochromic dyes have enabled, we herein present some solvatochromic dyes, with a particular focus on those based on pyrene and Nile red. As these dyes exhibit preferable photophysical properties in terms of fluorescence microscopy applications and unique distribution/localization in cellular compartments, some have already found applications in cell biological and biophysical studies. The goal of this review is to provide information to researchers who have never used solvatochromic dyes or who have not discovered applications of such dyes in biological studies.

High-resolution structure of a naturally red-shifted LOV domain

LOV domains are widespread photosensory modules that have also found applications in fluorescence microscopy, optogenetics, and light-driven generation of reactive oxygen species. Many of these applications require stable proteins with altered spectra. Here, we report a flavin-based fluorescent protein CisFbFP derived from Chloroflexus islandicus LOV domain-containing protein. We show that CisFbFP is thermostable, and its absorption and fluorescence spectra are red-shifted for ∼6 nm, which has not been observed for other cysteine-substituted natural LOV domains. We also provide a crystallographic structure of CisFbFP at the resolution of 1.2 Å that reveals alterations in the active site due to replacement of conservative asparagine with a serine. Finally, we discuss the possible effects of presence of cis-proline in the Aβ-Bβ loop on the protein's structure and stability. The findings provide the basis for engineering and color tuning of LOV-based tools for molecular biology.

Varifocal Metalens for Optical Sectioning Fluorescence Microscopy.

Fluorescence microscopy with optical sectioning capabilities is extensively utilized in biological research to obtain three-dimensional structural images of volumetric samples. Tunable lenses have been applied in microscopy for axial scanning to acquire multiplane images. However, images acquired by conventional tunable lenses suffer from spherical aberration and distortions. Here, we design, fabricate, and implement a dielectric Moiré metalens for fluorescence imaging. The Moiré metalens consists of two complementary phase metasurfaces, with variable focal length, ranging from ∼10 to ∼125 mm at 532 nm by tuning mutual angles. In addition, a telecentric configuration using the Moiré metalens is designed for high-contrast multiplane fluorescence imaging. The performance of our system is evaluated by optically sectioned images obtained from HiLo illumination of fluorescently labeled beads, as well as ex vivo mice intestine tissue samples. The compact design of the varifocal metalens may find important applications in fluorescence microscopy and endoscopy for clinical purposes.

An autofluorescence-based survey of late follicular atresia in ovaries of a teleost fish (Thunnus thynnus).

In this study, the authors examined late atretic follicles in the ovaries of Atlantic bluefin tuna, Thunnus thynnus (Linnaeus 1758), at different times of the year using transmitted light and epifluorescence microscopy. Atresia (degeneration and resorption of developing ovarian follicles) is a natural process involved in fecundity downregulation in teleosts and is substantially enhanced in stressful conditions. Early (α and β) atretic stages of yolked oocytes have a relatively short duration in seasonally reproducing species, whereas later (γ and δ) atretic follicles (LAF) persist for longer time in the ovary, serving as a sign of previous vitellogenic activity. LAF can thus be used as reliable markers of maturity during non-reproductive periods. Lipofuscin granules accumulate in the cytoplasm of LAF cells as a result of lysosomal digestion of oocyte components. Taking advantage of the well-known autofluorescent properties of lipofuscins, LAF may be identified in unstained histological sections under fluorescence microscopy using appropriate excitation and emission wavelengths. The authors explore in this study the applicability of fluorescence microscopy to provide a fast and effective method to assess late atresia in fishes. This method may be particularly useful to determine sexual maturity in individuals sampled long after the spawning season, where LAF are difficult to detect in standard histological sections. Furthermore, LAF autofluorescence provides a rapid way to quantify late atresia in fishes using image analysis.

Fluorophore Multimerization as an Efficient Approach towards Bright Protein Labels

AbstractCell analysis techniques like flow cytometry and fluorescence microscopy are widely used to explore cells in real‐time and provide important insights into a variety of cellular processes. These techniques require bright and specific staining reagents to generate strong fluorescence signal and enable reliable read‐out, making the choice of fluorescent labels a key aspect of experimental design. Much research has been done in the field of fluorophore development to generate bright small‐molecule dyes, fluorescent proteins, and emitting nanoparticles. However, it remains challenging to modify biomolecules with multiple emitters and avoid self‐quenching of such fluorophores at the same time. Herein, we present advances in multimerization‐based methods for the generation of fluorescent labels with high brightness and discuss their advantages and limitations in the context of flow cytometry and fluorescence microscopy applications.

Low noise, self-phase-modulation-enabled femtosecond fiber sources tunable in 740-1236 nm for wide two-photon fluorescence microscopy applications

We have demonstrated widely tunable Yb:fiber-based laser sources, aiming to replace Ti:sapphire lasers for the nJ-level ultrafast applications, especially for the uses of nonlinear light microscopy. We investigated the influence of different input parameters to obtain an expansive spectral broadening, enabled by self-phase modulation and further reshaped by self-steepening, in the normal dispersion regime before the fiber damage. We also discussed the compressibility and intensity fluctuations of the demonstrated pulses, to reach the transform-limited duration with a very low intensity noise. Most importantly, we have demonstrated clear two-photon fluorescence images from UV-absorbing fluorophores to deep red dye stains.

Low-Light Photodetectors for Fluorescence Microscopy

Over the years, fluorescence microscopy has evolved and has become a necessary element of life science studies. Microscopy has elucidated biological processes in live cells and organisms, and also enabled tracking of biomolecules in real time. Development of highly sensitive photodetectors and light sources, in addition to the evolution of various illumination methods and fluorophores, has helped microscopy acquire single-molecule fluorescence sensitivity, enabling single-molecule fluorescence imaging and detection. Low-light photodetectors used in microscopy are classified into two categories: point photodetectors and wide-field photodetectors. Although point photodetectors, notably photomultiplier tubes (PMTs), have been commonly used in laser scanning microscopy (LSM) with a confocal illumination setup, wide-field photodetectors, such as electron-multiplying charge-coupled devices (EMCCDs) and scientific complementary metal-oxide-semiconductor (sCMOS) cameras have been used in fluorescence imaging. This review focuses on the former low-light point photodetectors and presents their fluorescence microscopy applications and recent progress. These photodetectors include conventional PMTs, single photon avalanche diodes (SPADs), hybrid photodetectors (HPDs), in addition to newly emerging photodetectors, such as silicon photomultipliers (SiPMs) (also known as multi-pixel photon counters (MPPCs)) and superconducting nanowire single photon detectors (SSPDs). In particular, this review shows distinctive features of HPD and application of HPD to wide-field single-molecule fluorescence detection.

Unbiased analysis of obesity related, fat depot specific changes of adipocyte volumes and numbers using light sheet fluorescence microscopy.

In translational obesity research, objective assessment of adipocyte sizes and numbers is essential to characterize histomorphological alterations linked to obesity, and to evaluate the efficacies of experimental medicinal or dietetic interventions. Design-based quantitative stereological techniques based on the analysis of 2D-histological sections provide unbiased estimates of relevant 3D-parameters of adipocyte morphology, but often involve complex and time-consuming tissue processing and analysis steps. Here we report the application of direct 3D light sheet fluorescence microscopy (LSFM) for effective and accurate analysis of adipocyte volumes and numbers in optically cleared adipose tissue samples from a porcine model of diet-induced obesity (DIO). Subcutaneous and visceral adipose tissue samples from DIO-minipigs and lean controls were systematically randomly sampled, optically cleared with 3DISCO (3-dimensional imaging of solvent cleared organs), stained with eosin, and subjected to LSFM for detection of adipocyte cell membrane autofluorescence. Individual adipocytes were unbiasedly sampled in digital 3D reconstructions of the adipose tissue samples, and their individual cell volumes were directly measured by automated digital image analysis. Adipocyte numbers and mean volumes obtained by LSFM analysis did not significantly differ from the corresponding values obtained by unbiased quantitative stereological analysis techniques performed on the same samples, thus proving the applicability of LSFM for efficient analysis of relevant morphological adipocyte parameters. The results of the present study demonstrate an adipose tissue depot specific plasticity of adipocyte growth responses to nutrient oversupply. This was characterized by an exclusively hypertrophic growth of visceral adipocytes, whereas adipocytes in subcutaneous fat tissue depots also displayed a marked (hyperplastic) increase in cell number. LSFM allows for accurate and efficient determination of relevant quantitative morphological adipocyte parameters. The applied stereological methods and LSFM protocols are described in detail and can serve as a guideline for unbiased quantitative morphological analyses of adipocytes in other studies and species.

Synthesis, characterization, and photocatalytic activity of ZnS and Mn-doped ZnS nanostructures

Semiconductor quantum dots such as zinc sulfide have interesting potential applications, due to their size-dependent optical properties. These nanostructures can be used for applications in agriculture, environmental chemistry, and fluorescence microscopy. The great rise in nanotechnology has sparked the scientific community’s interest in nanomaterials for the use of photodegradation in aquatic bodies. Quantum Dots (QDs) such as ZnS nanoparticles (NPs) can absorb electromagnetic radiation and generate subsequently reactive oxygen species (ROS) directly in aqueous phase. The presence of ROS in aquatic environments can be used to destroy organic contaminants by photocatalysis. Previous studies had evidenced that the presence of impurities (i.e. copper, manganese, nickel) in the crystalline structures of QDs can enhance their optical properties and consequently their catalytic capacity. Because of this, the present investigation was focused on generating water stable ZnS nanoparticles with catalytic properties. In this work, we have synthesized pure and doped ZnS nanoparticles using a reflux method. The morphology of these QDs was characterized by transmission electron microscopy (TEM). We also studied the photocatalytic properties of these nanostructures. A red shift was observed in the photoluminescence peak of pure ZnS nanoparticles when they were doped with heavy metals. Pure ZnS NPs and Mn-doped ZnS NPs showed luminescent peaks at 444 nm and 596 nm, respectively. Photodegradation studies were evaluated in the presence of organic dyes like Tropaeolin O (TO) and different concentrations of quantum dots (250 ppm and 500 ppm). The photodegradation of TO was dependent on the QDs concentration and exposure time. The destruction of organic dyes in the presence of photo-excited ZnS nanoparticles is envisioned as a fast and clean technology. The destruction of organic dyes in the presence of photo-excited ZnS nanoparticles is envisioned as a fast and clean technology.

Application of Two-Photon Microscopy to Study Sclerotium cepivorum Berk Sclerotia Isolated from Naturally Infested Soil and Produced In Vitro

The danger of Sclerotium cepivorum lies in the strength of its survival structure: sclerotia. Sclerotia comprising hardened mycelium contains food reserves that allow it to remain dormant for long period, which makes the sclerotia-infested soil useless to grow any crop of the Allium species, including onion and garlic. This paper would be the first report on the application of two-photon fluorescence microscopy to the analysis of the structure of sclerotia from S. cepivorum. For this study and, in order to test the method, two different types of sclerotia were used: (1) sclerotia isolated from naturally infested soil and (2) sclerotia produced in vitro (from 20-day-old cultures). Both types of sclerotia were processed by cryopreservation and eight µm histological cuts were used to obtain an autofluorescence image. For both sclerotia, the fluorescence spectrum has three peak signals at their wall. Sclerotia from infested soil presented fluorescence peaks at 400-436, 436-475, and 515-575nm, while signals from sclerotia produced in vitro presented fluorescence peaks at 400-442, 500-600, and 655-700nm. Peaks at the violet electromagnetic region (400-436 and 400-442) are like that of the signals reported by the melanin. This study showed that two-photon microscopy is a novel and valuable tool for the study of sclerotia structure and their fluorescence signal, and the possibility of using it as a specific marker to direct detection in the field should be explored.