Fluorescence Emission Difference Microscopy 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.

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.

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.

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.

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.

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.

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.

Enhancement of fluorescence emission difference microscopy using conjugated vortex phase modulation.

In this paper, we propose and demonstrate an improved fluorescence emission difference microscopy (FED) method, exploiting programmable phase modulation for image enhancement. The main novelty of the proposed approach lies in the matched size and intensity of two excitation spots. The proposed method improves the FED performance on image quality via artefact elimination. We demonstrate the feasibility of this method through theoretical studies and experimental tests. The experimental results of nanobeads and cells validate the practical performance of this method, which can enable reliable observations at superresolution in biomedical studies. LAY DESCRIPTION: In this paper, we propose a method to improve the imaging quality of regular fluorescence emission difference (FED) microscopy. In regular FED imaging, a solid and a doughnut excitation beam are successively used to acquire two images which are then subtracted with each other to improve the resolution of confocal microscopy. The doughnut beam can be generated by modulating the excitation beam with a vortex phase mask. Note that both of the excitation beam and the vortex phase mask must have the same handed direction in regular FED microscopy. However, some negative values may be produced and some information may be lost due to the subtraction process in regular FED imaging, which is mainly caused by the mismatched size and intensity of these two excitation spots. To address this issue, we propose conjugated FED (cFED) microscopy which additionally uses a conjugated vortex phase mask to modulate the solid beam to extend its focal spot size to be matched with the doughnut spot, which means the handed direction of the solid beam and the vortex phase mask is different. Besides, in order not to damage the resolution, the doughnut beam needs to be saturated to some degree. The experiment results show that, at the same resolution level, the negative values and the information loss in cFED image are all less than that of regular FED image.

Integrated dual-color stimulated emission depletion (STED) microscopy and fluorescence emission difference (FED) microscopy

We report on the design and implementation of an integrated dual-color stimulated emission depletion (STED) microscopy and fluorescence emission difference (FED) microscopy. This highly integrated system combines the respective advantages of STED and FED microscopies, enabling acquisition of super-resolution STED images with specific fluorophores and high laser power, or resolution-enhanced FED imaging for ordinary fluorescent dyes and other materials with moderate demand for laser power. The functional integration enables researchers to select the more appropriate microscopy according to practical scenario and requirements, giving full play to their respective strengths and satisfying the imaging demands maximally. The system’s resolution is characterized by dual-color imaging of fluorescent particles. Its practical applicability is verified through imaging various kinds of samples, including several fixed cells stained with different organic dyes and a given organic material—organometallic halide perovskite.

Non-bleaching fluorescence emission difference microscopy using single 808-nm laser excited red upconversion emission.

Optical super-resolution microscopy has become a powerful technique to help scientists to monitor the sample of interest at nanoscale. Fluorescence emission difference (FED) microscopy, a very facile super-resolution method, does not require high depleting laser intensity and is independent on the species of agents, which makes FED microscopy possess great potential. However, to date, the biomarkers applied in FED microscopy usually suffer from a photo-bleaching problem. In this work, by introducing Er3+ activated upconverting nanoparticles with red-color emission and non-photobleaching properties, we demonstrate nonbleaching super-resolution imaging with FED microscopy. The dopant neodymium ions (Nd3+) can work as highly efficient sensitizing ions and enable near infrared 808-nm CW laser excitation of relatively low power, which would potentially reduce high intensity/short-wavelength light induced tissue damage. Both simulations and experiments on monodispersed NaYF4:Nd3+/Yb3+/Er3+@NaYF4:Nd3+ UCNPs also indicate that the easy saturation of the multiphoton properties of these UCNPs is beneficial to resolution enhancement in FED microscopy.

Compact three-dimensional super-resolution system based on fluorescence emission difference microscopy

A compact microscope system for three-dimensional (3-D) super-resolution imaging is presented. The super-resolution capability of the system is based on a size-reduced effective 3-D point spread function generated through the fluorescence emission difference (FED) method. The appropriate polarization direction distribution and manipulation allows the panel active area of the spatial light modulator to be fully utilized. This allows simultaneous modulation of the incident light by two kinds of phase masks to be performed with a single spatial light modulator in order to generate a 3-D negative spot. The system is more compact than standard 3-D FED systems while maintaining all the advantages of 3-D FED microscopy. The experimental results demonstrated the improvement in 3-D resolution by nearly 1.7 times and 1.6 times compared to the classic confocal resolution in the lateral and axial directions, respectively.

Image scanning fluorescence emission difference microscopy based on a detector array.

We propose a novel imaging method that enables the enhancement of three-dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications.

Progress of point-wise scanning superresolution methods

Optical microscope has been giving impetus to the development of modern technology. As the advancement of these techniques, high resolution microscopy becomes crucial in biological and material researches. However, the diffraction limit restricts the resolution of conventional microscopy. In 1968, confocal microscopy, the first pointwise scanning superresolution method, appeared. It improves the imaging resolution, enhances the contrast, and thus breaks through the diffraction limit. Since then many superresolution methods have come into being, among which the pointwise scanning superresolution method earns reputation for its high imaging resolution and contrast. The stimulated emission depletion microscopy becomes the most prominent method with an achievable resolution of about 2.4 nm and then widely used. Besides, the newly developed fluorescence emission difference microscopy (FED) and the saturated absorption competition microscopy (SAC) have their advantages of non-constraint on fluorescent dyes, low saturated beam power, simplified optical setups, while they achieve a resolution of lower than /6. Further explorations of FED will be keen on vivo biological observations by using it, while that of SAC can concentrate on enhancing the resolution on a nanoscale and reducing the signal-to-noise ratio. In addition, the Airyscan technique in which a detector array is used for image acquisition, can serve as a complementary tool to further enhance the imaging quality of pointwise scanning superresolution method. The detector-array enables both the narrowed size of pinhole and the increasing of the acquired signal intensity by 1.84 folds. The other methods, e.g. superoscillation lens and high-index resolution enhancement by scattering, have the potentialities to obtain superresolved image in material science or deep tissues. After being developed in the past three decades, the superresolution methods now encounter a new bottleneck. Further improvement of the current methods is aimed at imaging depth, and being used more practically and diversely. In this review, we detailedly describe the above pointwise scanning superresolution methods, and explain their principles and techniques. In addition, the deficiencies and potentialities of these methods are presented in this review. Finally, we compare the existing methods and envision the next generation of the pointwise scanning superresolution methods.

Comparison of multi-mode parallel detection microscopy methods

Four microscopy resolution enhancement methods based on parallel detection were investigated in this study: confocal microscopy with four pinhole sizes, fluorescence emission difference microscopy (FED) based on parallel detection, Airyscan microscopy, and virtual k-vector modulation optical microscopy (Vikmom). These methods use different algorithms to process parallel detection data and achieve resolution improvement. We investigated these methods first by performing simulations and then experimentally. In this report, the basic theories of these methods are briefly introduced. Then, analyses and comparisons of their imaging performances, especially in terms of resolution improvement, imaging speed, and signal-to-noise ratio, are presented. Finally, the results of our comparative study are summarized.

Resolution enhancement of saturated fluorescence emission difference microscopy.

Fluorescence emission difference microscopy (FED) obtains resolution-enhanced images by subtracting acquired solid and doughnut confocal images. Because of the mismatch of the outer contours of the two subtraction components, negative values are inevitable in the conventional FED method, giving rise to deformations. In this study, by using a saturation effect, we obtain imaging results with a profile-extended solid and center-shrunken doughnut point spread function. Owing to the nonlinear effect, two better-matched saturated images not only eliminate the deformations, but also enhance the resolving ability and signal to noise ratio compared to conventional FED. Simulations based on the saturated model of rhodamine 6G, as well as experiments on biological samples, are presented to verify the capability of the proposed concept, while experimental results show the unprecedented resolving ability of the saturated FED method.

Saturated virtual fluorescence emission difference microscopy based on detector array

Virtual fluorescence emission difference microscopy (vFED) has been proposed recently to enhance the lateral resolution of confocal microscopy with a detector array, implemented by scanning a doughnut-shaped pattern. Theoretically, the resolution can be enhanced by around 1.3-fold compared with that in confocal microscopy. For further improvement of the resolving ability of vFED, a novel method is presented utilizing fluorescence saturation for super-resolution imaging, which we called saturated virtual fluorescence emission difference microscopy (svFED). With a point detector array, matched solid and hollow point spread functions (PSF) can be obtained by photon reassignment, and the difference results between them can be used to boost the transverse resolution. Results show that the diffraction barrier can be surpassed by at least 34% compared with that in vFED and the resolution is around 2-fold higher than that in confocal microscopy.

3D fluorescence emission difference microscopy based on spatial light modulator

We report three-dimensional fluorescence emission difference (3D-FED) microscopy using a spatial light modulator (SLM). Zero phase, 0–2[Formula: see text] vortex phase and binary 0-pi phase are loaded on the SLM to generate the corresponding solid, doughnut and z-axis hollow excitation spot, respectively. Our technique achieves super-resolved image by subtracting three differently acquired images with proper subtractive factors. Detailed theoretical analysis and simulation tests are proceeded to testify the performance of 3D-FED. Also, the improvement of lateral and axial resolution is demonstrated by imaging 100[Formula: see text]nm fluorescent beads. The experiment yields lateral resolution of 140[Formula: see text]nm and axial resolution of approximate 380[Formula: see text]nm.