Fluorescence Emission Difference Research Articles

Deep learning enhanced fluorescence emission difference microscopy

Fluorescence emission difference microscopy (FED) obtains super-resolution microscopic images by extracting the information of the intensity differences between the solid and doughnut confocal images. Due to the mismatch of the outer contour of the solid and doughnut point spread function, FED suffers the problem of information loss. Here, we present a framework for deep-learning-enhanced FED (DL-FED) microscopy through cycle-consistent generative adversarial network (CycleGAN) based image reconstruction. Using this framework, we effectively avoid the information loss and enhance the spatial resolution of the fluorescence images acquired by the standard FED. We also demonstrate that the standard FED images can be transformed to match the results of Airyscan-based FED and saturated-FED, which can effectively enhance the signal-to-noise ratio and avoid the photo-bleaching effect. The validity of DL-FED is demonstrated by simulations and experiments based on fluorescent nanoparticles and biological cells. Featuring the potential to realize a high imaging speed, this approach may be widely applied in live cells investigations.

Single multimode fibre for in vivo light-field-encoded endoscopic imaging

Super-resolution microscopy is typically not applicable to in situ imaging through a narrow channel due to the requirement for complex optics. Although multimode fibres (MMFs) have emerged as a potential platform for cost-effective and precise endoscopic imaging, they suffer from extreme sensitivity to bending and other external conditions. Here we demonstrate imaging through a single thin MMF for in vivo light-field encoded imaging with subcellular resolution. We refer to the technique as spatial-frequency tracking adaptive beacon light-field-encoded (STABLE) endoscopy. Spatial-frequency beacon tracking provides up to 1 kHz disorder tracking frequency, thus ensuring stable imaging through long-haul MMFs under fibre bending and various operating conditions. The full-vector modulation and fluorescence emission difference are combined to enhance the imaging signal-to-noise ratio and achieve a subdiffraction resolution of 250 nm. We integrate STABLE in a white-light endoscope and demonstrate cross-scale imaging in a bronchus model and in vivo imaging in mice models. The high-resolution and resilience to observation in a minimally invasive manner paves the way to the expansion of MMF in endoscopy to the study of disease mechanisms in biomedical sciences and clinical studies.

Sparse denoising and adaptive estimation enhances the resolution and contrast of fluorescence emission difference microscopy based on an array detector.

An array detector allows a resolution gain for confocal microscopy by combining images sensed by a set of photomultipliers tubes (or sub-detectors). Several methods have been proposed to reconstruct a high-resolution image by linearly combining sub-detector images, especially the fluorescence emission difference (FED) technique. To improve the resolution and contrast of FED microscopy based on an array detector, we propose to associate sparse denoising with spatial adaptive estimation. We show on both calibration slides and real data that our approach applied to the full stack of spatially reassigned detector signals, enables us to achieve a higher reconstruction performance in terms of resolution, image contrast, and noise reduction.

Chemical-Anatomical Characterization of Stems of Asparagaceae Species with Potential Use for Lignocellulosic Fibers and Biofuels

During the last decades, the possibility of using species resistant to droughts and extreme temperatures has been analyzed for use in the production of lignocellulosic materials and biofuels. Succulent species are considered to identify their potential use; however, little is known about Asparagaceae species. Therefore, this work aimed to characterize chemically-anatomically the stems of Asparagaceae species. Stems of 10 representative species of Asparagaceae were collected, and samples were divided into two. One part was processed to analyze the chemical composition, and the second to perform anatomical observations. The percentage of extractives and lignocellulose were quantified, and crystalline cellulose and syringyl/guaiacyl lignin were quantified by Fourier transform infrared spectroscopy. Anatomy was observed with epifluorescence microscopy. The results show that there were significant differences between the various species (p < 0.05) in the percentages of extractives and lignocellulosic compounds. In addition, there were anatomical differences in fluorescence emission that correlated with the composition of the vascular tissue. Finally, through the characterization of cellulose fibers together with the proportion of syringyl and guaiacyl, it was obtained that various species of the Asparagaceae family have the potential for use in the production of lignocellulosic materials and the production of biofuels.

Fluorescence emission difference microscopy based on polarization modulation

In this paper, we propose a new fluorescence emission difference microscopy (FED) technique based on polarization modulation. An electro-optical modulator (EOM) is used to switch the excitation beam between the horizontal and vertical polarization states at a high frequency, which leads to solid- and donut-shaped beams after spatial light modulation. Experiment on the fluorescent nanoparticles demonstrates that the proposed method can achieve [Formula: see text] spatial resolution. Using the proposed system, the dynamic imaging of subcellular structures in living cells over time is achieved.

Multicolor expansion fluorescence emission difference microscopy to reveal potential organelle contacts

Biologists have long been interested in the submicroscopic structures of cells and interactions between organelles. However, due to resolution limitations, investigating cell biology using fluorescence microscopy remains a challenge. In recent decades, super-resolution microscopy has been developed as an essential solution. Recently, a new microscopic technique called expansion microscopy has been developed, which circumvents this resolution limit by increasing the size of the biological sample, thus allowing for super-resolution of the enlarged structure. In this study, we combined expansion microscopy with fluorescence emission difference microscopy (ExFED) and achieved a spatial resolution of approximately 40 nm in mammalian cells. Delicate subcellular and sub-organelle structures were investigated, and potential contacts were detected. The results indicated that ExFED is a simple and powerful super-resolution method for studying biological specimens.

Dendron-Polymer Hybrids as Tailorable Responsive Coronae of Single-Walled Carbon Nanotubes.

Functional composite materials that can change their spectral properties in response to external stimuli have a plethora of applications in fields ranging from sensors to biomedical imaging. One of the most promising types of materials used to design spectrally active composites are fluorescent single-walled carbon nanotubes (SWCNTs), noncovalently functionalized by synthetic amphiphilic polymers. These coated SWCNTs can exhibit modulations in their fluorescence spectra in response to interactions with target analytes. Hence, identifying new amphiphiles with interchangeable building blocks that can form individual coronae around the SWCNTs and can be tailored for a specific application is of great interest. This study presents highly modular amphiphilic polymer-dendron hybrids, composed of hydrophobic dendrons and hydrophilic polyethylene glycol (PEG) that can be synthesized with a high degree of structural freedom, for suspending SWCNTs in aqueous solution. Taking advantage of the high molecular precision of these PEG-dendrons, we show that precise differences in the chemical structure of the hydrophobic end groups of the dendrons can be used to control the interactions of the amphiphiles with the SWCNT surface. These interactions can be directly related to differences in the intrinsic near-infrared fluorescence emission of the various chiralities in a SWCNT sample. Utilizing the susceptibility of the PEG-dendrons toward enzymatic degradation, we demonstrate the ability to monitor enzymatic activity through changes in the SWCNT fluorescent signal. These findings pave the way for a rational design of functional SWCNTs, which can be used for optical sensing of enzymatic activity in the near-infrared spectral range.

Differential fluorescence microscopy by using a dynamic cylindrical-vector field.

Fluorescence emission difference (FED) microscopy, as an emerging super-resolution imaging modality, uses double-exposure and subtraction between double-exposed fluorescence images to achieve high spatial resolution beyond the diffraction limit. Here we report on a new FED imaging approach with a single-exposure scheme based on dynamic cylindrical-vector fields, where the fluorescence excitation beam can be switched between radial and azimuthal polarization states at a designated high radio frequency. Lateral spatial resolution of ${\sim} \lambda/4$ is achieved. Being able to integrate with lock-in amplifier detection, the proposed method will find promising applications for high-speed fluorescence imaging with improved signal-to-noise ratio.

Three-Dimension Resolution Enhanced Microscopy Based on Parallel Detection

Pixel reassignment image scanning microscopy (PRISM) is a useful tool to improve the resolution of confocal laser scanning microscopy (CLSM) only equipped with a detector array. However, while it can improve the lateral resolution, it has little effect on the axial resolution. Here, new microscopy has been proposed which combines three-dimension fluorescence emission difference microscopy (3D FED) with PRISM to further improve three-dimension resolution. We call this method three-dimension pixel reassignment fluorescence emission difference microscopy (3D-PRFED). Detailed theoretical analysis and simulation are presented in this paper. Additionally, the performance of lateral and axial resolution improvement of this method has been demonstrated by imaging 100 nm fluorescent beads and nuclear pore complexes samples. Experiment results show that this method in our system can improve lateral resolution by a factor of 1.85 and axial resolution by a factor of 1.48 compared with CLSM.

Fluorescent Silica Hybrid Film-Forming Materials Based on Salicylaldazine

Fluorescent film-forming materials were obtained by embedding salicylaldazine (SAA) in silica hybrids generated by sol–gel processes from different silane precursors in acid catalysis. Tuned local environments for the fluorophore were generated in the hosting network by modifying silica sols with organic groups through the co-condensation of tetraethylortosilicate (TEOS) and different alkoxysilanes hydrolysis products. The photophysical properties of the luminescent hybrid films were studied in direct relationship with structural, textural, and surface properties and based on interactions between SAA species and the silica hosting network. Film-forming materials were studied in order to determine differences in absorption and fluorescence emission due to the environments around the fluorophore. The variations recorded in the fluorescence emission spectra of the hybrid films were related to interactions established between the fluorophore species and their sterically hindered surroundings of the host hybrid silica, where free molecular motions are restricted. The influence of the type and amount of network modifier and of the fluorophore loading on the transparency of the films and fluorescence intensity was also investigated. The study carried out led to the elucidation of the necessary conditions for obtaining luminescent film-forming materials with high luminescence intensity and transparency useful for the design of new light concentrators.

3D resolution enhancement in saturated competition microscopy

To overcome the diffraction barrier, super-resolution microscopy is contrived and has witnessed scientific developments in varying fields, especially in last few decades, such as stochastic optical reconstruction microscopy, stimulated emission depletion microscopy (STED), mirror-enhanced super-resolution microscopy (MEANS), and fluorescence emission difference microscopy (FED). Recently, saturated competition microscopy (SAC) was developed to realize high sub-diffraction resolution in either fluorescent or non-fluorescent imaging. Compared with STED, SAC features non-constraint in fluorescent dye selection. Nevertheless, the lateral resolution is limited in consideration of photobleaching side effects. Also, the axial resolution enhancement of SAC has not been demonstrated. In this study, a method, combining FED, MEANS, and SAC, is presented to improve the three-dimensional (3D) resolution. The numerical study reveals that the lateral resolution is close to 0.085λ and axial resolution can be enhanced to 0.184λ. In addition, the SNR is improved simultaneously. The availability to improve 3D resolution of SAC is believed to be significant for biological imaging in the future.

3D resolution improvement in confocal microscopy by mirror refection interference and fluorescence emission difference

A method based on mirror refection interference and fluorescence emission difference is provided to improve the three-dimensional resolution and SNR in confocal microscopy. Theoretical analysis and experimental results have been performed to verify the effectiveness of the method. Our numerical simulations predict that lateral resolution close to 0.285λ and axial resolution close to 0.205λ are possible for practical confocal microscopy with visible light. Furthermore, the SNR has also been improved. The experimental results of both fluorescent beads and cell microtubules are presented to verify the applicability and effectiveness of our method. The ability to increase the lateral resolution and axial resolution using mirror refection interference and fluorescence emission difference without increasing the laser power is of great importance for imaging fluorescent biological specimens, which cannot tolerate high laser power.

Novel NBQ-48 as marker of hypoxic cells in 2D and 3D colon cancer cells.

This study presents the applicability of a novel nitro-substituted heterocyclic compound NBQ48, member of the family of compounds identified as 3 nitrobenzazolo[3, 2-a] quinolinium chloride salts (NBQS) as a screening tool to identify hypoxic tumor cells. The applicability was tested on COLO 205 colon cancer cells 2D and 3D cultures treated with NBQ48 to assess the formation of a bio-reduction fluorescent metabolite under hypoxic conditions in contrast, to those under aerobic environment. Hypoxic environment was created applying a controlled hypoxic gas chamber. Prior to testing the applicability of NBQ48 as a hypoxic fluorescent marker, cytotoxic studies where performed to identify a low-toxicity dose at 24 hours under aerobic and hypoxic environments that would allow the bio-reduction process with little toxicity. The differences in fluorescence emission after treatment was measured by fluorometer and fluorescence microscopy. Results indicated that cell treatments up to 24 hours with NBQ48 under aerobic environment did not reach an IC50 dose in COLO205 cells, however under hypoxic environment the IC50 reached at 100μM. The significant fluorescence increment after 24 and 48 hrs in 2D and 3D cultures treated with NBQ48 (75uM) at hypoxic in contrast to aerobic environments clearly demonstrated the need of a low oxygen content for the bio-reduction of the parent NBQ48. This study confirms the applicability of NBQ48 as markers for hypoxia in metabolically active 2D and 3D cultures. This hydrophilic hypoxic marker could additionally aid researchers in testing hypoxia activated pro-drugs for therapeutic applications in cancer as well as on other diseases where cellular hypoxia is a significant risk factor.

Dynamic live-cell super-resolution imaging with parallelized fluorescence emission difference microscopy

The investigation of the cell biology with fluorescence microscopy remains challenging because of the requirement of both high temporal and spatial resolution. In this paper, parallelized fluorescence emission difference microscopy (pFED) is presented to provide 2-fold increasing imaging speed than conventional FED and achieve high spatial resolution (0.2 λ–0.3 λ) with a novel system design. The super-resolution images are obtained by aligning and subtracting two images, that are acquired simultaneously using parallelized scanning solid- and donut-shaped excitation beams with a lateral offset between two foci. Herein, we demonstrate the general applicability of pFED with time-lapse imaging of various subcellular dynamics in live cells over an extended period.

Image scanning difference microscopy.

Here, we propose a novel imaging method, which is called image scanning difference microscopy (ISDM), for superresolution imaging. In ISDM, we implement a detector array composed of 19 avalanche photodiodes (APD) rather than single-point detector in standard confocal microscopy for reconstructing superresolved images with higher signal-to-noise ratio (SNR). Combining with our former proposed fluorescence emission difference (FED) method, we have achieved a lateral resolution of 111 nm (∼λ/6) without the damage of image quality, the highest FED resolution to the best of our knowledge. With its simple setup and remarkable performance, we believe that ISDM can become a versatile observation tool in biology and other fundamental studies. LAY DESCRIPTION: Fluorescence emission difference (FED) microscopy is a really simple and generalisable superresolved fluorescence microscopy method based on PSF engineering and difference algorithm recently. Compared to stimulated-emission-depletion fluorescence microscopy (STED), FED don't need complicated system or precise alignment and polarisation, available for wide variety of dyes and has low photobleaching and phototoxicity for living cells. However, the distortion caused by negative value is one of the biggest obstacles to the further development of FED. In light of this, we propose a novel superresolution imaging method based on the FED method with parallel detection system, which is called image scanning difference microscopy (ISDM). Our method has achieved a significant breakthrough in FED, increasing the resolution further while reducing artefacts generated by negative values, which cannot be accomplished through combining other methods. In addition, ISDM does not require complex setup and optical alignment, long time imaging and imposing no constraint on dyes. Importantly, we realised a transverse resolution of ∼λ/6 (triple diffraction limit) with single wavelength, single incident path and low light intensity, which has never been achieved in any other far-field superresolution microscopy.

Resolution enhancement of confocal fluorescence microscopy via two illumination beams

Confocal fluorescence microscopy is an effective imaging technique, but its resolution is limited by the diffraction-limit. Fluorescence emission difference (FED) method is a useful way to improve the resolution of confocal fluorescence microscopy, but the negative values generated during subtraction process might cause loss of valid information. In this paper, we propose one effective method to enhance the resolution of confocal fluorescence microscopy without generating significant negative values. The proposed method combines digital processing and FED method, obtaining the final images with higher resolution and less information loss.

Fluorescence emission difference microscopy by superoscillation excitation.

A superresolution fluorescence emission difference (FED) microscopy based on superoscillation excitation is investigated. The solid superoscillation excitation spot is produced by the radially polarised Laguerre-Gaussian beam (LG), and the donut superoscillation excitation spot is produced by the same LG beam with spiral phase modulation. We show that the superoscillation excitation can enhance the spatial resolution of FED microscopy. When the ratio of the pupil radius and the second moment radius of the LG3,1 beam is 0.85 and the subtractive factor is 0.3, the resolution can be enhanced about 2 times than the general confocal microscopy. Compared with the general FED microscopy, the resolution can be enhanced about 1.1 times. Our simulations also demonstrate that two-view Richardson-Lucy (RL) deconvolution method can reduce the effect of side lobes, which is related to subtractive factor and pinhole. LAY DESCRIPTION: The fluorescence emission difference (FED) microscopy is a high-resolution light microscopy, which is based on the subtraction of images produced by two different confocal imaging modes. Here, the solid excitation spot is used in one imaging mode, and the donut excitation spot is in another imaging mode. There are various ways that the solid and donut excitation spots can be structured, but superoscillation excitation spots are still not used in the FED microscopy. Considering that the main lobe of superoscillation can be arbitrarily small in principle, we exploit the radially polarised Laguerre-Gaussian beam (LG) and spiral phase plate to produce superoscillation solid and donut spots. We present a detailed analysis about the performance of FED microscopy via superoscillation excitation. Our analysis is based on vector diffraction theory and supported by simulations of imaging. We find the proposed FED microscopy has better resolution than the confocal microscopy or the general FED microscopy excited by Gaussian beam. Especially, two-view Richardson-Lucy (RL) deconvolution method can reduce the negative impact of strong side lobes, which is related to the subtractive factor and the size of pinhole located at the detector.

Streptavidin cooperative allosterism upon binding biotin observed by differential changes in intrinsic fluorescence

While the binding of biotin by streptavidin does not appear to be cooperative in the traditional sense of altered binding strength, it has been suggested that it may be cooperative in terms of differential structural changes in the protein. In this work we present intrinsic tryptophan fluorescence data as evidence of a cooperative structural change. The technique involves examination of the differences in fluorescence emission corresponding to distinct tryptophan populations accompanying protein-ligand binding. Specifically we note that the 335 nm emission population (i.e. more hydrophobic) saturates prior to the saturation of the 350 nm emission population commonly used in the standard binding activity assay. We also note that the wavelength of maximum emission, total integrated fluorescence emission and full width at half maximum during the titration of ligand into streptavidin also reach saturation before the expected 4:1 stoichiometric end point. This suggests that the binding of the first 3 biotins effect greater structural changes in the protein than the final ligand.

基于环形光瞳提高荧光辐射差分超分辨显微镜的成像分辨率

基于环形光瞳提高荧光辐射差分超分辨显微镜的成像分辨率

Phycoerythrin-phycocyanin aggregates and phycoerythrin aggregates from phycobilisomes of the marine red alga Polysiphonia urceolata

Phycoerythrin-phycocyanin aggregates and phycoerythrin aggregates showing special spectral characteristics were prepared from the partly dissociated products of phycobilisomes from Polysiphonia urceolata. The absorption difference spectra between the aggregates and phycoerythrins showing normal spectral characteristics show peaks at 583 nm. The fluorescence emission difference spectra between the phycoerythrin aggregates and normal phycoerythrins show peaks at 602 nm. The special spectral characteristics of the PE aggregates disappeared when the PE aggregates were dissociated in deionized water. The intact phycobilisomes show similar characteristics as phycoerythrin-phycocyanin aggregates. When the peak values at 583 nm in absorption difference spectra were compared with those at 615 nm, it can be found that the amount of the 583 nm chromophores in the phycoerythrin-phycocyanin aggregates and intact phycobilisomes is similar to the amount of PCB chromophores in phycocyanins. It can be concluded that the 583 nm chromophores are at the interface between phycoerythrins and phycocyanins in phycobilisomes and transfer the light energy absorbed by other chromophores in phycoerythrins to the PCB chromophores in phycocyanins. A rod linker polypeptide with molecular weight of 40 kDa was found in the phycoerythrin-phycocyanin aggregates and phycoerythrin aggregates, and it is believed to play roles in the spectral red shift of the aggregates.