Fig 3 - uploaded by Justin E Jureller
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(a) Single frame two-photon fluorescence multifocal raster scan image of a packed monolayer of 500 nm diameter microspheres at 1 fps. The unit cell boundaries are clearly oversampled, yielding artifacts in a brickwork pattern. (b) Single frame SS-MMM fluorescence image of the same area. The brickwork artifacts in (a) are not observed.
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Multiparticle tracking with scanning confocal and multiphoton fluorescence imaging is increasingly important for elucidating biological function, as in the transport of intracellular cargo-carrying vesicles. We demonstrate a simple rapid-sampling stochastic scanning multifocal multiphoton microscopy (SS-MMM) fluorescence imaging technique that enab...
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... points. Triangular drive waveforms are preferred to simple square and step drive waveforms due to the minimization of stop-start impulses to the scanner. Stochastic scanning enables high-frequency content at all points in the scan pattern. Therefore stochastic scanning is mechanically more efficient than rastering at a sub-resonant frequency. Fig. 3(a) shows a single frame 2-photon fluorescence image of a monolayer of 500 nm diameter fluorescent microspheres acquired with raster scanning at 1 fps. Boundaries between periodic replica cells are clearly over-sampled and are observed as bright edges of the single beam scan area. The slow axis edges (horizontal) are deemphasized versus ...
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... of 500 nm diameter fluorescent microspheres acquired with raster scanning at 1 fps. Boundaries between periodic replica cells are clearly over-sampled and are observed as bright edges of the single beam scan area. The slow axis edges (horizontal) are deemphasized versus the fast axis edges (vertical), but still noticeable for this galvanometer. Fig. 3(b) shows a sin- gle frame 2-photon fluorescence image of the same area obtained by stochastic scanning. The sampling is much more uniform and the image does not display the edge sampling artifacts. This relatively slow imaging rate (1 fps) represents the best-case-scenario for raster scanning; imaging at faster rates will only increase ...
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... pattern's edge. The overall effect would be to decrease the instantaneous peak power found in the foci, and therefore reduced 2-photon fluorescence. In practice, this effect was small, but noticeable at the edge of the pattern. Identical reductions in intensity toward the edge of the image are observed for both raster and stochastic scanning in Fig. ...
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Indirect-imaging methods involve at least two steps, namely optical recording and computational reconstruction. The optical-recording process uses an optical modulator that transforms the light from the object into a typical intensity distribution. This distribution is numerically processed to reconstruct the object’s image corresponding to differe...
Citations
... The grating period is 2 µm and the pixel size is 10 nm for 3x5 beam splitter. The maximal diffraction angle is 27°for the [1,2] diffraction order, so the fan-out angle is 54°. The optimal DOE 3D profile with height H=455 nm is shown in Fig. 7. ...
... Applications of fan-out elements include multi-focal microscopy [1,2], camera calibration [3], optical system distortion measurement [4], optical interconnects [5], and structured light projectors [6,7]. In particular, wide angle DOEs are used in promising field of applications [7][8][9] with recent progress in fabrication technology realizing nanoscale features. ...
Diffractive optical elements are ultra-thin optical components required for a variety of applications because of their high design flexibility. We introduce a gradient-based optimization method based on a step-transition perturbation approach which is an efficient approximation method using local field perturbations due to sharp surface profile transitions. Step-transition perturbation approach be available to calculate the gradient of figure of merit straightforwardly, we implemented optimization method based on this gradient. This fast and accurate inverse design creates binary (2-level) diffractive elements with small features generating the wide angle beam arrays. The results of the experimental characterization confirm that the optimization based on the perturbation method is valid for 1-to-117 fan-out grating generating beam pattern of linear array.
... Diffractive optical elements (DOEs) permit the generation of the spatial distribution of light beams by using a single element, and they are used for a variety of applications due to their high design flexibility, compact size, and mass productivity. For instance, diffractive beam splitters, often also referred to as fan-out gratings, have various applications, ranging from optical interconnects [1], multifocal microscopy [2,3], camera calibration [4], optical system distortion measurement [5] to structured light projectors [6,7]. For many applications, the goal is to propagate all the light in the desired orders and avoid losses. ...
Diffractive optical elements are ultra-thin optical components required for constructing very compact optical 3D sensors. However, the required wide-angle diffractive 2D fan-out gratings have been elusive due to design challenges. Here, we introduce a new strategy for optimizing such high-performance and wide-angle diffractive optical elements, offering unprecedented control over the power distribution among the desired diffraction orders with only low requirements with respect to computational power. The microstructure surfaces were designed by an iterative gradient optimization procedure based on an adjoint-state method, capable to account for application-dependent target functions while ensuring compatibility with existing fabrication processes. The results of the experimental characterization confirm the simulated tailored power distributions and optical efficiencies of the fabricated elements.
... To improve imaging speed, electrical scanning technologies such as galvanometer scanning and resonance mirror scanning have been developed, and many types of fast excitation methods to increase imaging speed have been developed. [9][10][11][12][13][14][15] Multifocal multiphoton microscopy (MMM) was reported by Hell's group in 1998. 11 The laser's full power is distributed into multiple beamlets that are focused to form a multifocal array. ...
Using the combination of a reflective blazed grating and a reflective phase-only diffractive spatial light modulator (SLM), scanless multitarget-matching multiphoton excitation fluorescence microscopy (SMTM-MPM) was achieved. The SLM shaped an incoming mode-locked, near-infrared Ti:sapphire laser beam into an excitation pattern with addressable shapes and sizes that matched the samples of interest in the field of view. Temporal and spatial focusing were simultaneously realized by combining an objective lens and a blazed grating. The fluorescence signal from illuminated areas was recorded by a two-dimensional sCMOS camera. Compared with a conventional temporal focusing multiphoton microscope, our microscope achieved effective use of the laser power and decreased photodamage with higher axial resolution.
... Confocal and multi-photon excitation methods [1][2][3][4][5][6][7]offer fine optical sectioning capabilities to reject out-of-focus background, but the price to pay for improved image quality in confocal or multi-photon endoscopic techniques is a point-by-point scan time [8]. Efforts to improve scanning efficiency by optimizing the scanning algorithm [9,10] or increasing the number of focal points [11] are ongoing. However, these methods can increase endoscopic system complexity and do not eliminate the need for moving parts; therefore, ongoing efforts are focused to eliminate scanning and enhance acquisition speed for 3D imaging. ...
A wide-field multi-plane endoscopic system incorporating multiplexed volume holographic gratings and Talbot illumination to simultaneously acquire optically sectioned fluorescence images of tissue structures from different depths is presented.
... Furthermore, accumulation effects appear from our data to have a major effect on development perturbations. Further improvement in the reduction of phototoxic effects might thus be obtained by increasing the scanning speed on the sample [70] and adapting the scanning pattern to increase the delay between two successive illuminations on the same point, e.g. by using random scanning patterns [71]. At shorter time scales (1 ns-1 ms), it has also been shown that reducing the repetition rate of the excitation source could significantly reduce photobleaching and photodamages [23,72,73]. ...
Light-induced toxicity is a fundamental bottleneck in microscopic imaging of live embryos. In this article, after a review of photodamage mechanisms in cells and tissues, we assess photo-perturbation under illumination conditions relevant for point-scanning multiphoton imaging of live Drosophila embryos. We use third-harmonic generation (THG) imaging of developmental processes in embryos excited by pulsed near-infrared light in the 1.0-1.2 µm range. We study the influence of imaging rate, wavelength, and pulse duration on the short-term and long-term perturbation of development and define criteria for safe imaging. We show that under illumination conditions typical for multiphoton imaging, photodamage in this system arises through 2- and/or 3-photon absorption processes and in a cumulative manner. Based on this analysis, we derive general guidelines for improving the signal-to-damage ratio in two-photon (2PEF/SHG) or THG imaging by adjusting the pulse duration and/or the imaging rate. Finally, we report label-free time-lapse 3D THG imaging of gastrulating Drosophila embryos with sampling appropriate for the visualisation of morphogenetic movements in wild-type and mutant embryos, and long-term multiharmonic (THG-SHG) imaging of development until hatching.
... The third method is multifocal multiphoton microscopy (MMM), which is an intermediate approach between the two previously described techniques [17][18][19][20][21][22] . With a lenslet array or diffractive optical element (DOE) 23,24 , a number of foci are generated simultaneously and scanned together. ...
Multifocal multiphoton microscopy (MMM) achieves fast imaging by simultaneously scanning multiple foci across different regions of specimen. The use of imaging detectors in MMM, such as CCD or CMOS, results in degradation of image signal-to-noise-ratio (SNR) due to the scattering of emitted photons. SNR can be partly recovered using multianode photomultiplier tubes (MAPMT). In this design, however, emission photons scattered to neighbor anodes are encoded by the foci scan location resulting in ghost images. The crosstalk between different anodes is currently measured a priori, which is cumbersome as it depends specimen properties. Here, we present the photon reassignment method for MMM, established based on the maximum likelihood (ML) estimation, for quantification of crosstalk between the anodes of MAPMT without a priori measurement. The method provides the reassignment of the photons generated by the ghost images to the original spatial location thus increases the SNR of the final reconstructed image.
... When they are used in the MMM system, oversampling or subsampling on the boundaries of each small region may occur and artifacts in brickwork pattern may be yielded, which will compromise the quantitative analysis of the research object. In order to obtain high-speed 3D imaging and study dynamic particle tracking, Jureller et al. developed a stochastic scanning MMM (SS-MMM)°uorescence imaging technique, 9 in which a 10 Â 10 hexagonal focus array was produced using a DOE and then scanned with white noise driven galvanometers. With the SS-MMM system, uniform, rapid and continuous scanning of the sample was obtained. ...
Multifocal multiphoton microscopy (MMM) has greatly improved the utilization of excitation light and imaging speed due to parallel multiphoton excitation of the samples and simultaneous detection of the signals, which allows it to perform three-dimensional fast fluorescence imaging. Stochastic scanning can provide continuous, uniform and high-speed excitation of the sample, which makes it a suitable scanning scheme for MMM. In this paper, the graphical programming language — LabVIEW is used to achieve stochastic scanning of the two-dimensional galvo scanners by using white noise signals to control the x and y mirrors independently. Moreover, the stochastic scanning process is simulated by using Monte Carlo method. Our results show that MMM can avoid oversampling or subsampling in the scanning area and meet the requirements of uniform sampling by stochastically scanning the individual units of the N × N foci array. Therefore, continuous and uniform scanning in the whole field of view is implemented.
... Interference on out-of-focus planes occurs between adjacent points when they are too close, degrading the resolution of the imaging system. In recent years, multifocal multiphoton microscopy (MMM) has emerged as a useful tool for high-speed-image acquisition with high resolution [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. In MMM, a single femtosecond (fs) laser beam is spatially or temporally split into N sub-beams to generate multiple foci on the sample plane. ...
We developed a novel addressable multiregional multiphoton microscope
that employs a fast one-dimensional discrete-line scanning approach
based on a spatial light modulator (SLM). The phase-only SLM shapes an
incoming mode-locked, near-infrared Ti:sapphire laser beam into multiple
specific discrete-lines, which are designed according to the sizes and
locations of the target samples. Only the target-sample areas of are
scanned one-dimensionally, resulting in an efficient use of the laser's
power. Compared with conventional multiphoton microscopies, this
technique shortens scanning time and minimizes photodamage by
concentrating scanning energy and dwell time on the areas of interest.
Additionally, our discrete-line-focus design eliminates the cross-talk
that occurs in conventional one-dimensional line-scanning multiphoton
microscopes, thus enhancing the lateral and axial resolutions of the
line-scanning imaging system.
... Nowadays microstructures have a very wide range of applications. They are used for beam shaping, splitting and steering [1], in optical interconnections [2], optical tweezers [3], multiphoton spectroscopy [4], lithographic fabrication of photonic crystals [5], object contouring [6], biological microscopy [7], measurement of moving object [8], characterization of micro optical elements, systems with CD and DVD [9], in X-ray microscopy [10], etc. Therefore the quality of produced microstructures is very important. E-beam lithography (EBL) is the most commonly used technique for micro-or nanolithography. ...
The thermal imprint process of polymer micro-patterning is widely applied in areas such as manufacturing of optical parts, solar energy, bio-mechanical devices and chemical chips. Polycarbonate (PC), as an amorphous polymer, is often used in thermoforming processes because of its good replication characteristics. In order to obtain replicas of the best quality, the imprint parameters (e.g., pressure, temperature, time, etc.) must be determined. Therefore finite element model of the hot imprint process of lamellar periodical microstructure into PC has been created using COMSOL Multiphysics. The mathematical model of the hot imprint process includes three steps: heating, imprinting and demolding. The material properties of amorphous PC strongly depend on the imprint temperature and loading pressure. Polycarbonate was modelled as an elasto-plastic material, since it was analyzed below the glass transition temperature. The hot imprint model was solved using the heat transfer and the solid stress-strain application modes with thermal contact problem between the mold and polycarbonate. It was used for the evaluation of temperature and stress distributions in the polycarbonate during the hot imprint process. The quality of the replica, by means of lands filling ratio, was determined as well.
