Laser scanning confocal microscopy cannot distinguish fluorescence signals of different labels, and there are overlapping and interference of different fluorescence signals in the acquired images. In order to solve this problem, a laser scanning confocal microscopy spectral imaging system based on filters was designed, to realize spectral screening and detection of different fluorescence signals. Spectral screening of the optical signals from the laser scanning confocal microscopy was performed using dichroic filters and detected through the blue-violet channel, the green channel, the yellow channel and the red-orange channel, respectively. The spectral imaging system operates in the wavelength range of 400nm≤λ≤720nm. The green channel and the yellow channel jointly achieve signal detection in the 475nm≤λ≤625nm wavelength range. In addition, the green channel and the yellow channel can further screen the signal spectrum through the continuously variable filters. The minimum spectral screening resolution in the 475nm≤λ≤625nm wavelength range is better than 5nm. Spectral calibration light source is white LED, and the spectral analysis was performed by a fiber optic spectrometer. The spectral calibration results were tested using a low-pressure mercury lamp, and the experimental results verified the accuracy of the spectral calibration results. The spectral imaging system proposed in this paper, has the advantages of high spectral flexibility and high optical efficiency, meeting the requirements of engineering applications.
Imaging three-dimensional, subcellular structures with high axial resolution has always been the core purpose of fluorescence microscopy. However, trade-offs exist between axial resolution and other important technical indicators, such as temporal resolution, optical power density, and imaging process complexity. We report a new imaging modality, fluorescence interference structured illumination microscopy (FI-SIM), which is based on three-dimensional structured illumination microscopy for wide-field lateral imaging and fluorescence interference for axial reconstruction. FI-SIM can acquire images quickly within the order of hundreds of milliseconds and exhibit even 30 nm axial resolution in half the wavelength depth range without z-axis scanning. Moreover, the relatively low laser power density relaxes the requirements for dyes and enables a wide range of applications for observing fixed and live subcellular structures.
KEYWORDS: Modulation, Simulations, Point spread functions, Light sources and illumination, Molecules, Microscopy, 3D image processing, Voxels, 3D modeling, Phase shifts
In recent years, modulated illumination localization microscopy (MILM) methods have been proposed to provide around two-fold improvement in lateral localization precision over conventional single molecule localization microscopy methods with the same photon budget. However, MILM with laterally modulated illumination was so far reported in two-dimensional imaging modalities. To fully exploit its three-dimensional (3D) imaging potential, we propose a 3D Single-Molecule Modulated Illumination Localization Estimator (3D-SMILE) that uses the raw data measured from MILM, which has enabled a high localization precision that reaches the theoretical Cramér-Rao lower bound (CRLB) in all three dimensions. 3D-SMILE is based an optimal joint fitting algorithm implemented on a graphics processing unit (GPU) for acceleration. We have shown in simulations that the average lateral localization precision of 3D-SMILE has been improved by more than 3.5 folds over 3D-SMLM over an imaging depth range of around 1.2 μm.
Direct laser writing (DLW) enables arbitrary three-dimensional nanofabrication. However, the diffraction limit poses a major obstacle for realizing nanometer-scale features. Furthermore, it is challenging to improve the fabrication efficiency using the currently prevalent single-focal-spot systems, which cannot perform high-throughput lithography. To overcome these challenges, a parallel peripheral-photoinhibition lithography system with a sub-40-nm two-dimensional feature size and a sub-20-nm suspended line width was developed in our study, based on two-photon polymerization DLW. The lithography efficiency of the developed system is twice that of conventional systems for both uniform and complex structures. The proposed system facilitates the realization of portable DLW with a higher resolution and throughput.
Direct laser writing technology has potential applications in the fabrication of micro and nanostructures such as photonic crystals and photonic chips. However, current single-channel direct laser writing systems are mainly limited to low-flux systems, which have disadvantages such as low direct writing efficiency and slow writing speed. To overcome the limitations, we proposed a method based on screen division multiplexing of the spatial light modulator to parallelly fabricate the nanostructures. The spatial light modulator is divided into two parts to achieve a single column of 20 beams array. After optimizing the uniformity of the 20-beam, a 20-channel high-throughput parallel laser direct writing is thus achieved. This scheme directly writes repetitive patterns by an order of magnitude faster than single-beam laser direct writing, which greatly improves the speed of laser direct writing and has important applications in the direct writing of large-area repetitive devices.
Stimulated emission depletion (STED) nanoscopy is one of the most well-developed nanoscopy techniques that can provide subdiffraction spatial resolution imaging. Here, we introduce dual-modulation difference STED microscopy (dmdSTED) to suppress the background noise in traditional STED imaging. By applying respective time-domain modulations to the two continuous-wave lasers, signals are distributed discretely in the frequency spectrum and thus are obtained through lock-in demodulation of the corresponding frequencies. The background signals can be selectively eliminated from the effective signal without compromise of temporal resolution. We used nanoparticle, fixed cell, and perovskite coating experiments, as well as theoretical demonstration, to confirm the effectiveness of this method. We highlight dmdSTED as an idea and approach with simple implementation for improving the imaging quality, which substantially enlarges the versatility of STED nanoscopy.
A recently developed strategy to generate optical nonlinearities from various emitters localized in multilayer core/shell optical nanocrystals represents significant progress and great potential.
Recently, a new technique called MINFLUX was promoted and attained ~1-nanometer precision. However, MINFLUX is incapable of discerning two molecules within the diffraction-limited region unless with the help of on-off switching scheme of SMLM which yet entails time-consuming processes. Here, we produce a novel kind of focal spot pattern, called sub-diffraction dark spot, to localize molecules within the sub-diffraction region of interest. In our proposed technique nominated as sub-diffracted dark spot localization microscopy (SDLM), multiple molecules within the diffraction-limited region could be distinguished without the requirement of fluorescent switches. We have numerically presented the SDLM modality and some impacts, like intensity, are investigated. Simulative localization framework has been implemented on randomly-distributed and specifically-structured samples. SDLM is evidenced to have high localization accuracy and stability in densely-packed fluorescent solution.
KEYWORDS: Particles, Signal to noise ratio, Detection and tracking algorithms, Image processing, Microscopy, Algorithm development, Motion models, Point spread functions
With the development of super-resolution fluorescence microscopy, complex dynamic processes in living cells can be observed and recorded with unprecedented temporal and spatial resolution. Single particle tracking is the most important step to explore the relationship between the spatio-temporal dynamics of subcellular molecules and their functions. Although previous studies have developed single particle tracking algorithms to quantitatively analyze particle dynamics in cell, traditional tracking methods have poor performance when dealing with intersecting trajectories. This can be attributed to two main reasons: 1) They do not have point compensation process for overlapping points; 2) They use inefficient motion prediction models. In this paper, we presented a novel Fan-shaped Tracker (FsT) algorithm to reconstruct the trajectories of subcellular molecules in living cells. We proposed a customized point compensation method for overlapping points based on the fan-shape motion trend of the particles to solve the merging trajectory problem. Furthermore, we compared the performance of our Fan-shaped Tracker with five state-of-the-art tracking algorithms in simulated time-lapse movies with variable imaging quality. Our results showed that the Fan-shaped Tracker achieves better performance than other reported methods as we systematically evaluated using a set of standard evaluation parameters. We anticipate that our FsT method will have vast applications in tracking of moving objects in cell.
Nonlinear focal modulation microscopy (NFOMM) is a really simple and effective super-resolution imaging method based on phase modulation with high-intensity illumination, which can extend the effective spatial-frequency bandwidth of the imaging system and achieve a transverse resolution of ~ 60 nm (~λ/10). However, multiple images under different illumination pattern is needed, which result in limited imaging speed. A novel interleaved reconstruction method is proposed to increase the frame rate without any change in the raw data of NFOMM. Since allowing easy integration with confocal microscopes, we anticipate this method will be a useful observation tool in the biological community and other research field.
Super-resolution optical microscopy has enabled the observation of ultra-fine structures and features beyond the diffraction limit, among which nonlinear absorption has been a useful tool to investigate physical and biological characteristics in sub-100 nanometer range. Saturation competition microscopy (SAC), based on nonlinear absorption principle, has been demonstrated to obtain high resolution in either fluorescence or non-fluorescence imaging of biological applications. Furthermore, fluorescent nanodiamonds (FNDs) have been widely researched as nitrogenvacancy centers (NVCs) in FNDs are important medium in quantum entanglement. Here, we report on the characterization of NVCs using pulse SAC (pSAC) microscopy. Resolution of λ/6 has been reached and the experimental results shows that it has better signal-to-background ratio (SBR) with lower illumination intensity in contrast to stimulated emission depletion microscopy (STED).
Structured illumination microscopy(SIM) has shown priority in bio-research field due to its low photo-toxicity and high imaging speed. In addition to sinusoidal patterns, other types of illumination patterns have been reported to applied in structured illumination microscopy. Here we propose a method based on joint Richardson-Lucy deconvolution which reconstructs both the illumination patterns and object simultaneous to extend the application of structured illumination microscopy.
A new frequency-shifting confocal microscopy (FSCM) illuminated by an azimuthally polarized Bessel-Gaussian (BG) beam is investigated. The solid excitation spot is produced by the BG beam modulated by a spiral phase plate, and the donut excitation spot is directly obtained by the same BG beam. Through vector diffraction theory and two-view RL reconstruction algorithm, the optical transfer functions of two confocal imaging modes and the simulation imaging of FSCM are presented. The results show that, two illumination modes produced by the azimuthally polarized BG beam can enhance the spatial resolution of FSCM, the spatial resolution of reconstructed image is mainly depended on the illumination mode with higher frequency transfer efficiency, the small pinhole is helpful to improve the contrast and spatial resolution of image. When the iterations number is about 100, the reconstructed image has good quality. This FSCM is helpful to quickly realize super resolution and high contrast in cell imaging.
Light sheet fluorescence microscopy (LSFM) is widely used in biological imaging because of its low photobleaching and phototoxicity. The axial resolution of LSFM is determined and also limited by the thickness of the light sheet and the numerical aperture (NA) of the detection objective. We propose a novel method, light sheet modulation fluorescence microscopy (LSMFM) which is able to achieve a promising axial resolution enhancement of light sheet microscopy.
In this paper, a novel microscopic imaging technology, termed light field Fourier ptychographic microscopy (LFFPM), is presented which enables the reconstruction of wide-field, high-resolution images with the depth information by utilizing a light-emitting-diode (LED) illumination matrix.Conventional light field microscope lacks the ability of high lateral resolution because it sacrifices its lateral resolution to its angular resolution. Compared to conventional light field microscope, LFFPM enhances the lateral resolution with no need for the object to be in focus. Using LFFPM, the result shows that the lateral resolution is more than 2 times better than light field microscopy in simulation and 1.5 times in the experiment. Furthermore, the simulation result also shows that the ability of LFFPM to recovery the information of the phase and amplitude.Thus, this technology has a wide prospect of various biomedical applications such as neuroanatomy and haematology.
Light sheet fluorescence microscopy (LSFM) with fluorescence emission difference (FED) obtains axial resolution-enhanced images by the subtraction of signals excited by different light sheets, Gaussian light sheet and negative light sheet. Negative values are inevitable in FED LSFM with conventional subtraction coefficient, due to mismatches in the outer contours of the raw signals excited by different light sheets, causing image distortion. In this paper, we propose a method based on tangent-function subtraction coefficient to reduce image distortion. This tangent-function subtraction coefficient is introduced to minimize image distortion without lowering the axial resolution. The value of the variable subtraction coefficient is obtained from the intensity value of raw signals. We performed experiments using fluorescent beads with a diameter of 1 micron and compared the subtraction results with constant and tangent-function subtraction coefficient. The experimental results demonstrated that the proposed tangent-function subtraction coefficient has an advantage over the conventional constant coefficient.
Light sheet fluorescence microscopy is an imaging method in which the sample is illuminated from the side by a wide and relatively thin light sheet. This method minimizes the out-of-focus excitation, provides a good balance between spatial resolution and temporal resolution, and mitigates photobleaching and photodamage. Unfortunately, the axial resolution of light sheet fluorescence microscopy is much lower than lateral resolution, which limits the achievement of isotropic imaging. We propose to combine the subtraction method with light sheet fluorescence microscopy to improve the axial resolution. The experimental results demonstrate that the proposed method can produce higher axial resolution than that of conventional light sheet fluorescence microscopy. In addition, a way to reduce negative values in subtraction method is also discussed.
Assessing tissues’ inhomogeneous optical properties is helpful for diagnosis, but high-cost measurement and experimental setups limit its development, data collecting and applications. In this paper, a portable microscope is proposed to assess the inhomogeneous optical properties of the sample. With a LED illumination, accurate quantitative phase (QP) map can be recovered from 5 intensity images captured at different axial positions. Then based on the scattering-phase theorem and statistical dispersion relation (SDR), the inhomogeneous optical properties of the sample can be quantitatively assessed from these QP maps. In contrast to DHM and SLIM, our setup is cost-effective, use-flexible, and with a small amount of data acquisition, thus having the potential to promote the development of assessing tissues’ inhomogeneous optical properties, especially in resource-limited areas.
We introduce a phase mask with a left-handed azimuthal polarization to generate a special shape of point spread function. The proposed point spread function has low-intensity and small size in the central lobe. This central lobe of the point spread function is used to obtain an image on the detector, and therefore the lateral resolution is improved. The proposed method can achieve a remarkable improvement in the lateral resolution and the depth of field compared with conventional confocal scanning microscopy. The image simulation demonstrated that the images obtained by this proposed method have better imaging quality.
We present an alternative approach to realize structured illumination microscopy (SIM), which is capable for live cell imaging. The prototype utilizes two sets of scanning galvo mirrors, a polarization converter and a piezo-platform to generate a fast shifted, s-polarization interfered and periodic variable illumination patterns. By changing the angle of the scanning galvanometer, we can change the position of the spots at the pupil plane of the objective lens arbitrarily, making it easy to switch between widefield and total internal reflection fluorescent-SIM mode and adapting the penetration depth in the sample. Also, a twofold resolution improvement is achieved in our experiments. The prototype offers more flexibility of pattern period and illumination orientation changing than previous systems.
Assessing tissues’ inhomogeneous optical properties is helpful for label-free diagnosis. In this paper, a method and a setup are proposed to assess the tissues’ inhomogeneous optical properties based on quantitative phase imaging (QPI) and dark-field microscopy using LED illumination. In the bright-field and coherent dark-field imaging, quantitative phase (QP) can be recovered from multi-frame intensity images captured at different axial positions by Transport-intensity-equation (TIE) and multi-height Gerchberg-Saxton(G-S) phase retrieval algorithms. In the incoherent dark-field imaging, some scattering samples can be observed easily without any computation and the resolution can be improved in contrast to bright-field illumination. According to the scattering-phase theorem and the statistical dispersion relation (SDR), tissues’ inhomogeneous optical properties can be assessed. In our experiments, we find, it is a good idea to combine QP maps and dark-field images to assess inhomogeneous optical properties of bio-samples. In contrast to the DHM and interferometers, our setup is cost-effective and use-flexible, thus having the potential to serve as a very powerful tool in biological applications.
A stable optical system is required to acquire a high-quality image. A motionless lensless setup is designated to obtain high-resolution and large field of view images. The sample is sequentially illuminated with multiple random phase patterns, and the recorded images are subtracted from the system calibration images correspondingly. The resultant images are propagated to the sample plane. The summation of all images yields a final image with resolution of ∼4 μm, field of view of ∼15 mm2, and better signal-to-noise ratio. This technique provides a compact, stable, and cost-effective optical system.
KEYWORDS: Optical transfer functions, Wavefronts, Image restoration, Imaging systems, Signal to noise ratio, Digital signal processing, Image compression, Phase shift keying, Modulation transfer functions, Point spread functions
The random selection of phase mask parameters will cause the degradation of imaging quality, which can be fixed through the optimization process. We introduce an evaluation function based on the use of multitarget optimization to obtain optimal phase mask parameters. The proposed method gives the optimal phase mask parameters, which produce together an imaging quality at all defocus positions as best as possible. The cubic phase mask and the general cubic phase mask are both used to perform optimization with a proposed evaluation function. The simulation result gives quite interesting values for the two types of these phase masks.
Most of the quantitative phase microscopy systems are unable to provide depth-resolved information for measuring complex biological structures. Optical diffraction tomography provides a non-trivial solution to it by 3D reconstructing the object with multiple measurements through different ways of realization. Previously, our lab developed a reflection-mode dynamic speckle-field phase microscopy (DSPM) technique, which can be used to perform depth resolved measurements in a single shot. Thus, this system is suitable for measuring dynamics in a layer of interest in the sample. DSPM can be also used for tomographic imaging, which promises to solve the long-existing “missing cone” problem in 3D imaging. However, the 3D imaging theory for this type of system has not been developed in the literature. Recently, we have developed an inverse scattering model to rigorously describe the imaging physics in DSPM. Our model is based on the diffraction tomography theory and the speckle statistics. Using our model, we first precisely calculated the defocus response and the depth resolution in our system. Then, we further calculated the 3D coherence transfer function to link the 3D object structural information with the axially scanned imaging data. From this transfer function, we found that in the reflection mode excellent sectioning effect exists in the low lateral spatial frequency region, thus allowing us to solve the “missing cone” problem. Currently, we are working on using this coherence transfer function to reconstruct layered structures and complex cells.
Diffraction Phase Microscopy (DPM) is a widely used quantitative phase imaging method, whose common-path nature endows it with low noise and high sensitivity. Current applications of DPM include biological topography as well as biological dynamics for its nondestructive feature. Many different forms of DPM based on the original idea have appeared according to the different demands. In this paper, both the principle and the DPM classification will be given a comprehensive description. Furthermore, the future trend of DPM development is also discussed.
Total internal reflection fluorescence microscopy (TIRFM) has been widely used in biomedical research to visualize cellular processes near the cell surface. In this study, a novel multi-angle ring-illuminated TIRFM system, equipped with two galvo mirrors that are on conjugate plan of a 4f optical system was developed. Multi-angle TIRFM generates images with different penetration depths through the controlled variation of the incident angle of illuminating laser. We presented a method to perform three-dimensional (3-D) reconstruction of microtubules from multi-angle TIRFM images. The performance of our method was validated in simulated microtubules with variable signal-to-noise ratios (SNR) and the axial resolution and accuracy of reconstruction were evaluated in selecting different numbers of illumination angles or in different SNR conditions. In U373 cells, we reconstructed the 3-D localization of microtubules near the cell surface with high resolution using over a hundred different illumination angles. Theoretically, the presented TIRFM setup and 3-D reconstruction method can achieve ∼40 nm axial resolution in experimental conditions where SNR is as low as 2, with ∼35 different illumination angles. Moreover, our system and reconstruction method have the potential to be used in live cells to track membrane dynamics in 3-D.
In the past decade, various quantitative phase microscopy (QPM) techniques have emerged, driven by the need to study biological samples non-invasively. However, the fundamental limit for phase noise is scarcely discussed in the literature. In a typically off-axis phase microscope system, the phase noise is limited to a few milliradians using a moderate camera. Common-path QPMs offer much reduced phase noise compared to typical Mach-Zehnder-based systems. However, further scaling down the phase noise becomes difficult. Here we propose a high-sensitivity common-path QPM that promises to reduce the phase noise by a factor of 10 (assuming the mechanical noise is negligible). This is achieved by a specifically designed signal filter, leaving only the subtle phase fluctuations coming from the dynamics sample scattering. By working at photon shot-noise limited detection, we can magnify the subtle phase contrast which is proportional to the camera well depth. We expect this system to have the height sensitivity similar to an atomic force microcopy, while measuring biological structures with a full field of view in a single-shot. We plan to use this system to study cell dynamics, particularly lamellipodial height fluctuations as well as stiffer cell membrane fluctuations.
We have designed and built a time-gated continuous wave stimulated emission depletion (CW-STED) nanoscopy to visualize microstructures beyond the diffraction limit. An off-line time-gating detection was performed with the help of time-correlated single-photon counting technique. Experimental results showed that before time-gating, the resolution of our system was about 75 nm with a depletion beam (592 nm) power of 200 mW. By using the off-line time-gating detection, the resolution was further improved to 38 nm. Biological samples were also used to test the performance of our time-gated CW-STED, and a resolution of 70 nm was achieved with a depletion beam (592 nm) power of 85 mW. Detailed principles of time-gated CW-STED were discussed in the text. The time-gated STED provides a better resolution with finite laser power.
Stimulated emission depletion microscopy (STED) has been proved to be a feasible and straightforward method of breaking the conventional diffraction barrier in the far-field. In this paper, we design and setup a home-built high speed STED microscope. Both the excitation beam (488nm) and the depletion beam (592nm) are provided by continuous wave lasers. By using a pair of galvo mirrors (TILL Yanus IV Digital Scan Head), we realize a pixel dwell time down to 2 μs in the experiment which enables an acquisition speed of 2 frames per second in an imaging field of 5*5 μm with an individual pixel size of 10nm. The image acquisition process is controlled by the software Imspector. In the manuscript, we give a clear description on how to build the microscope and also conduct several experiments to evaluate its performance in practice. A spatial resolution of <100nm, which is well beyond the diffraction barrier has been demonstrated in both nanoparticles and biomedical samples. Featuring a superresolution together with a high imaging speed, our STED microscope has big potential to be widely applied in related scientific researches.
We present here a stimulated emission based fluorescence lifetime imaging (FLIM) scheme using a pair of synchronized diode lasers operating at gain switched pulse mode. The two semiconductor lasers, with wavelengths at 635 nm and 700 nm,
serve as the excitation and the stimulation light sources for the ATTO647N labeled sample, respectively. FLIM is readily
achieved with their relative time delay controlled electronically. The coherent nature of the stimulated emission signal also allows FLIM at long working distance. In this way, a high performance all-semiconductor FLIM module is realized in a flexible, compact, and cost effective configuration.
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