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This PDF file contains the front matter associated with SPIE Proceedings Volume 9717, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.
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We present a new type of adaptive lens with 18 actuators that can correct up the 4th order of aberration. The Multi-actuator Adaptive Lens (M-AL) can guarantee a good level of aberration correction for many applications and, with respect to deformable mirror, it allows the realization of more compact and simple optical systems. The adaptive lens is based on the use of piezoelectric actuators and, without any obstruction or electrodes in the clear aperture, can guarantee a fast response time, in the order of about 10ms. The clear aperture of the M-AL allows its use in “classical” Adaptive Optics configuration together with a wavefront sensor. To introduce a further simplification to the optical system design we show that the adaptive lens can be also driven with a wavefront sensorless control algorithm during in vivo optical coherence tomography of the human retina and for two-photon excitation fluorescence microscopy. In the experimental setup we used two aberration correcting devices a commercial adaptive lens (AL) with a high dynamic range to correct for defocus and the Multi-actuator Adaptive Lens (M-AL) to correct for the Zernike aberrations up to the 4th order. Experimental results show that the ocular aberrations of human eyes can be successfully corrected with our M-AL for pupils of 5mm and that retinal cones can be readily imaged.
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When a light propagates through highly disordered medium, its optical parameters such as amplitude, phase and polarization states are completely scrambled because of multiple scattering events. Since the multiple scattering is a fundamental optical process that contains extremely high degrees of freedom, optical information of a transmitted light is totally mingled. Until recently, the presence of multiple scattering in an inhomogeneous medium is considered as a major obstacle when manipulating a light transmitting through the medium. However, a recent development of wavefront shaping techniques enable us to control the propagation of light through turbid media; a light transmitting through a turbid medium can be effectively controlled by modulating the spatial profile of the incident light using spatial light modulator.
In this work, stand-alone scattering optical device is proposed; a holographic photopolymer film, which is much economic compared to the other digital spatial light modulators, is used to record and reconstruct permanent wavefront to generate optical field behind a scattering medium. By employing our method, arbitrary optical field can be generated since the scattering medium completely mixes all the optical parameters which allow us to access all the optical information only by modulating spatial phase profile of the impinging wavefront. The method is experimentally demonstrated in both the far-field and near-field regime where it shows promising fidelity and stability. The proposed stand-alone scattering optical device will opens up new avenues for exploiting the randomness inherent in disordered medium.
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AO for Microscopy and Optical Coherence Tomography I
In-vivo imaging of the eye’s fundus is widely used to study eye’s health. State of the art Adaptive Optics devices can resolve features up to a lateral resolution of 1.5 um. This resolution is still above what is needed to observe sub-cellular structures such as cone cells (1-1.25 um diameter). This limit in resolution is due to the small numerical aperture of the eye when the pupil is fully dilated (max 0.24).
In our work, we overcome this limit using a non-standard illumination scheme. A laser beam is shined on the lateral choroid layer, whose scattered light is illuminating the eye’s fundus. Thanks to a Spatial Light Modulator the scattered light from the choroid layer can be manipulated to produce a scanning focus spot on the fundus. The intensity of the reflected light from the fundus is collected from the pupil and used for reconstructing the image.
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This proceeding reports early results in the development of a new technique for adaptive optics in confocal microscopy. The term adaptive optics refers to the branch of optics in which an active element in the optical system is used to correct inhomogeneities in the media through which light propagates. In its most classical form, mostly used in astronomical imaging, adaptive optics is achieved through a closed loop in which the actuators of a deformable mirror are driven by a wavefront sensor. This approach is severely limited in fluorescence microscopy, as the use of a wavefront sensor requires the presence of a bright, point like source in the field of view, a condition rarely satisfied in microscopy samples. Previously reported approaches to adaptive optics in fluorescence microscopy are therefore limited to the inclusion of fluorescent microspheres in the sample, to use as bright stars for wavefront sensors, or time consuming sensorless optimization procedures, requiring several seconds of optimization before the acquisition of a single image.
We propose an alternative approach to the problem, implementing sensorless adaptive optics in a Programmable array microscope. A programmable array microscope is a microscope based on a digital micromirror device, in which the single elements of the micromirror act both as point sources and pinholes.
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Wavefront sensorless schemes for correction of aberrations induced by biological specimens require a time invariant property of an image as a measure of fitness. Image intensity cannot be used as a metric for Single Molecule Localization (SML) microscopy because the intensity of blinking fluorophores follows exponential statistics. Therefore a robust intensity-independent metric is required. We previously reported a Fourier Metric (FM) that is relatively intensity independent. The Fourier metric has been successfully tested on two machine learning algorithms, a Genetic Algorithm and Particle Swarm Optimization, for wavefront correction about 50 μm deep inside the Central Nervous System (CNS) of Drosophila. However, since the spatial frequencies that need to be optimized fall into regions of the Optical Transfer Function (OTF) that are more susceptible to noise, adding a level of denoising can improve performance. Here we present wavelet-based approaches to lower the noise level and produce a more consistent metric. We compare performance of different wavelets such as Daubechies, Bi-Orthogonal, and reverse Bi-orthogonal of different degrees and orders for pre-processing of images.
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AO for Microscopy and Optical Coherence Tomography II
The development of fluorescence microscopy, which allows live-cell imaging with high labeling specificity, has made the visualization of cellular architecture routine. However, for centuries, the spatial resolution of optical microscopy was fundamentally limited by diffraction. The past two decades have seen a revolution in far-field optical nanoscopy (or “super-resolution” microscopy). The best 3D resolution is achieved by optical nanoscopes like the isoSTED or the iPALM/4Pi-SMS, which utilize two opposing objective lenses in a coherent manner. These system are, however, also more complex and the required interference conditions demand precise aberration control. Our research involves developing novel adaptive optics techniques that enable high spatial and temporal resolution imaging for biological applications. In this talk, we will discuss how adaptive optics can enhance dual-objective lens nanoscopes. We will demonstrate how adaptive optics devices provide unprecedented freedom to manipulate the light field in isoSTED nanoscopy, allow to realize automatic beam alignment, suppress the inherent side-lobes of the point-spread function, and dynamically compensate for sample-induced aberrations. We will present both the theoretical groundwork and the experimental confirmations.
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Adaptive optics (AO) is necessary to correct aberrations when imaging the mouse eye with high numerical aperture. In order to obtain cellular resolution, we have implemented wavefront sensorless adaptive optics for in vivo fluorescence imaging of mouse retina. Our approach includes a lens-based system and MEMS deformable mirror for aberration correction. The AO system was constructed with a reflectance channel for structural images and fluorescence channel for functional images. The structural imaging was used in real-time for navigation on the retina using landmarks such as blood vessels. We have also implemented a tunable liquid lens to select the retinal layer of interest at which to perform the optimization. At the desired location on the mouse retina, the optimization algorithm used the fluorescence image data to drive a modal hill-climbing algorithm using an intensity or sharpness image quality metric. The optimization requires ~30 seconds to complete a search up to the 20th Zernike mode. In this report, we have demonstrated the AO performance for high-resolution images of the capillaries in a fluorescence angiography. We have also made progress on an approach to AO with pupil segmentation as a possible sensorless technique suitable for small animal retinal imaging. Pupil segmentation AO was implemented on the same ophthalmic system and imaging performance was demonstrated on fluorescent beads with induced aberrations.
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The accurate focusing of ultrashort laser pulses is extremely important in multiphoton microscopy. Using adaptive optics to manipulate the incident ultrafast beam in either the spectral or spatial domain can introduce significant benefits when imaging. Here we introduce pulse front adaptive optics: manipulating an ultrashort pulse in both the spatial and temporal domains. A deformable mirror and a spatial light modulator are operated in concert to modify contours of constant intensity in space and time within an ultrashort pulse. Through adaptive control of the pulse front, we demonstrate an enhancement in the measured fluorescence from a two photon microscope.
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Optical imaging of biological samples or living tissue structures requires light delivery to a region of interest and then collection of scattered light or fluorescent light in order to reconstruct an image of the object. When the coherent illumination light enters bulky biological object, each of scattering center (single molecule, group of molecules or other sample feature) acts as a secondary light source. As a result, scattered spherical waves from these secondary sources interact with each other, generating cross-talk noise between optical channels (eigenmodes). The cross-talk effect have serious impact on the performance of the imaging systems. In particular it reduces an ability of optical system to transfer high spatial frequencies thereby reducing its resolution. In this work we present a fast method to eliminate all unwanted waves combination, that overlap at image plane, suppressing recovery of high spatial frequencies by using the spatio-temporal optical coherence manipulation (STOC, [1]). In this method a number of phase mask is introduced to illuminating beam by spatial light modulator in a time of single image acquisition. We use a digital mirror device (DMD) in order to rapid cross-talk noise reduction (up to 22kHz modulation frequency) when imaging living biological cells in vivo by using full-field microscopy setup with double pass arrangement. This, to our best knowledge, has never been shown before.
[1] D. Borycki, M. Nowakowski, and M. Wojtkowski, Opt. Lett. 38, 4817 (2013).
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AO for Microscopy and Optical Coherence Tomography III
This overview talk will focus on forward-looking scientific needs and physical limits to images of neuronal processes. The challenge in nervous systems is that the basic unit for "switching" events in the nervous system occurs on the one micrometer scale of synaptic spines, while computations involve communication between individual neurons across the full expanse of cortex, which is ten millimeters for mouse cortex. I will address hoped-for advances in optical microscopy, within the context of existing and proposed contrast mechanisms of neuronal function, that span the four orders of magnitude of length scales for neuronal processing
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The biggest obstacle for deep tissue imaging is the scattering of light due to the heterogeneous distribution of biological tissue. In this respect, multiphoton microscopy has an inherent advantage as the scattering is significantly reduced by the use of longer excitation wavelengths. However, as we go deeper into the brain, effects of scattering still accumulate resulting in a loss of resolution and increased background noise. Adaptive optics is an ideal tool of choice to correct for such distortions of the excitation wavefront; the incident light can be tuned to cancel out the wavefront distortion experienced while propagating into greater depths resulting in a diffraction limited focus at the depth of interest.
However, the biggest limitation of adaptive optics for in vivo brain imaging is its limited corrected field-of-view (FOV). For typical multiphoton laser scanning microscopes, the wavefront corrector for adaptive optics is placed at the pupil plane. This means that a single correction wavefront is applied to the entire scanned FOV which results in inefficient correction as the correction is averaged over the entire FOV. In this work, we demonstrate a novel approach to measure and display different correction wavefronts over different segments of the FOV. The application of the different correction wavefronts for each segment is realized in parallel resulting in fast aberration corrected imaging over a large FOV for high resolution in vivo brain imaging.
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AO for Microscopy and Optical Coherence Tomography IV
Adaptive optics is a strategy to compensate for sample-induced aberrations in microscopy applications. Generally, it requires the presence of "guide stars" in the sample to serve as localized reference targets. We describe an implementation of conjugate adaptive optics that is amenable to widefield (i.e. non-scanning) microscopy, and can provide aberration corrections over potentially large fields of view without the use of guide stars. A unique feature of our implementation is that it is based on wavefront sensing with a single-shot partitioned-aperture sensor that provides large dynamic range compatible with extended samples. Combined information provided by this sensor and the imaging camera enable robust image de-blurring based on a rapid estimation of sample and aberrations obtained by closed-loop feedback. We present the theoretical principle of our technique and experimental demonstrations using both trans-illumination and fluorescence microscopes. Finally, we apply our technique to mouse brain imaging.
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Active optics can facilitate two-photon microscopic imaging deep in tissue. We are investigating fast focus control mirrors used in concert with an aberration correction mirror to control the axial position of focus and system aberrations dynamically during scanning. With an adaptive training step, sample-induced aberrations may be compensated as well. If sufficiently fast and precise, active optics may be able to compensate under-corrected imaging optics as well as sample aberrations to maintain diffraction-limited performance throughout the field of view. Toward this end we have measured a Boston Micromachines Corporation Multi-DM 140 element deformable mirror, and a Revibro Optics electrostatic 4-zone focus control mirror to characterize dynamic performance. Tests for the Multi-DM included both step response and sinusoidal frequency sweeps of specific Zernike modes. For the step response we measured 10%-90% rise times for the target Zernike amplitude, and wavefront rms error settling times. Frequency sweeps identified the 3dB bandwidth of the mirror when attempting to follow a sinusoidal amplitude trajectory for a specific Zernike mode. For five tested Zernike modes (defocus, spherical aberration, coma, astigmatism and trefoil) we find error settling times for mode amplitudes up to 400nm to be less than 52 us, and 3 dB frequencies range from 6.5 kHz to 10 kHz. The Revibro Optics mirror was tested for step response only, with error settling time of 80 μs for a large 3 um defocus step, and settling time of only 18 μs for a 400nm spherical aberration step. These response speeds are sufficient for intra-scan correction at scan rates typical of two-photon microscopy.
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An aberrated imaging system PSF is broadened; this broadening is responsible of the blurring of the images. A lot of effort has been carried out to correct the effects of aberrations on OCT images for eye examination or biological samples. We have worked on quantifying the effect of geometrical aberrations on Full-Field OCT images and found that there is mostly no loss of resolution but a decrease of the signal level. This is obviously why we use these signals as metric to correct the wavefront distortion. Moreover we found that this absence of blurring, which is due to the fact that we record the dot product of a diffraction limited reference signal and the distorted sample signal, is specific to the use of an incoherent illumination and did not show up with OCT approaches that use spatially coherent sources. More precisely the loss in signal is roughly proportional to the square root of the Strehl ratio: for example, a Strehl ratio of 1/9, which is considered to give a low quality image, would only be 1/3 in Full-Field OCT while keeping the sharpness of the image. Using both an USAF resolution target and a transmissive SLM we have demonstrated this unique feature of sharpness conservation. It was also confirmed by using biological samples. We think that we can thus restrict the aberration corrections in eye examination to the main aberrations (e.g. focus and astigmatism) that will increase the speed of the correction.
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To reveal fast biological dynamics in deep tissue, we combine two wavefront engineering methods that were developed in our laboratory, namely optical phase-locked ultrasound lens (OPLUL) based volumetric imaging and iterative multiphoton adaptive compensation technique (IMPACT). OPLUL is used to generate oscillating defocusing wavefront for fast axial scanning, and IMPACT is used to compensate the wavefront distortions for deep tissue imaging. We show its promising applications in neuroscience and immunology.
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Stimulated emission depletion microscopy (STED) has become one of the powerful research tools in the field of superresolution microscopy. As its spatial resolution is gained by phase modulation of the light field, the aberrations produced by optical systems and specimens may have negative impact on the focusing properties of two beams, especially the STED beam, resulting in reduced spatial resolution. In thick samples, the aberration effect may play an even more critical role in affecting spatial resolution. Here, we report our recent effort in correcting the aberration in STED microscopy by using coherent optical adaptive technique (COAT) so that the resolution can be improved.
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Recently it has been shown that shaping the wavefront of an incident laser beam can significantly enhance the total transmission of light through strong scattering media [1]. This is done by coupling light to high transmission channels. However, optical absorption would modify such transmission channels. In a disordered system with uniform absorption, the maximal transmission channel changes from diffusive to ballistic-like transport [2]. This ballistic-like transport may enable new modes of imaging in absorbing media. If the absorption is distributed non-uniformly in space, the high transmission channels redirect the energy flows to circumvent the absorbing regions to minimize loss. Thus the attenuation of high transmission channels by inhomogeneous absorption becomes lower than that by homogeneous absorption [3]. Since the maximum transmission channel is the most efficient in bypassing the absorbing region, the ratio of its transmittance to the average transmittance increases with absorption, eventually exceeds the ratio without absorption. The finding that inhomogeneous absorption may have a weaker impact on open channels than homogeneous absorption is promising for practical applications.
[1] S. M. Popoff, A. Goetschy, S. F. Liew, A. D. Stone, and H. Cao. Phys. Rev. Lett. 112, 133903 (2014).
[2] S. F. Liew, S. M. Popoff, A. P. Mosk, W. L. Vos, and H. Cao. Phys. Rev. B 89, 224202 (2014).
[3] S. F. Liew and H. Cao. Opt. Express 23, 11043 (2015).
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The ability to non-invasively focus light through scattering media has significant applications in many fields ranging from nanotechnology to deep tissue sensing. Until recently, the multiple light scattering events that occur in complex media such as biological tissue have inhibited the focusing ability and penetration depth of optical tools. Through the use of optical wavefront shaping, the spatial distortions due to these scattering events can be corrected, and the incident light can be focused through the scattering medium. Here, we demonstrate that wavefront shaping can be used to non-invasively enhance the Raman signal of a material through a scattering medium. Raman signal enhancement was achieved using backscattered light and a continuous sequential algorithm. Our results show the potential of wavefront shaping as an important addition to non-invasive detection techniques.
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Second-harmonic generation (SHG) has proven to be an effective method to both image and detect structural
variations in fibrillar collagen. The ability to detect these differences is especially useful in studying diseases
like cancer and fibrosis.1 SHG techniques have historically been limited by their ability to penetrate and image
through strongly scattering tissues. Recently, optical wavefront shaping has enabled light to be focused through
highly scattering media such as biological tissue.2-4 This technology also enables us to examine the dependence
of second harmonic generation on the spatial phase of the pump laser. Here, we demonstrate that wavefront
shaping can be used to enhance the generation of second harmonic light from collagen fibrils even when scattering
is low or non-existent.
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Wavefront shaping applied on scattering light is a promising optical imaging method in biological systems. Normally, optimized modulation can be obtained by a Liquid-Crystal Spatial Light Modulator (LC-SLM) and CCD hardware iteration. Here we introduce an improved method for this optimization process. The core of the proposed method is to firstly detect the disturbed wavefront, and then to calculate the modulation phase pattern by computer simulation. In particular, phase retrieval method together with phase conjugation is most effective. In this way, the LC-SLM based system can complete the wavefront optimization and imaging restoration within several seconds which is two orders of magnitude faster than the conventional technique. The experimental results show good imaging quality and may contribute to real time imaging recovery in scattering medium.
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When waves travel through disordered media such as ground glass and skin tissues, they are scattered multiple times. Most of the incoming energy bounces back at the superficial layers and only a small fraction can penetrate deep inside. This has been a limiting factor for the working depth of various optical techniques. We present a systematic method to enhance wave penetration to the scattering media. Specifically, we measured the reflection matrix of a disordered medium with wide angular coverage for each orthogonal polarization states. From the reflection matrix, we identified reflection eigenchannels of the medium, and shaped the incident wave into the reflection eigenchannel with smallest eigenvalue, which we call anti-reflection mode. This makes reflectance reduced and wave penetration increased as a result of the energy conservation. We demonstrated transmission enhancement by more than a factor of 3 by the coupling of the incident waves to the anti-reflection modes. Based on the uneven distribution of eigenvalues of reflection eigenchannels, we further developed an iterative feedback control method for finding and coupling light to anti-reflection modes. Since this adaptive control method can keep up with sample perturbation, it promotes the applicability of exploiting reflection eigenchannels. Our approach of delivering light deep into the scattering media will contribute to enhancing the sensitivity of detecting objects hidden under scattering layers, which is universal problem ranging from geology to life science.
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We show how biological elements, like live bacteria species and Red Blood Cells (RBCs) can accomplish optical functionalities in DH systems. Turbid media allow coherent microscopy despite the strong light scattering these provoke, acting on light just as moving diffusers. Furthermore, a turbid medium can have positive effects on a coherent imaging system, providing resolution enhancement and mimicking the action of noise decorrelation devices, thus yielding an image quality significantly higher than the quality achievable through a transparent medium in similar recording conditions. Besides, suspended RBCs are demonstrated to behave as controllable liquid micro-lenses, opening new possibilities in biophotonics for endoscopy imaging purposes, as well as telemedicine for point-of-care diagnostics in developing countries and low-resource settings.
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A major limiting factor of optical imaging in biological applications is the diffusion of light by tissue, preventing focusing at depths greater than ~1 mm in the body. To overcome this issue, phase-based wavefront shaping alters the phase of sections of the incident wavefront to counteract aberrations in phase caused by scattering. This enables focusing through scattering media beyond the optical diffusion limit and increases signal compared to amplitude-based compensation. However, in previous studies, speed of optimization has typically been limited by the use of a liquid crystal spatial light modulator (SLM) for measurement and display. SLMs usually have refresh rates of less than 100 Hz and require much longer than the speckle correlation time of tissue in vivo, usually on the order of milliseconds, to determine the optimal wavefront. Here, we present a phase-based iterative wavefront shaping method based on an onaxis digital micromirror device (DMD) in conjunction with an electro-optic modulator (EOM) for measurement and a fast SLM for display. By combining phase modulation from an EOM with the modal selection of the DMD, we take advantage of DMDs higher refresh rate, approximately 23 kHz, for iterative phase measurement. The slower SLM requires one update for display following the rapid determination of the optimal wavefront via the DMD, allowing for high-speed wavefront shaping. Using this system, we are able to focus through scattering media using 64 modes in under 8 milliseconds, on the order of the speckle correlation time for tissue in vivo.
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Our approach first consists in measuring a time-gated reflection matrix associated to a scattering medium using a spatial light modulator at the input and a CCD camera at the output. An interferometric arm allows to discriminate the scattered photons as a function of their time of flight. Inspired by previous works in acoustics, a random matrix approach then allows to get rid of multiple scattering. This improves by far the detection and imaging of targets embedded in or hidden behind a highly scattering medium. As proof of concept, we tackle with the issue of imaging ZnO micrometric beads across a highly scattering paper sheet whose optical thickness is of 12.5 ls, with ls the scattering mean free path. This experimental situation is particularly extreme, even almost desperate for imaging. The ballistic wave has to go through 25 ls back and forth, thus undergoing an attenuation of 10^-11 in intensity. For an incident plane wave, 1 scattered photon over 1000 billions is associated to the target beads. In optical coherence tomography, the single-to-multiple scattering ratio is of 5×10^-4 which prevents from any target detection and imaging. On the contrary, our approach allows to get rid of most of the multiple scattering contribution in this extreme situation. By means of the time-reversal operator, the ballistic echoes associated to each bead are extracted and allow to reconstruct a satisfying image of the targets. The perspective of this work is to apply this promising approach to in-depth imaging of biological tissues.
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In this paper, we demonstrate a fast binary intensity modulation based on the measurement of the binary TM. For each correction, the binary TM was calculated based on measurements of the intensity change at the target with a series of input masks. After preloading the measurement masks, the DMD can run at full speed during measurement. The system allows dynamic focusing at 12.5 Hz with 1024 input modes, and more than 60 times intensity enhancement. We demonstrate focusing light through a highly dynamic scattering sample, a live drosophila embryo.
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In this contribution we use wavefront shaping approaches for image correlation based flow-field measurements for the first time. Aberrations introduced by a single phase boundary in the detection beam path were explored. Variations of the optical path-length result in strong errors in position allocation and thus to an enhancement of the measurement uncertainty of the velocity. Our results show that the usage of wavefront shaping enables to reduce these errors and to strongly improve the quality of image correlation based flow-field measurements. First experimental and simulated results underline the importance of these approaches.
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We explore the spatial profile of the ensemble average of the energy density of eigenchannels of the transmission matrix within random diffusive media using computer simulations and nonperturbative diagrammatic technique. A symmetrical profile with a peak in the middle of the sample is found for the fully transmitting eigenchannel and is shown to be closely related to a position dependent diffusion coefficient of the open media. We show that the average spatial profile of each transmission eigenchannel when normalized by the profile of the completely transmitting eigenchannel depends only upon the value of transmission through the corresponding eigenchannel. A universal expression for the average spatial profile is given in terms of the auxiliary localization lengths determined from transmission eigenvalues and position dependent diffusion coefficient. These lengths were first introduced by Dorokhov to describe the scaling of transmission and conductance through disordered media. Though direct measurement of energy distribution within a scattering medium is generally difficult, we demonstrate in microwave measurements that the integrated energy density stored in the media of each eigenchannel can be determined from the measurements of spectra of the transmission matrix. The derivative of the composite phase of the eigenchannels with respect to the angular frequency yields the contribution to the density of states (DOS) from the individual transmission eigenchannels. This is proportional to integrated energy stored and the dwell time of the transmission eigenchannel. The DOS determined from the transmission eigenchannel is shown to be in good agreement with DOS obtained by analyzing the field spectra into quasi-normal modes of the open medium. These results provide a path towards controlling the energy deposition within a scattering medium.
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Diffusion equation describes the energy density inside a scattering medium such as biological tissues and paint [1]. The solution of the diffusion equation is a sum over a complete set of eigensolutions that shows a characteristic linear decrease with depth in the medium. It is of particular interest if one could launch energy in the fundamental eigensolution, as this opens the opportunity to achieve a much greater internal energy density. For applications in optics, an enhanced energy density is vital for solid-state lighting, light harvesting in solar cells, low-threshold random lasers, and biomedical optics.
Here we demonstrate the first ever selective coupling of optical energy into a diffusion eigensolution of a scattering medium of zinc oxide (ZnO) paint. To this end, we exploit wavefront shaping to selectively couple energy into the fundamental diffusion mode, employing fluorescence of nanoparticles randomly positioned inside the medium as a probe of the energy density. We observe an enhanced fluorescence in case of optimized incident wavefronts, and the enhancement increases with sample thickness, a typical mesoscopic control parameter. We interpret successfully our result by invoking the fundamental eigensolution of the diffusion equation, and we obtain excellent agreement with our observations, even in absence of adjustable parameters [2].
References
[1] R. Pierrat, P. Ambichl, S. Gigan, A. Haber, R. Carminati, and R. Rotter, Proc. Natl. Acad. Sci. U.S.A. 111, 17765 (2014).
[2] O. S. Ojambati, H. Yilmaz, A. Lagendijk, A. P. Mosk, and W. L. Vos, arXiv:1505.08103.
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We recently showed how the correlations of a broadband and incoherent wave-field can directly yield the time-dependent Green's functions between scatterers of a complex medium [Badon et al., Phys. Rev. Lett., 2015]. In this study, we apply this approach to the imaging of optical transport properties in complex media. A parallel measurement of millions of Green's functions at the surface of several strongly scattering samples (ZnO, TiO2, Teflon tape) is performed. A statistical analysis of this Green’s matrix allows to investigate locally the spatio-temporal evolution of the diffusive halo within the scattering sample. An image of diffusion tensor is then obtained. It allows to map quantitatively the local concentration of scatterers and their anisotropy within the scattering medium. The next step of this work is to test this approach on biological tissues and illustrate how it can provide an elegant and powerful alternative to diffuse optical imaging techniques.
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We provide an approach to improve the quality of image reconstruction in wide-field imaging through turbid media (WITM). In WITM, a calibration stage which measures the transmission matrix (TM), the set of responses of turbid medium to a set of plane waves with different incident angles, is preceded to the image recovery. Then, the TM is used for estimation of object image in image recovery stage. In this work, we aim to estimate highly resolved angular spectrum and use it for high quality image reconstruction. To this end, we propose to perform a dense sampling for TM measurement in calibration stage with finer incident angle spacing. In conventional approaches, incident angle spacing is made to be large enough so that the columns in TM are out of memory effect of turbid media. Otherwise, the columns in TM are correlated and the inversion becomes difficult. We employ compressed sensing (CS) for a successful high resolution angular spectrum recovery with dense sampled TM. CS is a relatively new information acquisition and reconstruction framework and has shown to provide superb performance in ill-conditioned inverse problems. We observe that the image quality metrics such as contrast-to-noise ratio and mean squared error are improved and the perceptual image quality is improved with reduced speckle noise in the reconstructed image. This results shows that the WITM performance can be improved only by executing dense sampling in the calibration stage and with an efficient signal reconstruction framework without elaborating the overall optical imaging systems.
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We present a mathematical model for the generation of vortex-beams by using a square profile amplitude fork diffraction grating with arbitrary topological charge. The mathematical framework of aberrations in the forked-shape diffraction grating is analysed, and the resulting diffracted pattern is simulated. Three cases of desired distortions (aberrations) in the diffraction grating are considered, obtaining phase modulation from the amplitude grating. Experimental optical vortices are generated by using a transmission spatial light modulator, which is used as a dynamic diffraction grating, allowing us to aberrate it. We show the effect of aberrations in the experimental diffracted vortex-beams and compare it with the numerical simulation.
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Adaptive optics (AO) can shape aberrated optical wavefronts to physically restore the constructive interference needed for high-resolution imaging. With access to the complex optical field, however, many functions of optical hardware can be achieved computationally, including focusing and the compensation of optical aberrations to restore the constructive interference required for diffraction-limited imaging performance. Holography, which employs interferometric detection of the complex optical field, was developed based on this connection between hardware and computational image formation, although this link has only recently been exploited for 3D tomographic imaging in scattering biological tissues. This talk will present the underlying imaging science behind computational image formation with optical coherence tomography (OCT) — a beam-scanned version of broadband digital holography. Analogous to hardware AO (HAO), we demonstrate computational adaptive optics (CAO) and optimization of the computed pupil correction in 'sensorless mode' (Zernike polynomial corrections with feedback from image metrics) or with the use of 'guide-stars' in the sample. We discuss the concept of an 'isotomic volume' as the volumetric extension of the 'isoplanatic patch' introduced in astronomical AO. Recent CAO results and ongoing work is highlighted to point to the potential biomedical impact of computed broadband interferometric tomography. We also discuss the advantages and disadvantages of HAO vs. CAO for the effective shaping of optical wavefronts, and highlight opportunities for hybrid approaches that synergistically combine the unique advantages of hardware and computational methods for rapid volumetric tomography with cellular resolution.
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Shaped Beams for Light Sheet And Structured Illumination Microscopy
Of the large amount of the animal models available for cardiac research, the zebrafish is extremely valuable due to its transparency during early stages of development. In this work a dual illumination laser sheet microscope with simultaneous dual camera imaging is used to image the beating heart at 200 fps, dynamically and selectively focusing inside the beating heart through the use of a tunable lens. This dual color dynamic focusing enables imaging with cellular resolution at unprecedented high frame rates, allowing 3D imaging of the whole beating heart of embryonic zebrafish.
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Aberrations, scattering and absorption degrade the performance light-sheet fluorescence microscopes (LSFM). An adaptive optics system to correct for these artefacts and to optimize the light-sheet illumination is presented. This system allows a higher axial resolution to be recovered over the field-of-view of the detection objective. It is standard selective plane illumination microscope (SPIM) configuration modified with the addition of a spatial light modulator (SLM) and a third objective for the detection of transmitted light. Optimization protocols use this transmission light allowing the extension the depth-of-field and correction of aberrations whilst retaining a thin optical section.
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The combined use of a wavefront modulator and a scattering medium forms an "opaque lens" which forces the light to focus tightly. The adaptive focus has the same shape as the correlation function of the original speckle pattern and it can be generated at defined positions with resolution up to hundreds of nanometers. We have demonstrated that manipulating the speckle pattern spatial components can structure the shape of the focus. Exploiting selectively spatial-frequencies from the speckle components we realized opaque lenses able to produce sub-correlation foci and Bessel beams.
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Structured illumination enables high-resolution fluorescence imaging of nanostructures [1]. We demonstrate a new high-resolution fluorescence imaging method that uses a scattering layer with a high-index substrate as a solid immersion lens [2]. Random scattering of coherent light enables a speckle pattern with a very fine structure that illuminates the fluorescent nanospheres on the back surface of the high-index substrate. The speckle pattern is raster-scanned over the fluorescent nanospheres using a speckle correlation effect known as the optical memory effect. A series of standard-resolution fluorescence images per each speckle pattern displacement are recorded by an electron-multiplying CCD camera using a commercial microscope objective. We have developed a new phase-retrieval algorithm to reconstruct a high-resolution, wide-field image from several standard-resolution wide-field images. We have introduced phase information of Fourier components of standard-resolution images as a new constraint in our algorithm which discards ambiguities therefore ensures convergence to a unique solution. We demonstrate two-dimensional fluorescence images of a collection of nanospheres with a deconvolved Abbe resolution of 116 nm and a field of view of 10 µm × 10 µm. Our method is robust against optical aberrations and stage drifts, therefore excellent for imaging nanostructures under ambient conditions.
[1] M. G. L. Gustafsson, J. Microsc. 198, 82–87 (2000).
[2] H. Yilmaz, E. G. van Putten, J. Bertolotti, A. Lagendijk, W. L. Vos, and A. P. Mosk, Optica 2, 424-429 (2015).
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Recent advances in wavefront control, spatial light modulators, and computational power enable the use of a single multimode fiber as a fluorescence scanning microscope. We explore multimode fibers with different characteristics (diameter, index profile, etc.) and compare their performance regarding robustness against external perturbations and quality of the scanning focus.
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We recently described a method for confocal reflection imaging through fibers, as a way to increase contrast when imaging unstained biological specimens. Using a transmission matrix, focused spots can be created at the distal end of a fiber. The backscattered field coming back from the sample can be filtered using optical correlation to obtain spatial selectivity in the detection. In this proceedings article, we briefly review the working principle of this method, and we discuss how the scheme could be adapted to confocal fluorescence imaging. In particular, we show simulations of the achievable detection bandwidth when using step-index multimode fibers as imaging devices.
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Recent advances in wavefront shaping techniques have enabled so-called lensless endoscopes using fiber probes. Unlocking the full potential of such endoscopes call for the capability of optically sectioned and/or label free imaging. Or in other words, imaging through fibers must retain the functionality of a nonlinear microscope. This is a key challenge due to the temporal broadening of ultrashort pulses in fibers owing to modal dispersion.
Here, we detail the first ever demonstration of two photon fluorescence imaging at the distal tip of a conventional graded index (GRIN) multimode fiber. GRIN fibers possess a high mode density, excellent throughput and limited temporal broadening. These features, in addition to its ready availability, make them attractive candidates for ultrathin endoscopes. In our approach, we apply the transmission matrix formalism and treat these fibers akin to highly scattering media. This lets us retrieve combinations of input modes that would generate intense focal spots throughout the field of view. Furthermore, we identify a regime where the modal dispersion in the fiber is minimal and two-photon excitation with femtosecond light pulses is possible. This allows us to perform two-photon imaging with ultrashort pulses in an epi-detection configuration analogous to conventional nonlinear microscopes. Finally, these concepts are validated by acquiring optically sectioned two photon fluorescence images of 3D samples with cellular resolution. We believe this first report of an ultrathin rigid endoscope of only 125 µm thickness would further accelerate the development of novel tools for demanding applications in biological imaging and opto-genetics.
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The vast number of propagating solutions to the wave equation in multimode optical fibers represents a larger information capacity than provided by fiber bundles of the same diameter. Therefore, in the field of imaging, multimode fibers potentially allow the transmission of images with higher resolution. However, image transmission through multimode fibers is not direct as in the fiber bundle case, in which each of the fiber cores can relay a portion of the distal image. In multimode fiber transmission, a distribution of intensity is scrambled in time and space by the propagating modes, leading to a speckle-like pattern that does not resemble the initial distribution.
Here, we demonstrate two-photon excitation imaging of fluorescent beads through a multimode optical fiber. We show that our method maintains the advantages of two-photon excitation microscopy compared to single-photon excitation such as reduced photo-bleaching, deeper penetration depth and sectioning capability. Our method is based on time-gated digital phase conjugation, which allows the generation of focused pulses on the other side of a multimode fiber. To acquire an image, the focused femtosecond pulse is scanned in a three-dimensional mesh, producing two-photon excitation on each spatial location of the sample. By collecting the fluorescence through the fiber, a 3D two-photon image is reconstructed.
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We present near diffraction limited two photon fluorescence (TPF) imaging through a lensless, multi-core fiber (MCF) endoscope utilizing digital phase conjugation. The ultra-small size of MCFs make them desirable tools for imaging deep into the body. TPF imaging enables optical sectioning and is widely used in brain and biological imaging and is a desired modality for fiber endoscopes. Previous implementations of TPF imaging through MCFs focus and scan the light from individual cores for image formation. In such systems the resolution is limited by the MCF core spacing, although a lens may be used to improve the resolution at the expense of the field of view. Other, more recent work has improved the resolution limitation using custom built MCFs for focusing and scanning of ultrafast pulses using wavefront shaping. Here we present digital phase conjugation for ultrafast pulse focusing through a MCF for an imaging resolution independent of the MCF core spacing. Furthermore, the phase conjugation technique does not require the use of a lens at the fiber end for focus formation and is compatible with commercially available MCFs with a large number of cores. Here, we present a 3000 core MCF endoscope and demonstrate ultrafast pulse focusing with sufficient focus spot contrast and power for TPF endoscope imaging. We construct TPF images by digital scanning of the phase conjugated focus on the target object and collection of the emitted fluorescence through the MCF. This work demonstrates the viability of digital conjugation combined with commercially available MCFs for higher resolution lensless, two photon endoscopy.
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We demonstrate a single multi-mode fiber-based micro-endoscope for measuring blood flow speeds. We use the transmission-matrix wavefront shaping approach to calibrate the multi-mode fiber and raster-scan a focal spot across the distal fiber facet, imaging the cross-polarized back-reflected light at the proximal facet using a camera. This setup allows assessment of the backscattered photon statistics: by computing the mean speckle contrast values across the proximal fiber facet we show that spatially-resolved flow speed maps can be inferred by selecting an appropriate camera integration time. The proposed system is promising for minimally-invasive studies of neurovascular coupling in deep brain structures.
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Endoscopy can be used to obtain high-resolution images at large depths in biological tissues. Usually endoscopic devices have a diameter ranging from 1 to few millimeters. Using digital phase conjugation, it is possible to adapt ultrathin multimode fibers to endoscopic purposes. Recently, we demonstrated that a 330 μm diameter, water-filled silica capillary waveguide can guide high frequency ultrasound waves through a 3 cm thick fat layer, allowing optical resolution photoacoustic imaging. Here we demonstrate that using digital phase conjugation, the same water-filled capillary waveguide (3 cm long) can be used as an endoscopic probe to obtain both fluorescence and optical resolution photoacoustic imaging, with no optical or acoustic elements at the tip of the waveguide. We study the consequences of using digital phase conjugation combined with a capillary waveguide and we conclude with possible future improvements of our endoscopic approach.
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Wavefront Shaping For Photoacoustic and Acousto-Optical Imaging/TRUE
Focusing light deep inside scattering media plays a key role in such biomedical applications as high resolution optical imaging, control, and therapy. In recent years, wavefront shaping technologies have come a long way in controlling light propagation in complex media. A prominent example is time-reversed ultrasonically encoded (TRUE) focusing, which allows noninvasive introduction of “guide stars” inside biological tissue to guide light focusing. By measuring the optical wavefront emanating from an ultrasound focus created at the target location, TRUE determines the desired wavefront non-iteratively, and achieves focusing at the target position via a subsequent optical time reversal. Compared to digital counterparts that employ slow electronic spatial light modulators and cameras, analog TRUE focusing relies on nonlinear photorefractive crystals that inherently accommodate more spatial modes and eliminate the troublesome alignment and data transfer required by digital approaches. However, analog TRUE focusing suffers from its small gain, defined as the energy or power ratio between the focusing and probing beams in the focal volume. Here, by implementing a modified analog TRUE focusing scheme that squeezes the duration of the time-reversed photon packet below the carrier-recombination-limited hologram decay time of the crystal, we demonstrated a photon flux amplification much greater than unity at a preset focal voxel in between two scattering layers. Although the energy gain was still below unity, the unprecedented power gain will nevertheless benefit new biomedical applications.
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Optical focusing plays a central role in biomedical optical imaging, manipulation, and therapy. However, in scattering media, direct optical focusing becomes infeasible beyond ~10 mean free paths. To break this limit, time-reversed ultrasonically encoded (TRUE) optical focusing phase-conjugates ultrasonically tagged diffuse light back to the ultrasonic focus, thus forming a focus deep inside scattering media. In previous works, the speed of wavefront measurement was limited by the low frame rate of the camera used to record the four images required for phase-shifting holography. Moreover, most of the bits of a pixel value were used to represent an informationless background caused by the large amount of untagged light, increasing the amount of data to transfer and necessitating the use of costly high-resolution analog-to-digital converters (ADCs). Here, we developed a digital TRUE focusing system based on a lock-in camera (300×300 pixels), in which each pixel performs analog lock-in detection on chip. Since only the information of the signal, not that of the background, is digitized, the lock-in camera reduces the amount of data to transfer, and enables the use of cheap low-resolution ADCs. Using this lock-in camera, we were able to measure the wavefront of ultrasonically tagged light in less than 0.3 ms, and to achieve TRUE focusing in between two ground glass diffusers. Even when the signal-to-background ratio dropped to 6.32×10^-4, a phase sensitivity as low as 0.51 rad could still be realized, which is more than enough for digital optical phase conjugation.
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Optical scattering of biological tissue limits the penetration depth of conventional optical techniques, which rely on the detection of ballistic photons. Recent developed optical phase conjugation (OPC) technique breaks through this depth limit by shaping an optical wavefront that can “undo” the optical scattering. Assisted with an ultrasound focus, this technique enables optical focusing inside biological tissue in a freely addressable fashion. However, ultrasound modulation efficiency is low and the focusing resolution is limited by the ultrasound. Here we present a new technique, time-reversed ultrasound microbubble encoded (TRUME) optical focusing, which is able to provide high focusing efficiency and sub-ultrasound resolution. This technique achieves the wavefront solution by taking the difference of the optical fields captured outside the sample before and after ultrasound-driven microbubble destruction. A conjugated wavefront was then reconstructed and sent back to the sample to form a focus at the site of microbubble destruction. We experimentally demonstrate that a focus with ~2 um size was formed through a 2-mm thick biological tissue using this method. While the size the microbubble sets the resolution of an individual focus, the scale of the ultrasound focus limits the focusing addressability of this technique. Importantly, by utilizing the nonlinear destruction of microbubbles, the TRUME technique breaks the addressable focus resolution barrier imposed by the ultrasound focus. We experimentally demonstrate a 2-fold improvement in addressability using this effect. Since microbubbles are widely used as ultrasound contrast agents in human, this technique provides a promising solution for focusing light deep inside biological tissue.
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Wavefront shaping based on optoacoustic (photoacoustic) feedback has recently emerged as a promising tool to control the light distribution in optically-scattering media. In this approach, the phase of a short-pulsed light beam is spatially-modulated to create constructive light interference (focusing) at specific locations in the speckle pattern of the scattered wavefield. The optoacoustic signals generated by light absorption provide a convenient feedback mechanism to optimize the phase mask of the spatial light modulator in order to achieve the desired light intensity distribution. The optimization procedure can be done by directly considering the acquired signals or the reconstructed images of the light absorption distribution. Recently, our group has introduced a volumetric (three-dimensional) optoacoustic wavefront shaping platform that enables monitoring the distribution of light absorption in an entire volume with frame rates of tens of Hz. With this approach, it is possible to simultaneously control the volumetric light distribution through turbid media. Experiments performed with absorbing microparticles distributed in a three-dimensional region showcase the feasibility of enhancing the light intensity at specific points, where the size of particles is also essential to maximize the signal enhancement. The advantages provided by optoacoustic imaging in terms of spatial and temporal resolution anticipate new capabilities of wavefront shaping techniques in biomedical optics.
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Laser scanning microscopy allows 3D cross-sectional imaging inside biospecimens. However, certain aberrations produced can degrade the quality of the resulting images. We previously reported a transmissive liquid-crystal device that could compensate for the predominant spherical aberrations during the observations, particularly in deep regions of the samples. The device, inserted between the objective lens and the microscope revolver, improved the image quality of fixed-mouse-brain slices that were observed using two-photon excitation laser scanning microscopy, which was originally degraded by spherical aberration. In this study, we developed a transmissive device that corrects primary coma aberration and astigmatism, motivated by the fact that these asymmetric aberrations can also often considerably deteriorate image quality, even near the sample surface. The device's performance was evaluated by observing fluorescent beads using single-photon excitation laser scanning microscopy. The fluorescence intensity in the image of the bead under a cover slip tilted in the y-direction was increased by 1.5 times after correction by the device. Furthermore, the y- and z-widths of the imaged bead were reduced to 66% and 65%, respectively. On the other hand, for the imaged bead sucked into a glass capillary in the longitudinal x-direction, correction with the device increased the fluorescence intensity by 2.2 times compared to that of the aberrated image. In addition, the x-, y-, and z-widths of the bead image were reduced to 75%, 53%, and 40%, respectively. Our device successfully corrected several asymmetric aberrations to improve the fluorescent signal and spatial resolution, and might be useful for observing various biospecimens.
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The measurement of a wavefront is a powerful tool for characterizing optical systems. The most commonly used wavefront measurement technique is the method of local-light aberrometry. The conventional version of this kind of measurement principle is the Hartmann-Shack wavefront sensor. This method returns the result of the matrix of spatially-resolved gradients of the wavefront. However, the last and crucial step of the wavefront analysis is the reconstruction of the wavefront from the measured data packets. The issues of the measurement preparation and design are interesting in the same volume. The work presented here describes the comparison between a Fourier-Iteration algorithm and the Zernike approximation method for the wavefront reconstruction in relation to the measurement design. In the context of this work, the term "design of the measurement" refers to the issue of the number and relative positions of the measurement points. In this work, the behavior of the wavefront reconstruction method using Monte-Carlo simulations was analyzed. The optimum point distribution was found and a validation parameter to describe the impact of measurement errors on the analysis results was determined. Based on this parameter, a Monte-Carlo based simulation to make the design of the experiment with the highest accuracy was realized. The technique of white noise injection was implemented in the reconstruction routine and the propagation of errors was analyzed. The presented comparison technique was applied to determine the optimum measurement positions over the beam's surface.
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