Mitochondria are extremely important organelles in the regulation of bone marrow and brain activity. However, live imaging of these subcellular features with high resolution in scattering tissues like brain or bone has proven challenging. In this study, we create a next-generation two-photon fluorescence microscope that leverages low-order wavefront correction by Shack-Hartmann wavefront sensor based on different metrics to achieve fast imaging of subcellular organelles of highly scattering living mice. Metrics include maximum intensity, minimum full width at half maximum (FWHM), and maximum energy of the point spread function (PSF), enabling accuracy and robustness of sensorless correction of the system. Using AO increases the fluorescence intensity and FWHM of the PSF and achieves fast imaging of subcellular organelles with 400nm resolution through 85 μm of highly scattering tissue. This study demonstrates a promising tool for imaging mitochondria and other organelles in optically distorting biological environments, which could facilitate the study of a variety of diseases connected to mitochondrial morphology and activity in a range of biological tissues.
Multiphoton microscopy applied in bone tissue is susceptible to optical aberrations caused by heterogeneity in refractive index. Optical clearing can be applied to alleviate some of these aberrations, but it is invasive and causes deviations from normal tissue biology. We recover diffraction limited imaging by means of a high spatial frequency digital micromirror device (DMD), and binary wavefront modulation. A genetic algorithm optimizes the DMD pattern by evaluating the intensity of the Second Harmonic Generation point spread function measured in the bone sample. We present a five-fold GFP intensity improvement, and a 29% spatial resolution increase within an ex vivo mouse sample.
Self-Interference Digital Holography (SIDH) enables imaging of incoherently emitting objects over large axial ranges with sub-diffraction resolution in all three dimensions, utilizing only three two-dimensional images. Our prior research has shown that point-like sources emitting as few as 4,200 photons can be reconstructed over a 10 μm axial range by light-sheet SIDH. This highlights the potential of combining SIDH with Single-Molecule Localization Microscopy (SMLM) to accomplish 3D imaging across a large axial range with nanometer precision, without the need for mechanical refocusing. Because SIDH captures the phase of the light field, aberrations are recorded in the hologram. We have developed a computational aberration correction method based on SIDH, capable of correcting optical aberrations over a large axial range without incorporating any adaptive elements into the imaging system. Our algorithm iteratively searches the coefficients of each Zernike mode via a one-dimensional parabolic fit. For each reconstructed image, a metric function value is calculated to evaluate the image quality. The optimal correction strength for the corresponding Zernike mode is determined by the peak of the parabolic fit, and the virtual phase mask is adjusted accordingly. The aberration correction can be applied directly to the holograms to further improve the localization precision of SIDH.
Light Sheet Fluorescence Microscopy (LSFM) is an ideal tool for imaging model organisms that are hundreds of microns in size, providing high resolution and fast frame rates. In order to extend LSFM to rapid 3D volumetric imaging, a popular method is to add an electrically tunable lens (ETL) in the detection path of the LSFM. But for larger fields of view and higher NA objectives, the ETL introduces aberrations in the system. Here, we developed an LSFM with adaptive optics and an ETL for rapid focusing. We demonstrate that the system enables imaging over a volume of 499 × 499 × 140 μm3 with a volumetric speed of 4 Hz. We apply the system to image neural activity in the zebrafish larvae to capture rare seizure events.
Light-Sheet Fluorescence Microscopy (LSFM) has demonstrated its effectiveness in imaging many model organisms. However, traditional LSFMs often exhibit low resolution and encounter striping artifacts. Here, we introduce a novel LSFM design to address these challenges of imaging large biological samples. Our system employs a single-objective light-sheet geometry, incorporating Structured Illumination Microscopy (SIM) to allow multi-direction light-sheet illumination. This combination aims to rectify striping artifacts and achieve improved lateral resolutions of better than 200 nm over a 277 × 277 × 500 μm3 volume. Our system is demonstrated through imaging beads, C. elegans and cerebellum organoids.
Self-interference digital holography (SIDH) can image incoherently emitting objects over large axial ranges with sub-diffraction resolution in all three dimensions from three two-dimensional images. By combining SIDH with single-molecule localization microscopy (SMLM), incoherently emitting objects can be localized with nanometer precision over a wide axial range without mechanical refocusing. Simulations show that SIDH can achieve sub-20 nm precision with only a few thousand photons. However, background light substantially degrades the performance of SIDH due to the relatively large size of the hologram. Therefore, to achieve the best results, the background must be reduced, and the hologram size must be optimized to increase the signal-to-noise ratio (SNR) and maximize the light efficiency of the SIDH optical system. To optimize the performance of SIDH, we performed simulations to study the optimal hologram radius (𝑅𝑅ℎ) for different levels of background photons. The results show that the reduction of the hologram size improves the localization precision of SIDH. For a given hologram size under different background noise levels, a lower background noise level provides a higher localization precision. By reducing the radius of the entry hologram to 1.4 mm and optimizing the SIDH design, we can achieve a localization precision of better than 60 nm laterally and 80 nm axially over a 10 μm axial range under the conditions of low signal level (6000 photons) with ten photons/pixel of background noise.
Light sheet fluorescence microscopy is an excellent tool for imaging larger and thicker model organisms such as zebrafish larvae. Samples can be imaged with high spatial and temporal resolution over large fields of view without photodamage. But light sheet microscopy still suffers from optical aberrations due to the thickness of the samples and the high resolution. We have previously demonstrated imaging of the zebrafish central nervous system and correction of aberrations using sensorless Adaptive Optics. Sensorless AO is slow because many images are required to achieve a correction. In this work, we demonstrate measuring the wavefront using a Shack-Hartmann wavefront sensor and a confocal spot as the guide star. This approach does not require special sample preparation or an additional laser. Here we demonstrate our approach by imaging fluorescent beads and inducing wavefront errors with the deformable mirror.
Light-Sheet Fluorescence Microscopy (LSFM) has proven to be an excellent tool for imaging the nervous system zebrafish and other model organisms. However, these LSFMs typically have low resolution and suffer from striping artifacts. Here, we present a light sheet system using a novel single-objective light-sheet geometry to allow for multi-direction light-sheet illumination with structured light. This geometry is designed to mitigate striping artifacts and achieve lateral resolutions of better than 200 nm over a 277 × 277 × 100 µm3 volume. We demonstrate the system by imaging the central nervous system of dissected Drosophila embryos as well as cerebellum organoids.
Localization based microscopy using self-interference digital holography (SIDH) provides three-dimensional (3D) positional information about point sources with nanometer scale precision. To understand the performance limits of SIDH, we calculate the Cram´er-Rao lower bound (CRLB) of the localization precision for SIDH. We further compare the calculated precision bounds to the 3D single molecule localization precision from different Point Spread Functions. SIDH results in almost constant localization precision in all three dimensions for a 20 µm axial range. For high signal-to-background ratio (SBR), SIDH on average achieves better localization precision. For lower SBR values, the large size of the hologram on the detector results in more overall noise, and PSF models perform better.
Light Sheet Microscopy has many advantages for imaging living model organisms. Its optical sectioning capability and high volumetric imaging speed over a large field of view make it especially favorable for recording highly dynamic biological events, such as neural signaling. The combination of an electrical tunable lens (ETL) and a scanning light sheet allows us to record image stacks at high speed without moving the sample or the detection objective. The performance of the light sheet microscope is affected by aberrations from the sample mounting and the sample itself as well as aberrations introduced by The ETL which limit the usable field of view and focusing range of the system. Here, we present the development of a light sheet microscope optimized for volumetric imaging of zebrafish larvae with adaptive optics correction for extended focusing range and increased image quality at a speed of 0.6Hz over 400 × 400 × 100μm3 using an electrical tunable lens.
With sub-diffraction resolution in three dimensions and good optical sectioning capability, three-dimensional superresolution structured illumination microscopy (3D-SRSIM) can provide eight-fold more information than conventional widefield microscopy. However, the application of SR-SIM is limited to single cells due to optical aberrations in thick tissues. The destructive impacts of aberrations include the decrease in spatial resolution and signal-to-noise ratio (SNR), the distortion of sample morphology, and, even worse, the failure of SIM reconstruction. There are several adaptive optics (AO) methods to correct the optical aberration, including direct wavefront sensing using a Shack Hartmann wavefront sensor (SHWFS). The SHWFS possesses good wavefront measurement accuracy and high-speed response but works best with an isolated guide-star. Therefore, combining SHWFS with widefield microscopy poses difficulties and remains challenging. To effectively apply the direct wavefront sensing method, we built a reconfigurable microscopy system that can switch to a confocal setup for measuring the wavefront where the fluorescence light emitted from the confocal illumination spot is used as the “guide-star” for wavefront measurements. We experimentally demonstrate that the confocal illumination based direct wavefront sensing AO method can precisely correct the sample induced optical aberrations and help to improve the image quality and fidelity of 3D-SIM imaging in thick samples, exhibiting enormous potential for in vivo biomedical research.
Light Sheet Microscopy has developed rapidly over the past decade and is the ideal approach for imaging model organisms such as zebrafish and other thick tissue specimens. Despite the superior optical sectioning capability, high imaging speed, and large field of view, the performance of light sheet microscopy still suffers from optical aberrations. We have implemented a scene-based Shack-Hartmann wavefront sensor for directly measuring the optical aberrations on the emission side of the light-sheet microscope. In this work, we show that our system is capable of AO correction using sensor based and sensorless based approaches. We demonstrate correction up-to one hundred microns deep in zebrafish and fruitfly embryos.
Structured illumination microscopy has been used with great success on single cell samples. However, it cannot be applied to multicellular thick samples because aberrations caused by the sample not only reduce the intrinsic resolution and contrast, they cause the SIM reconstruction to fail. Although the structured illumination pattern can still be projected onto the focal plane inside the sample when imaging deep into thick biological samples, the resulting raw fluorescent images are corrupted resulting in poor reconstructed SIM images with poor signal to noise ratio, degraded spatial resolution and artifacts that can call into question the reliability of the image. To image in thicker samples with diffraction limited resolution, adaptive optics can be used to correct the optical aberrations due to the sample. Here, we combine three-dimensional structured illumination microscopy (3DSIM) and adaptive optics (AO), demonstrating full three-dimensional aberration-free super-resolution imaging deep into thick multicellular samples. We applied a frequency-based metric function in image-based sensorless AO method for aberration corrections. In some cases, we also applied a customized dot-array illumination pattern to optimize the image spectrum. Through the imaging of various samples, we show that the image-based sensorless AO method performs a satisfying and robust correction of different aberrations with minimal photobleaching. The final three-dimensional image achieves a resolution of ~120 nm laterally and ~500 nm axially with optical sectioning, which is a two-fold resolution enhancement without any nonlinear deconvolution methods. AO-3DSIM provides a reliable solution for three-dimensional super-resolution imaging in vivo with improved fidelity.
KEYWORDS: Super resolution microscopy, Holograms, Photons, Signal to noise ratio, 3D image reconstruction, Digital holography, Michelson interferometers, Microscopes
Self-interference digital holography (SIDH) is a promising approach for three-dimensional imaging as it offers the ability to view a complete three-dimensional volume from a single image. SIDH has so far, largely been limited to image samples that emit a large number of photons. We report the use of a Michelson interferometer based SIDH setup which provides higher light throughput compared to previous systems that employed spatial light modulators (SLM). SIDH microscopy incorporating a Michelson interferometer in the proposed optical configuration and high-numerical-aperture oil immersion objectives can be used to perform super-resolution single-molecule localization microscopy (SMLM) and single-particle tracking (SPT) over large axial ranges. We demonstrate this by localizing a single 0.1 µm diameter fluorescent nanosphere using a custom built wide-field microscope. With 49,000 photons detected, the proposed system achieves a localization precision of 4.5 nm in x, 5 nm in y and 39.8 nm in z over a 20 µm axial range. Further, we also discuss the SNR requirements to image photon-limited light sources such as a single-molecules using SIDH.
Light sheet fluorescence microscopy (LSFM) is a powerful tool for investigating model organisms including zebrafish. However, due to scattering and refractive index variations within the sample, the resulting image often suffers from low contrast. Structured illumination (SI) has been combined with scanned LSFM to remove out-of-focus and scattered light using square-law detection. Here, we demonstrate that the combination of LSFM with linear reconstruction SI can further increase resolution and contrast in the vertical and axial directions compared to the widely adopted root-mean square reconstruction method while using the same input images. We apply this approach to imaging neural activity in 7-day postfertilization zebrafish larvae. We imaged two-dimensional sections of the zebrafish central nervous system in two colors at an effective frame rate of 7 frames per second.
Scattering is a major obstacle on the way of imaging deeper than a few mean-free-paths through bone. The high density of mineralization and collagen fibers deposition make bone a very inhomogeneous tissue that produces severe scattering. Although long wavelength excitation extends the mean-free-path for multi-photon microscopy, however imaging more than 150 microns through bone suffers from loss of resolution and intensity. We previously simulated the wavefront distortions caused by bone using phase accumulation ray tracing (PART) method. Our findings show that some low-order optical aberrations can be corrected using traditional adaptive optics systems such as a deformable mirror, however, a significant amount of high order aberrations are remaining, which require a secondary correction method to restore the point spread function at depth. In this work, we use a high-speed binary wavefront correction method using a digital light processor (DLP) to correct the wavefront in a hostile environment such as bone. We use the PART method to produce an initial estimate of the wavefront, and use a genetic algorithm to evolve it to an optimum using maximum intensity metric. The binary wavefront correction produces a factor of 21 enhancement and the initialization using PART method increases the enhancement 2.5 times.
Super-resolution imaging of volumes as large as whole cells in three-dimensions (3D) is required to reveal unknown features of cellular organization which cannot be resolved by conventional fluorescence microscopy. We propose a new 3D high resolution imaging technique based on the principles of single-molecule localization microscopy (SMLM) and fluorescence incoherent correlation holography (FINCH). FINCH enables hologram acquisition and three-dimensional (3D) imaging of large objects emitting incoherent light. This technique combines FINCH and SMLM to enable single-molecule volumetric imaging over large axial ranges without scanning the sample using a simple and robust setup, hence making it a viable solution for whole cell super-resolution imaging of biological samples. Here, we present the underlying theory and simulations demonstrating the extended depth of field. We image a single 0.2-μm fluorescent microsphere using this approach and discuss the signal-to-noise ratio (SNR) requirements for an experimental implementation.
Zebrafish are an important vertebrate model used to view the mechanisms underlying seizure disorders. Due to their relatively small size and transparency, larval zebrafish are an excellent model through which to view the occurence of seizure-like neural activity in vivo using light sheet fluorescence microscopy (LSFM). Although LSFM possesses good optical sectioning capability and high speed, the resolution and contrast degrade as the imaging plane is moved deeper into the sample due to refractive index variations. We have developed a system that combines a structured illumination light sheet microscope with adaptive optics in the emission path to correct optical aberrations and increase the resolution when imaging deep into the sample. We show that our system can record neural activity fast enough to capture seizure events, and is able to correct optical aberrations throughout the sample.
Near infrared and infrared multi-photon imaging through or inside bone is an emerging field that promises to help answer many biological questions that require minimally invasive intravital imaging. Neuroscience researchers especially have begun to take advantage of long wavelength imaging to overcome multiple scattering and image deep inside the brain through intact or partially intact bone. Since the murine model is used in many biological experiments, here we investigate the optical aberrations caused by mouse cranial bone, and their effects on light propagation. We previously developed a ray tracing model that uses second harmonic generation in collagen fibers of bone to estimate the refractive index structure of the sample. This technique is able to rapidly provide initial information for a closed loop adaptive optics system. However, the ray tracing method does not account for refraction or scattering. Here, we extend our work to investigate the wavefront aberrations in bone using a full electromagnetic model. We used Finite-Difference Time-Domain modeling of light propagation in refractive index bone datasets acquired with second harmonic generation imaging. In this paper we show modeled wavefront phase from different originating points across the field of view.
Light-sheet microscopy is an ideal imaging modality for long-term live imaging in model organisms. However, significant optical aberrations can be present when imaging into an organism that is hundreds of microns or greater in size. To measure and correct optical aberrations, an adaptive optics system must be incorporated into the microscope. Many biological samples lack point sources that can be used as guide stars with conventional Shack-Hartmann wavefront sensors. We have developed a scene-based Shack-Hartmann wavefront sensor for measuring the optical aberrations in a light-sheet microscopy system that does not require a point-source and can measure the aberrations for different parts of the image. The sensor has 280 lenslets inside the pupil, creates an image from each lenslet with a 500 micron field of view and a resolution of 8 microns, and has a resolution for the wavefront gradient of 75 milliradians per lenslet. We demonstrate the system on both fluorescent bead samples and zebrafish embryos.
Zebrafish are a promising vertebrate model for elucidating how neural circuits generate behavior under normal and pathological conditions. The Baraban group first demonstrated that zebrafish larvae are valuable for investigating seizure events and can be used as a model for epilepsy in humans. Because of their small size and transparency, zebrafish embryos are ideal for imaging seizure activity using calcium indicators. Light-sheet microscopy is well suited to capturing neural activity in zebrafish because it is capable of optical sectioning, high frame rates, and low excitation intensities. We describe work in our lab to use light-sheet microscopy for high-speed long-time imaging of neural activity in wildtype and mutant zebrafish to better understand the connectivity and activity of inhibitory neural networks when GABAergic signaling is altered in vivo. We show that, with light-sheet microscopy, neural activity can be recorded at 23 frames per second in twocolors for over 10 minutes allowing us to capture rare seizure events in mutants. We have further implemented structured illumination to increase resolution and contrast in the vertical and axial directions during high-speed imaging at an effective frame rate of over 7 frames per second.
Superresolution microscopy is rapidly becoming an essential tool in the biological sciences allowing imaging biological structure at length scales below 250 nm. Currently, superresolution microscopy has been applied successfully on single cells achieving resolutions of 100nm down to 20nm over a few microns of depth. When superresolution microscopy is applied in thicker samples the resolution rapidly degrades. Optical aberrations and scattering distort and reduce the point spread function causing different superresolution techniques to fail in different ways. I will discuss our work on combining structured illumination microscopy and stochastic optical reconstruction microscopy with adaptive optics to achieve sub-diffraction resolution in thick tissue.
Optical aberrations significantly affect the resolution and signal-to-noise ratio of deep tissue microscopy. As multiphoton microscopy is applied deeper into tissue, the loss of resolution and signal due to propagation of light in a medium with heterogeneous refractive index becomes more serious. Efforts in imaging through the intact skull of mice cannot typically reach past the bone marrow (∼150 μm of depth) and have limited resolution and penetration depth. Mechanical bone thinning or optical ablation of bone enables deeper imaging, but these methods are highly invasive and may impact tissue biology. Adaptive optics is a promising noninvasive alternative for restoring optical resolution. We characterize the aberrations present in bone using second-harmonic generation imaging of collagen. We simulate light propagation through highly scattering bone and evaluate the effect of aberrations on the point spread function. We then calculate the wavefront and expand it in Zernike orthogonal polynomials to determine the strength of different optical aberrations. We further compare the corrected wavefront and the residual wavefront error, and suggest a correction element with high number of elements or multiconjugate wavefront correction for this highly scattering environment.
Multiphoton imaging through the bone to image into the bone marrow or the brain is an emerging need in the scientific community. Due to the highly scattering nature of bone, bone thinning or removal is typically required to enhance the resolution and signal intensity at the imaging plane. The optical aberrations and scattering in the bone significantly affect the resolution and signal to noise ratio of deep tissue microscopy. Multiphoton microscopy uses long wavelength (nearinfrared and infrared) excitation light to reduce the effects of scattering. However, it is still susceptible to optical aberrations and scattering since the light propagates through several layers of media with inhomogeneous indices of refraction. Mechanical removal of bone is highly invasive, laborious, and cannot be applied in experiments where imaging inside of the bone is desired. Adaptive optics technology can compensate for these optical aberrations and potentially restore the diffraction limited point spread function of the system even in deep tissue. To design an adaptive optics system, a priori knowledge of the sample structure assists selection of the proper correction element and sensing methods. In this work we present the characterization of optical aberrations caused by mouse cranial bone, using second harmonic generation imaging of bone collagen. We simulate light propagation through the bone, calculate aberrations and determine the correction that can be achieved using a deformable mirror.
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.
Widefield and confocal fluorescence microscopy using a single objective suffer from poor resolution and a strong anisotropy between the lateral and axial resolution. Coherently combining the excitation and emission from two coaxial objectives improves the axial resolution up to sevenfold, but leaves the lateral resolution unchanged. Here we investigate the coherent combination of three objectives to create a point spread function (PSF) that is isotropic with higher resolution in the plane of the objectives. We develop a theoretical framework for simulating the performance of interferometric imaging with three objectives. Using three identical objectives with a large working distance and 0.9 numerical aperture (NA), the full-width half maximum of the confocal PSF is 135 nm compared to the lateral FWHM of 274 nm for imaging with a single objective at a wavelength of 515 nm.
Optical Aberrations are a major challenge in imaging biological samples. In particular, in single molecule localization (SML) microscopy techniques (STORM, PALM, etc.) a high Strehl ratio point spread function (PSF) is necessary to achieve sub-diffraction resolution. Distortions in the PSF shape directly reduce the resolution of SML microscopy. The system aberrations caused by the imperfections in the optics and instruments can be compensated using Adaptive Optics (AO) techniques prior to imaging. However, aberrations caused by the biological sample, both static and dynamic, have to be dealt with in real time. A challenge for wavefront correction in SML microscopy is a robust optimization approach in the presence of noise because of the naturally high fluctuations in photon emission from single molecules. Here we demonstrate particle swarm optimization for real time correction of the wavefront using an intensity independent metric. We show that the particle swarm algorithm converges faster than the genetic algorithm for bright fluorophores.
Binary amplitude modulation promises to allow rapid focusing through strongly scattering media with a large number of segments due to the faster update rates of digital micromirror devices (DMDs) compared to spatial light modulators (SLMs). While binary amplitude modulation has a lower theoretical enhancement than phase modulation, the faster update rate should more than compensate for the difference – a factor of π2 /2. Here we present two new algorithms, a genetic algorithm and a transmission matrix algorithm, for optimizing the focus with binary amplitude modulation that achieve enhancements close to the theoretical maximum. Genetic algorithms have been shown to work well in noisy environments and we show that the genetic algorithm performs better than a stepwise algorithm. Transmission matrix algorithms allow complete characterization and control of the medium but require phase control either at the input or output. Here we introduce a transmission matrix algorithm that works with only binary amplitude control and intensity measurements. We apply these algorithms to binary amplitude modulation using a Texas Instruments Digital Micromirror Device. Here we report an enhancement of 152 with 1536 segments (9.90%×N) using a genetic algorithm with binary amplitude modulation and an enhancement of 136 with 1536 segments (8.9%×N) using an intensity-only transmission matrix algorithm.
Although Single Molecule Localization (SML) techniques have pushed the resolution of fluorescence microscopy beyond the diffraction limit, the accuracy of SML has been limited by the brightness of the fluorophores. The introduction of Quantum Dots (QD) for SML promises to overcome this barrier, and the QD Blueing technique provides a novel approach to SML microscopy. QDs have a higher quantum yield and absorption cross-section, making them brighter, thereby providing a higher accuracy of localization. The blueing technique is also faster and more quantitative than other SML techniques such as dSTORM. The initial bleaching step required by dSTORM is not necessary and each QD is imaged only once as its emission spectrum moves through the blueing window in contrast to dSTORM where the same molecule might be imaged multiple times. Single color QD Blueing has been demonstrated. However in biological imaging, multi-color imaging is essential for understanding the samples under study. Here we introduce two color superresolution microscopy using QD Blueing on biological samples. We demonstrate simultaneous imaging of microtubules and mitochondria in HepG2 cells with a localization accuracy of 40nm. We further show how QD Blueing can be optimized through the control of the sample mounting medium.
Structured illumination microscopy provides twice the linear resolution of conventional fluorescence microscopy, but in thick samples, aberrations degrade the performance and limit the resolution. Here, we demonstrate structured illumination microscopy through 35 µm of tissue using adaptive optics (AO) to correct aberrations resulting in images with a resolution of 140 nm. We report a 60% minimum improvement in the signal-to-noise ratio of the structured illumination reconstruction through thick tissue by correction with AO.
Stochastic Optical Reconstruction Microscopy (STORM) requires a high Strehl ratio point spread function (PSF) to
achieve high resolution, especially in the presence of background fluorescence. The PSF is degraded by aberrations
caused by imperfections in the optics, the refractive index mismatch between the sample and coverslip, and the refractive
index variations of the sample. These aberrations distort the shape of the PSF and increase the PSF width directly
reducing the resolution of STORM. Here we discuss the use of Adaptive Optics (AO) to correct aberrations, maintaining
a high Strehl ratio even in thick tissue. Because the intensity fluctuates strongly from frame to frame, image intensity is
not a reliable measure of PSF quality, and the choice of a robust optimization metric is critical. We demonstrate the use
of genetic algorithms with single molecule imaging for optimization of the wavefront and introduce a metric that is
relatively insensitive to image intensity. We demonstrate the correction of the wavefront from measurements of single
quantum dots.
KEYWORDS: Adaptive optics, Microscopy, Optical transfer functions, Point spread functions, Image resolution, Wavefronts, Signal to noise ratio, Photons, Tissues, Molecules
μLinear Structured Illumination Microscopy (SIM) provides a two-fold increase over the diffraction limited resolution.
SIM produces excellent images with 120nm resolution in tissue culture cells in two and three dimensions. For SIM to
work correctly, the point spread function (PSF) and optical transfer function (OTF) must be known, and, ideally, should
be unaberrated. When imaging through thick samples, aberrations will be introduced into the optical system which will
reduce the peak intensity and increase the width of the PSF. This will lead to reduced resolution and artifacts in SIM
images. Adaptive optics can be used to correct the optical wavefront restoring the PSF to its unaberrated state, and AO
has been used in several types of fluorescence microscopy. We demonstrate that AO can be used with SIM to achieve
120nm resolution through 25m of tissue by imaging through the full thickness of an adult C. elegans roundworm. The
aberrations can be corrected over a 25μm × 45μm field of view with one wavefront correction setting, demonstrating that
AO can be used effectively with widefield superresolution techniques.
KEYWORDS: Microscopy, Optical transfer functions, Signal to noise ratio, Luminescence, Multiphoton microscopy, Multiphoton fluorescence microscopy, 3D image processing, Microscopes, Information operations, Filtering (signal processing)
Structured Illumination Microscopy is a simple and effective method to remove out-of-focus light in widefield
fluorescence microscopy. Neil et al. originally proposed a simple square-law method for calculating the optically
sectioned image from the three raw images with the structured illumination pattern super-imposed. However, the Neil
method does not make the most efficient use of the three raw images. The three structured illumination images can also
be used to separate three copies of the image covering shifted regions of frequency space in a similar manner to that
developed by Gustafsson et al. These can then be combined using a generalized Wiener filter to create an image with a
well-behaved optical transfer function in which the missing cone has been filled in, providing optical sectioning. Here,
we compare the Neil and Gustafsson methods and show that the Gustafsson method provides an image with higher
fidelity and a better Signal to Noise Ratio (SNR) at low photon counts. We apply the two methods to images of
fluorescent beads and GFP labeled septins in Aspergillus nidulans.
Phase Diversity is a powerful technique for estimating wavefront aberrations from images of extended scenes.
Phase Diversity was developed for two-dimensional imaging, typically using defocus as the phase diverse aberration.
Here we discuss different approaches for extending phase diversity to three-dimensional imaging for
biological applications. We show the results of using phase diversity to determine wavefront aberrations on
simulated images.
Three-dimensional live imaging in cell biology is hindered by optical aberrations which degrade the resolution
and signal-to-noise ratio as the focal plane is moved deeper into the sample. The solution to this problem is
to use adaptive optics to correct the aberrations. In this paper, we discuss our work on applying adaptive
optics to wide-field fluorescence microscopy. We demonstrate correction of depth-aberrations and focusing using
a deformable mirror in open-loop operation. We then discuss the use of phase retrieval and phase diversity in
adaptive optics.
A three-dimensional wide-field image of a small fluorescent bead contains more than enough information to accurately
calculate the wavefront in the microscope objective back pupil plane using the phase retrieval technique. The phase-retrieved
wavefront can then be used to set a deformable mirror to correct the point-spread function (PSF) of the
microscope without the use of a wavefront sensor. This technique will be useful for aligning the deformable mirror in a
widefield microscope with adaptive optics and could potentially be used to correct aberrations in samples where small
fluorescent beads or other point sources are used as reference beacons. Another advantage is the high resolution of the
retrieved wavefont as compared with current Shack-Hartmann wavefront sensors. Here we demonstrate effective
correction of the PSF in 3 iterations. Starting from a severely aberrated system, we achieve a Strehl ratio of 0.78 and a
greater than 10-fold increase in maximum intensity.
Adaptive optics (AO) improves the quality of astronomical imaging systems by using real time measurement of the
turbulent medium in the optical path using a guide star (natural or artificial) as a point source reference beacon [1]. AO
has also been applied to vision science to improve the view of the human eye. This paper will address our current
research focused on the improvement of fluorescent microscopy for biological imaging utilizing current AO technology.
A Shack-Hartmann wavefront sensor (SHWS) is used to measure the aberration introduced by a Drosophila
Melanogaster embryo with an implanted 1 micron fluorescent bead that serves as a point source reference beacon.
Previous measurements of the wavefront aberrations have found an average peak-to-valley and root-mean-square (RMS)
wavefront error of 0.77 micrometers and 0.15 micrometers, respectively. Measurements of the Zernike coefficients
indicated that the correction of the first 14 Zernike coefficients is sufficient to correct the aberrations we measured. Here
we show that a MEMS deformable mirror with 3.5 microns of stroke and 140 actuators is sufficient to correct these
aberrations. The design, assembly and initial results for the use of a MEMS deformable mirror, SHWS and implanted
fluorescent reference beacon for wavefront correction are discussed.
Linear Structured Illumination is a powerful technique for increasing the resolution of a fluorescence microscope by a
factor of two beyond the diffraction limit. Previously this technique has only been used to image fixed samples because
the implementation, using a mechanically rotated fused silica grating, was too slow. Here we describe a microscope
design, using a ferroelectric spatial light modulator to structure the illumination light, capable of linear structured
illumination at frame rates up to 11Hz. We show live imaging of GFP labeled Tubulin and Kinesin in Drosophila S2
cells.
Adaptive optics (AO) improves the quality of astronomical imaging systems by using real time measurement of the
turbulent medium in the optical path using a guide star (natural or artificial) as a point source reference beacon. AO has
also been applied to vision science to improve the current view of the human eye. This paper will address our current
research focused on the improvement of fluorescent microscopy for biological imaging utilizing current AO technology.
A Shack-Hartmann wavefront sensor (SHWS) was used to measure the aberration introduced by a Drosophila Melanogaster embryo with an implanted 1 micron fluorescent bead that serves as a point source reference beacon. The measurements show an average peak-to-valley and root-mean-square (RMS) wavefront error of 0.77 micrometers and
0.15 micrometers, respectively. The Zernike coefficients have been measured for these aberrations which indicate that
the correction of the first 14 Zernike coefficients should be sufficient to correct the aberrations we have obtained. These
results support the utilization of SHWS for biological imaging applications and that a MEMS deformable mirror with 1 micron of stroke and 100 actuators will be sufficient to correct these aberrations. The design, assembly and initial results for the use of a MEMS deformable mirror, SHWS and implanted fluorescent reference beacon for wavefront correction will also be discussed.
Depth aberrations are a major source of image degradation in three-dimensional microscopy, causing a significant loss of resolution and intensity deep into the sample. These aberrations occur because of an inevitable mismatch between the sample refractive index and the immersion medium index. We have built a wide-field fluorescence microscope that incorporates a large-throw deformable mirror to correct for depth aberrations in 3D imaging. We demonstrate a corrected point spread function imaging beads in water with an oil immersion lens and a twofold improvement in peak signal intensity. We apply this new microscope to imaging biological samples, and show sharper images and improved deconvolution.
In this paper, we will present the recent progress of electrically-pumped directly-modulated tunable 1550 nm VCSEL development at Bandwidth9. The device is fabricated from an all epitaxial VCSEL structure grown on an InP substrate, with a monolithically integrated tuning arm for continuous wavelength tuning. We have demonstrated over 1 mW CW output power and over 20 nm tuning range in C-band and error free transmission performance at 2.5 Gbps over 100 km SMF-28 fiber. The reliability test data of the tunable VCSELs shows a projected failure rate of less than 400 FITS.
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