SignificanceIn the realm of cerebrovascular monitoring, primary metrics typically include blood pressure, which influences cerebral blood flow (CBF) and is contingent upon vessel radius. Measuring CBF noninvasively poses a persistent challenge, primarily attributed to the difficulty of accessing and obtaining signal from the brain.AimOur study aims to introduce a compact speckle contrast optical spectroscopy device for noninvasive CBF measurements at long source-to-detector distances, offering cost-effectiveness, and scalability while tracking blood flow (BF) with remarkable sensitivity and temporal resolution.ApproachThe wearable sensor module consists solely of a laser diode and a board camera. It can be easily placed on a subject’s head to measure BF at a sampling rate of 80 Hz.ResultsCompared to the single-fiber-based version, the proposed device achieved a signal gain of about 70 times, showed superior stability, reproducibility, and signal-to-noise ratio for measuring BF at long source-to-detector distances. The device can be distributed in multiple configurations around the head.ConclusionsGiven its cost-effectiveness, scalability, and simplicity, this laser-centric tool offers significant potential in advancing noninvasive cerebral monitoring technologies.
Fourier ptychographic microscopy (FPM) has its strength in tackling the trade-off between resolution and field-of-view of imaging systems by computational methods. Here, we present a time-efficient and physics-based algorithm for FPM image stack reconstruction using implicit neural representation and tensor low-rank approximation. The method is free of any pre-training process and can be easily adapted to various computational microscopes. Compared to the conventional FPM methods for image stack reconstruction, the proposed method can be several times faster than conventional FPM methods on the same graphics processing units (GPU) and significantly reduce data volume for storage. The proposed method has potential applications in digital pathology and its downstream data-driven tasks, and can be beneficial to data collaboration in biological sciences.
The level of computational power we can currently access, has significantly changed the way we think about, process and interact with information. In this talk, I will discuss some of our recent computational microscopy and deep learning work, that showcase some of these shifts in the context of pathology and life science research.
The level of computational power we can currently access has significantly changed the way we think about, process and interact with image information. In this talk, I will give a broad survey of the exciting work going on in the field of computational imaging – ranging from physical-based computational microscopy methods to machine-learning driven image rendering. As a case study, I will discuss how computational imaging is impacting digital pathology in predictable and surprising ways.
Measuring blood flow on the head is an indirect yet crucial way to access or quantify brain activity non-invasively, useful for diagnosing traumatic brain injuries by looking at the changes of blood flow. However, measuring the cerebral blood flow (CBF) remains challenging, principally due to the ability to reach and collect signal from the brain. In this talk, I will present our efforts in building an affordable, compact, and portable laser device for real-time Cerebral Blood Flow (CBF) monitoring. Our technique draws upon the speckle visibility spectroscopy technique, using the spatial ensemble of the speckle field to evaluate blood flow dynamics.
We design and implement a novel imaging technique that integrates bimodal phase and 3D fluorescence capabilities through aperture segmentation. This approach involves capturing four distinct fluorescence images, mirroring the principles of the Fourier light field microscope and the multi-view reflector microscope, enabling accurate 3D sample reconstruction. Additionally, four brightfield images are acquired for quantitative phase and amplitude reconstruction based on the Kramers-Kronig relations. By combining the strengths of phase imaging, such as digital refocusing, extended depth of field, and non-invasiveness, with the specificity of fluorescence imaging, this method offers a unique imaging solution. Imaging maize roots highlights its exceptional depth of field extension, while imaging a mixture of bacterial cells with and without fluorescent protein tags demonstrates its unique bimodal capabilities.
For imaging, an ideal lens should give images with high-resolution across a large field-of-view (FOV). However, designing and manufacturing such lens is almost impossible due to the intrinsic properties of real materials. Fourier ptychography microscopy (FPM), a computational imaging method, attracts board interests as it improves over imperfections of a real objective. With the aid of computation, FPM can provide aberration-free, high-resolution images over a large FOV. However, its iterative reconstruction is non-convex and may not converge to a real solution. Moreover, its aberration correction algorithm does not work well under large aberrations. In this talk, I will present a new imaging method, termed analytical multiangle illumination microscopy (AMIM), that performs complex field reconstructions using all analytical methods. By using critical-angle and darkfield measurements, AMIM extracts the aberration and reconstructs the complex field in a purely analytical way. We show that AMIM works well with extremely large aberrations and can reconstruct the complex field in a non-iterative way.
Diffusing Wave Spectroscopy (DWS) employs non-invasive techniques to study dynamic behaviors within scattering mediums. This involves detecting speckle light patterns from the moving media and gauging speckle decorrelation time for dynamic quantification. A specific form of DWS is Speckle Visibility Spectroscopy (SVS), which employs spatial ensembles by utilizing a high-pixel-count cameras to capture multiple speckles in a single frame. To further increase SNR at low signal level, one combines interferometric techniques with SVS to form interferometric SVS (iSVS).
In this talk, I will present the benefits and constraints associated with the utilization of interferometric detection (iSVS) over direct detection (SVS). Surprisingly, we found that iSVS does not always outperform SVS, but it does emerge as the prevailing choice in most cases.
KEYWORDS: Neural networks, Wavefronts, Coherence imaging, Biological imaging, Data modeling, Holography, Convolution, Super resolution, Image restoration, Education and training
Large-scale computational imaging can provide remarkable space-bandwidth product that is beyond the limit of optical systems. In coherent imaging (CI), the joint reconstruction of amplitude and phase further expands the information throughput and sheds light on label-free observation of biological samples at micro- or even nano-levels. The existing large-scale CI techniques usually require scanning/modulation multiple times to guarantee measurement diversity and long exposure time to achieve a high signal-to-noise ratio. Such cumbersome procedures restrict clinical applications for rapid and low-phototoxicity cell imaging. In this work, a complex-domain-enhancing neural network for large-scale CI termed CI-CDNet is proposed for various large-scale CI modalities with satisfactory reconstruction quality and efficiency. CI-CDNet is able to exploit the latent coupling information between amplitude and phase (such as their same features), realizing multidimensional representations of the complex wavefront. The cross-field characterization framework empowers strong generalization and robustness for various coherent modalities, allowing high-quality and efficient imaging under extremely low exposure time and few data volume. We apply CI-CDNet in various large-scale CI modalities including Kramers–Kronig-relations holography, Fourier ptychographic microscopy, and lensless coded ptychography. A series of simulations and experiments validate that CI-CDNet can reduce exposure time and data volume by more than 1 order of magnitude. We further demonstrate that the high-quality reconstruction of CI-CDNet benefits the subsequent high-level semantic analysis.
This report is the second part of a comprehensive two-part series aimed at reviewing an extensive and diverse toolkit of novel methods to explore brain health and function. While the first report focused on neurophotonic tools mostly applicable to animal studies, here, we highlight optical spectroscopy and imaging methods relevant to noninvasive human brain studies. We outline current state-of-the-art technologies and software advances, explore the most recent impact of these technologies on neuroscience and clinical applications, identify the areas where innovation is needed, and provide an outlook for the future directions.
Digital refocusing is a key feature of Fourier ptychographic microscopy (FPM). It is currently performed by determining and removing the defocus aberration during the iterative phase retrieval process. We examine the feasibility of digitally refocusing an FPM image by numerically propagating the recovered complex FPM image after the phase retrieval process has been completed – in effect, disentangling the defocus correction process from the iterative phase retrieval process. If feasible, this type of postreconstruction digital refocusing can significantly reduce the FPM computational load and provide a quick and efficient way for refocusing microscopy images on the fly. We report that such an approach is infeasible for large defocus distances because the raw FPM dataset associated with a defocused sample is illconditioned for the FPM’s phase-retrieval process, and it will not output a complex-valued image that corresponds to any physically relevant image wavefront. When the defocus distance is small, the FPM can output an approximately correct image wavefront. However, this wavefront does not contain a global defocus phase term and, therefore, cannot be further focused using the digital refocusing application of a reverse global phase term. In totality, this means that postreconstruction digital refocusing does not serve a meaningful function for any defocus distance. To verify our analysis, we performed a series of experiments, and the results showed that the postreconstruction digital refocusing method is not a viable digital refocusing method.
Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics’ agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, in this article we review an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion article, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed and provide an outlook for the future directions.
We reported a novel non-interferometric and non-iterative computational imaging method, synthetic aperture imaging based on Kramers-Kronig relations (KKSAI), to reconstruct complex wave-field. By collecting images through a modified microscope system with pupil modulation capability, we show that the phase and amplitude profile of the sample at pupil limited resolution can be extracted from as few as two intensity images by exploiting Kramers-Kronig relations. KKSAI reconstruction is non-iterative, free of parameter tuning and applicable to a wider range of samples. Simulation and experiment results have proved that it has much lower computational burden and achieves the best reconstruction quality when compared with two existing phase imaging methods.
Non-line-of-sight (NLOS) imaging is a rapidly developing research direction that has significant applications in autonomous vehicles, remote sensing, etc. Existing NLOS methods primarily depend on time gated measurements and/or sophisticated signal processing to extract information from the scattered light. Here, we introduce a new method that directly manipulates the light to counter the wall’s scattering. This method operates by actively focusing light onto the target in a NLOS path using wavefront shaping. By raster scanning that focus, we can actively image the occluded object. The focus thus formed is near diffraction limited and can be substantially smaller than the object itself, thereby enabling us to perform NLOS imaging with unprecedented resolution. We demonstrate that a resolution of ∼ 0.6 mm at a distance of 0.55 m is achievable in our experiment.
Diffusing wave spectroscopy (DWS) is a well-known set of methods to measure the temporal dynamics of dynamic samples. In DWS, dynamic samples scatter the incident coherent light, and the information of the temporal dynamics is encoded in the scattered light. To record and analyze the light signal, there exist two types of methods – temporal sampling methods and speckle ensemble methods. Temporal sampling methods, including diffuse correlation spectroscopy (DCS), use one or multiple large bandwidth detectors to well sample and analyze the temporal light signal to infer the sample temporal dynamics. Speckle ensemble methods, including speckle visibility spectroscopy (SVS), use a high-pixel-count camera sensor to capture a speckle pattern and use the speckle contrast to infer sample temporal dynamics. In this paper, we theoretically and experimentally demonstrate that the decorrelation time (τ ) measurement accuracy or SNR of the two types of methods has a unified and similar fundamental expression based on the number of independent observables (NIO) and the photon flux. Given a time measurement duration, NIO in temporal sampling methods is constrained by the measurement duration, while speckle ensemble methods can outperform by using simultaneous sampling channels to scale up NIO significantly. In the case of optical brain monitoring, the interplay of these factors favors speckle ensemble methods. We illustrate that this important engineering consideration is consistent with the previous research on blood pulsatile flow measurements, where a speckle ensemble method operating at 100-fold lower photon flux than a conventional temporal sampling system can achieve a comparable SNR.
Fluorescence imaging is indispensable to biomedical research, and yet it remains challenging to image through dynamic scattering samples. Wavefront shaping has enabled fluorescence imaging through scattering media. However, the translation of these techniques into in vivo applications has been hindered by the lack of high speed solutions to counter the fast speckle decorrelation of dynamic tissue. Here, we report an ultrasound enabled optical imaging method that instead leverages the dynamic nature to perform imaging. The method utilizes the correlation between dynamic speckle encoded fluorescence and ultrasound modulated light signal that originate from the same location within a sample. We successfully imaged fluorescent targets within a dynamic scattering medium.
We report a single-shot surface three-dimensional (3-D) imaging method that uses optical coherence as a contrast mechanism to acquire the vertical (z-direction) information of an object. The illumination of the imaging system comes from a light source with the optical coherence length similar to the depth of field (DoF) of the optical system. Holographic recording is used to retrieve the coherence visibility factor, which is then converted to z-direction information. In the experiment, we compare the imaging results of our method to conventional incoherent imaging results, showing that this contrast mechanism is able to provide additional information. We also validate our 3D imaging results by using axial scanning full-field optical coherence tomography.
Significance: Ultrasound-assisted optical imaging techniques, such as ultrasound-modulated optical tomography, allow for imaging deep inside scattering media. In these modalities, a fraction of the photons passing through the ultrasound beam is modulated. The efficiency by which the photons are converted is typically referred to as the ultrasound modulation’s “tagging efficiency.” Interestingly, this efficiency has been defined in varied and discrepant fashion throughout the scientific literature.
Aim: The aim of this study is the ultrasound tagging efficiency in a manner consistent with its definition and experimentally verify the contributive (or noncontributive) relationship between the mechanisms involved in the ultrasound optical modulation process.
Approach: We adopt a general description of the tagging efficiency as the fraction of photons traversing an ultrasound beam that is frequency shifted (inclusion of all frequency-shifted components). We then systematically studied the impact of ultrasound pressure and frequency on the tagging efficiency through a balanced detection measurement system that measured the power of each order of the ultrasound tagged light, as well as the power of the unmodulated light component.
Results: Through our experiments, we showed that the tagging efficiency can reach 70% in a scattering phantom with a scattering anisotropy of 0.9 and a scattering coefficient of 4 mm − 1 for a 1-MHz ultrasound with a relatively low (and biomedically acceptable) peak pressure of 0.47 MPa. Furthermore, we experimentally confirmed that the two ultrasound-induced light modulation mechanisms, particle displacement and refractive index change, act in opposition to each other.
Conclusion: Tagging efficiency was quantified via simulation and experiments. These findings reveal avenues of investigation that may help improve ultrasound-assisted optical imaging techniques.
Due to the chromatic dispersion properties inherent in all optical materials, even the best designed multi-spectral objective will exhibit residual chromatic aberration effect. Here we show that the aberration correction ability of Fourier Ptychographic Microscopy (FPM) is well matched and well suited for post-image acquisition correction of these effects to render in-focus images. We show that an objective with significant spectral focal shift (up to 0.02 μm/nm) and spectral field curvature (up to 0.05 μm/nm drift at off-axis position of 800μm) can be computationally corrected to render images with effectively null spectral defocus and field curvature. This approach of combining optical objective design and computational microscopy provides a good strategy for high quality multi-spectral imaging over a broad spectral range, and eliminating the need for mechanical actuation solutions.
Normal development of the visual system in infants relies on clear images being projected onto the retina, which can be disrupted by lens opacity caused by congenital cataract. This disruption, if uncorrected in early life, results in amblyopia (permanently decreased vision even after removal of the cataract). Doctors are able to prevent amblyopia by removing the cataract during the first several weeks of life, but this surgery risks a host of complications which can be equally visually disabling. Here, we investigated the feasibility of focusing light noninvasively through highly scattering cataractous lenses to stimulate the retina, thereby preventing amblyopia. This approach would allow the cataractous lens removal surgery to be delayed and hence greatly reduce the risk of complications from early surgery. Employing a wavefront shaping technique named time-reversed ultrasonically encoded (TRUE) optical focusing in reflection mode, we focused 532 nm light through a highly scattering ex vivo adult human cataractous lens of 112 mean free path thick. This work demonstrates a potential clinical application of wavefront shaping techniques.
Demands of multi-well plate readers has been on the rise for drug discovery and cell line development applications, as it can obtain fluorescence, absorbance, and morphology information from cell cultures grown in tens to hundreds of conditions. Existing systems typically only house one camera, requiring slow mechanical actuation to cover a large area on the multi-well plate, or to sacrifice speed for area, forgoing the precious spatial information. We mitigate the time-resolution trade off with the Fourier ptychographic microscopy (FPM) technology by simultaneously capturing 96 high-resolution phase images (>20,000 cells per plate) with CMOS-based cameras with custom-designed microscope objectives. By illuminating the samples with a permutation of lighting conditions, we achieve synthetic numerical aperture (NA) of 0.3 at an extended depth-of-field of 20 micrometer for at most 96 conditions at one time. Unlike our previous invention of the 6-well plate reader (EmSight), the same illumination condition can be shared among adjacent cameras. Therefore, image acquisition and data transfer can be performed in a massively parallel manner. Along with computational acceleration with graphical processing units (GPUs), all these approaches reduces the plate-to-image turnover from hours to minutes – an eight-fold reduction in time over existing mechanical-scanning plate readers. In addition to providing phase imaging, the system is also capable of fluorescence imaging at the native resolution of the objectives. We anticipate that our high-throughout 96-camera imaging system will help advance the high content analysis of cell cultures beyond hundreds of test conditions, thus facilitates more in-depth characterization of biological screens and drug testing.
Fourier Ptychography has shown that we can computationally correct physical aberrations. Thereby, allow us to move beyond the traditional strategy of accomplishing high quality imaging through the exacting refinement of the physical microscope system. I will report on the use of Fourier Ptychography to implement high quality parallel imaging with plastic molded lenses for 96 well plate imaging.
The problem of optical scattering was long thought to fundamentally limit the depth at which light could be focused through turbid media such as fog or biological tissue. However, recent work in the field of wavefront shaping has demonstrated that by properly shaping the input light field, light can be noninvasively focused to desired locations deep inside scattering media. This has led to the development of several new techniques which have the potential to enhance the capabilities of existing optical tools in biomedicine. Unfortunately, extending these methods to living tissue has a number of challenges related to the requirements for noninvasive guidestar operation, speed, and focusing fidelity. Of existing wavefront shaping methods, time-reversed ultrasonically encoded (TRUE) focusing is well suited for applications in living tissue since it uses ultrasound as a guidestar which enables noninvasive operation and provides compatibility with optical phase conjugation for high-speed operation. In this paper, we will discuss the results of our recent work to apply TRUE focusing for optogenetic modulation, which enables enhanced optogenetic stimulation deep in tissue with a 4-fold spatial resolution improvement in 800-micron thick acute brain slices compared to conventional focusing, and summarize future directions to further extend the impact of wavefront shaping technologies in biomedicine.
Normal development of the visual system in infants relies on clear images being projected onto the retina, which can be disrupted by lens opacity caused by congenital cataract. This disruption, if uncorrected in early life, results in amblyopia (permanently decreased vision even after removal of the cataract). Doctors are able to prevent amblyopia by removing the cataract during the first several weeks of life, but this surgery risks a host of complications, which can be equally visually disabling. Here, we investigated the feasibility of focusing light noninvasively through highly scattering cataractous lenses to stimulate the retina, thereby preventing amblyopia. This approach would allow the cataractous lens removal surgery to be delayed and hence greatly reduce the risk of complications from early surgery. Employing a wavefront shaping technique named time-reversed ultrasonically encoded optical focusing in reflection mode, we focused 532-nm light through a highly scattering ex vivo adult human cataractous lens. This work demonstrates a potential clinical application of wavefront shaping techniques.
Differential phase contrast (DPC) is a non-interference quantitative phase imaging method achieved by asymmetric optical systems. Quantitative DPC images are achieved previously with asymmetric illumination systems. However, it works well for on-focus thin samples only. Considering the limitation, we develop a pupil modulation differential phase contrast (PMDPC) imaging method. Instead of modulating the illumination, we use a spatial light modulator (SLM) to modulate a 4f imaging system’s pupil plane. When half of the pupil plane is blocked by the SLM, a phase gradient image forms on the image plane. Using two such phase gradient images captured separately by applying complementary half-circle pupils on SLM, a DPC image can be constructed that carries the sample’s phase information. A quantitative phase image of the sample can be reconstructed after a deconvolution procedure. Further, we are able to combine this quantitative phase with the sample’s intensity image to obtain the complete complex object field which then allows us to post-process the image. We report experimentally that aberrations arising from the optical elements in the system can be corrected by deconvolving the reconstructed image with a pre-calibrated pupil function. We can also digitally extend the depth of field using angular spectrum propagation algorithm. With our PMDPC imaging setup where NA equals to 0.36, a quantitative phase image with periodic resolution of 1.73µm is obtained. The depth of field for a 20x, 0.4NA objective is extended digitally by 20 times to -50~50 micrometers.
Optical scattering of biological tissue limits the working depth of conventional biomedical optics, which relies on the detection of ballistic photons. Recent developed optical phase conjugation (OPC) technique breaks through this depth limit by harnessing the scattered photons and shaping an optical wavefront that can “undo” the optical scattering. The OPC system measures the complex light field exiting the tissue and reconstructs a phase conjugated copy of the measured wavefront, which propagates in the reversed direction to the source of the light. To focus light inside a scattering medium, an embedded light source or “guidestar” is often required. Therefore, developing guidestar mechanisms plays an important role in advancing the OPC technique for deep tissue optical focusing and imaging. In addition to having strong optical modulation efficiency and compact size, a favorable guidestar for biomedical applications should also have good biocompatibility, fast response time, and be noninvasive or require only minimally invasive procedure. While a number of guidestar mechanisms have been developed and showed promising for various biomedical applications, they all have their own limitations. We have been developing new guidestars and tailoring them to meet the need for biomedical imaging and therapies. We are going to present our recent progress in novel guidestar development, compare them with established guidestar mechanisms, and discuss their potential in biomedical applications.
Multi-day tracking of cells in culture systems can provide valuable information in bioscience experiments. We report the development of a cell culture imaging system, named EmSight, which incorporates multiple compact Fourier ptychographic microscopes with a standard multiwell imaging plate. The system is housed in an incubator and presently incorporates six microscopes, imaging an ANSI standard 6-well plate at the same time. By using the same low magnification objective lenses (NA of 0.1) as the objective and the tube lens, the EmSight is configured as a 1:1 imaging system that, providing large field-of-view (FOV) imaging (5.7 mm × 4.3 mm) onto a low-cost CMOS imaging sensor. The EmSight improves the image resolution by capturing a series of images of the sample at varying illumination angles; the instrument reconstructs a higher-resolution image by using the iterative Fourier ptychographic algorithm. In addition to providing high-resolution brightfield and phase imaging, the EmSight is also capable of fluorescence imaging at the native resolution of the objectives. We characterized the system using a phase Siemens star target, and show four-fold improved coherent resolution (synthetic NA of 0.42) and a depth of field of 0.2 mm. To conduct live, long-term dopaminergic neuron imaging, we cultured ventral midbrain from mice driving eGFP from the tyrosine hydroxylase promoter. The EmSight system tracks movements of dopaminergic neurons over a 21 day period.
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.
We present a method to acquire both fluorescence and high-resolution bright-field images with correction for the spatially varying aberrations over a microscope’s wide field-of-view (FOV). First, the procedure applies Fourier ptychographic microscopy (FPM) to retrieve the amplitude and phase of a sample, at a resolution that significantly exceeds the cutoff frequency of the microscope objective lens. At the same time, FPM algorithm is able to leverage on the redundancy within the set of acquired FPM bright-field images to estimate the microscope aberrations, which usually deteriorate in regions further away from the FOV’s center. Second, the procedure acquires a raw wide-FOV fluorescence image within the same setup. Lack of moving parts allows us to use the FPM-estimated aberration map to computationally correct for the aberrations in the fluorescence image through deconvolution. Overlaying the aberration-corrected fluorescence image on top of the high-resolution bright-field image can be done with accurate spatial correspondence. This can provide means to identifying fluorescent regions of interest within the context of the sample’s bright-field information. An experimental demonstration successfully improves the bright-field resolution of fixed, stained and fluorescently tagged HeLa cells by a factor of 4.9, and reduces the error caused by aberrations in a fluorescence image by 31%, over a field of view of 6.2 mm by 9.3 mm. For optimal deconvolution, we show the fluorescence image needs to have a signal-to-noise ratio of ~18.
The optical opacity of biological tissue has long been a challenge in biomedical optics due to the strong scattering nature of tissue in the optical regime. While most conventional optical techniques attempt to gate out multiply scattered light and use only unscattered light, new approaches in the field of wavefront shaping exploit the time reversible symmetry of optical scattering in order to focus light inside or through scattering media. While these approaches have been demonstrated effectively on static samples, it has proven difficult to apply them to dynamic biological samples since even small changes in the relative positions of the scatterers within will cause the time symmetry that wavefront shaping relies upon to decorrelate. In this paper we investigate the decorrelation curves of acute rat brain slices for thicknesses in the range 1-3 mm (1/e decorrelation time on the order of seconds) using multi-speckle diffusing wave spectroscopy (MSDWS) and compare the results with theoretical predictions. The results of this study demonstrate that the 1/L^2 relationship between decorrelation time and thickness predicted by diffusing wave spectroscopy provides a good rule of thumb for estimating how the decorrelation of a sample will change with increasing thickness. Understanding this relationship will provide insight to guide the future development of biophotonic wavefront shaping tools by giving an estimate of how fast wavefront shaping systems need to operate to overcome the dynamic nature of biological samples.
Circulating tumor cells (CTCs) are recognized as a candidate biomarker with strong prognostic and predictive potential in metastatic disease. Filtration-based enrichment technologies have been used for CTC characterization, and our group has previously developed a membrane microfilter device that demonstrates efficacy in model systems and clinical blood samples. However, uneven filtration surfaces make the use of standard microscopic techniques a difficult task, limiting the performance of automated imaging using commercially available technologies. Here, we report the use of Fourier ptychographic microscopy (FPM) to tackle this challenge. Employing this method, we were able to obtain high-resolution color images, including amplitude and phase, of the microfilter samples over large areas. FPM’s ability to perform digital refocusing on complex images is particularly useful in this setting as, in contrast to other imaging platforms, we can focus samples on multiple focal planes within the same frame despite surface unevenness. In model systems, FPM demonstrates high image quality, efficiency, and consistency in detection of tumor cells when comparing corresponding microfilter samples to standard microscopy with high correlation (R2=0.99932). Based on these results, we believe that FPM will have important implications for improved, high throughput, filtration-based CTC analysis, and, more generally, image analysis of uneven surfaces.
Researchers use ultrasound (US) to modulate diffusive light in a highly scattering medium like tissue. This paper analyzes the US–optical interaction in the scattering medium and derives an expression for the US-modulated optical radiance. The diffusion approximation to the radiative transport equation is employed to develop a Green’s function for US-modulated light. The predicted modulated fluence and flux are verified using finite-difference time-domain simulations. The Green’s function is then utilized to illustrate the modulated reflectance as the US–optical interaction increases in depth. The intent of this paper is to focus on high US frequencies necessary for high-resolution imaging because they are of interest for applications such as phase conjugation.
The ability to focus light in most tissue degrades quickly with depth due to high optical scattering. Researchers have investigated using both ultrasound (US) and light synergistically to overcome this difficulty. Ultrasound has been utilized to modulated light within tissue to create a diffusive wave at that is modulated at the US frequency. Recently, there has been interest in the modulated sidebands which reside at optical frequency plus or minus the US frequency. This paper will discuss a model for US-light interactions in a scattering medium. We will use this model to relate the radiance in the probe beam to the radiance in the diffusive wave. We will then employ the P-1 approximation to the radiative transport equation to find the fluence and flux of the modulated wave. We will use these parameters to write a diffusion equation for the modulated wave that can be described in terms of the incoming optical power, and the US intensity and geometry.
I will discuss our recent work on the use of digital optical phase conjugation and ultrasound tagging to accomplish timereversal
deep tissue optical focusing for fluorescence imaging and other applications.
We studied the use of Talbot pattern illumination in scanning optical microscopy (SOM). Unlike conventional illumination spots used in SOM, the focal spots in Talbot pattern are more complicated and do not have a simple Gaussian intensity distribution. To find out the resolution of SOM using Talbot pattern, we characterized the evolution of the full-width-at-half-maximum spot size of the Talbot focal spots by computer simulation. We then simulated the SOM imaging under Talbot pattern illumination using the razor blade and the U.S. Air Force target as the sample objects, and compared the results with those performed with Gaussian spots as illumination. Using several foci searching algorithms, the optimal focal distances were found to be shorter than the theoretical Talbot distances. The simulation results were consistent with the experiment results published previously. We then provide a practical guidance for searching for optimal focal distances in the SOM based on these studies.
The effects of strong scattering in tissue limit the depth to which light may be focused. However, it has been shown that scattering may be reduced utilizing adaptive optics with a focused ultrasound (US) beam guidestar. The optical signal traveling through the US beam waist is frequency shifted and may be isolated with demodulation. This paper utilizes a multiphysics simulation to model the optical and US interactions in both synthetic tissue and random scattering media. The results illustrate that optical energy may be focused within a turbid medium utilizing a US guidestar. The results also suggest that optical energy travels preferentially along optical channels within a turbid medium.
The on-chip detection of a weak optical signal in biological experiments can easily be complicated by the presence of an
overwhelming background signal, and as such, pre-detection background suppression is substantively important for
weak signal detection. In this paper, we report a structure that can be directly incorporated onto optical sensors to
accomplish background suppression prior to detection. This structure, termed surface-wave-enabled darkfield aperture
(SWEDA), consists of a central sub-wavelength hole surrounded by concentric grooves that are milled onto a gold layer.
Incoming light can be collected and converted into surface waves (SW) by the concentric grooves and then be recoupled
into propagating light through the central hole. We show that the SW-assisted optical component and the direct
transmission component of the central hole can cancel each other, resulting in near-zero transmission under uniform
illumination (observed suppression factor of 1230). This structure can therefore be used to suppress a light field's bright
background and allow sensitive detection of localized light field non-uniformity (observed image contrast enhancement
of 27dB). We also show that under a coherent background illumination, a CMOS pixel patterned with the proposed
structure achieves better SNR performance than an un-patterned single pixel.
We describe the amplitude and resolution trends of the signals acquired by turbidity suppression through optical phase conjugation (TSOPC) with samples that span the ballistic and diffusive scattering regimes. In these experiments, the light field scattered through a turbid material is written into a hologram, and a time-reversed copy of the light field is played back through the sample. In this manner, the wavefront originally incident on the sample is reconstructed. We examine a range of scattering samples including chicken breast tissue sections of increasing thickness and polyacrylamide tissue-mimicking phantoms with increasing scattering coefficients. Our results indicate that only a small portion of the scattered wavefront (<0.02%) must be collected to reconstruct a TSOPC signal. Provided the sample is highly scattering, all essential angular information is contained within such small portions of the scattered wavefront due to randomization by scattering. A model is fitted to our results, describing the dependence of the TSOPC signal on other measurable values within the system and shedding light on the efficiency of the phase conjugation process. Our results describe the highest level of scattering that has been phase conjugated in biological tissues to date.
We have developed a novel microscope technique that can achieve wide field-of-view (FOV) imaging and yet possess
resolution that is comparable to conventional microscope. The principle of wide FOV microscope system breaks the link
between resolution and FOV magnitude of traditional microscopes. Furthermore, by eliminating bulky optical elements
from its design and utilizing holographic optical elements, the wide FOV microscope system is more cost-effective. In
our system, a hologram was made to focus incoming collimated beam into a focus grid. The sample is put in the focal
plane and the transmissions of the focuses are detected by an imaging sensor. By scanning the incident angle of the
incoming beam, the focus grid will scan across the sample and the time-varying transmission can be detected. We can
then reconstruct the transmission image of the sample. The resolution of microscopic image is limited by the size of the
focus formed by the hologram. The scanning area of each focus spot is determined by the separation of the focus spots
and can be made small for fast imaging speed. We have fabricated a prototype system with a 2.4-mm FOV and 1-μm
resolution. The prototype system was used to image onion skin cells for a demonstration. The preliminary experiments
prove the feasibility of the wide FOV microscope technique, and the possibility of a wider FOV system with better resolution.
The light scattering effects of turbid media causing opacity may be undone via Optical Phase Conjugation (OPC). Here
we rigorously simulate light scattering through a macroscopic random using the pseudospectral time-domain (PSTD)
technique. The OPC phenomenon of multiply scattered light can be quantitatively analyzed which is not feasible
otherwise. Specifically, factors affecting the electromagnetic energy propagation and refocusing phenomenon is
analyzed. The reported simulation study allows accurate characterization of the optical properties of the OPC
phenomenon for practical biomedical applications.
We report the narrowest to-date (21 gauge, 820-µm-diam) handheld forward-imaging optical coherence tomography (OCT) needle endoscope and demonstrate its feasibility for ophthalmic OCT inspection. The probe design is based on paired-angle-rotation scanning (PARS), which enables a linear B-scan pattern in front of the probe tip by using two counterrotating angle polished gradient-index (GRIN) lenses. Despite its small size, the probe can provide a numerical apertune (NA) of 0.22 and an experimental sensitivity of 92 dB at 0.5 frame/s. The feasibility of retinal imaging is tested on enucleated ex vivo porcine eyes, where structural features including remnant vitreous humor, retina, and choroid can be clearly distinguished. Due to its imaging quality comparable to a commercial OCT system and compatibility with the current ophthalmic surgery standard, the probe can potentially serve as a better alternative to traditional visual inspection by white light illumination during vitreoretinal surgery (e.g., vitrectomy).
This paper reports a complete on-chip high resolution lensless imaging device based on the optofluidic
microscopy method, which can form a vital optical microscopy component in a wide range of lab-on-a-chip
systems. This imaging device does not use any lens elements and yet is capable of resolution comparable to
that of a conventional microscope with a 20× objective. We demonstrate the use of the device for
Caenorhabditis elegans and microsphere imaging at a resolution of ~ 1 μm with an imaging time of ~2 sec.
The fabrication of this on-chip imaging device is fully compatible with existing semiconductor and
microfluidic technologies, so the device can be massively fabricated and integrated into microsystems to
form compact and low-cost total analysis systems for biological and colloidal studies.
We demonstrate a novel method of two-dimensional differential interference contrast (DIC) microscopy. Our method is
cheaper, more compact, and more robust compared to conventional DIC microscopes; since it uses a simple variation of
Young's double-slit geometry, no expensive or complex optical components are needed. In addition, our method
quantitatively measures differential phase, unlike conventional DIC, which makes our device useful for optical
metrology and cell biology applications. The device consists of four circular holes arranged in a "plus" pattern, milled
into a metal layer 80 μm above a complimentary metal-oxide semiconductor (CMOS) image sensor. Light incident upon
the four-hole aperture is transmitted through the holes and creates an interference pattern on the CMOS sensor. This
pattern shifts as a function of the spatial phase gradient of the incident light. By capturing the amplitude and location of
the zero-order fringe of the interference pattern, the amplitude and differential phase of the incident light can be
measured simultaneously. In this article, we model the response of the device using both geometric optics and Huygens
principle. We then verify these models by experimentally measuring the responsivity of our device. A short analysis on
the algorithm used to calculate the fringe location follows. We then show a beam profiling application by measuring the
amplitude and spatial phase gradient of a Gaussian laser beam and an optical vortex. Finally, we show a DIC microscope
application; we image a phase mask of the letters "CIT".
Our group has reported the use of harmonically matched diffraction grating for full-field quantitative phase imaging. In
this paper, we show the improvement of this technique and the application in observing dynamics of transparent
samples. By using the grating as a beam splitter/combiner in an interferometer, we are able to obtain non-trivial phase
difference between the output ports of the grating. We have built a Mach-Zehnder interferometer using the holographic
grating with 600 and 1200 lines/mm spacing. Two CCD cameras at the output ports of the grating-based Mach-Zehnder
interferometer are used to record the full-field quadrature interferograms, which are subsequently processed to
reconstruct the phase image. Since the two quadrature interferograms are acquired at the same time, the imaging speed
of the system is limited only by the frame rate of the CCD cameras. We have demonstrated the capability of our system
by observing dynamics of transparent samples.
Neurologic complications have been reported with spinal transforaminal injections. Causes include intraneural injection,
plus embolization occlusion of the radicular artery with subsequent spinal cord infarction. 1 Optical coherence
tomography (OCT) is a non-invasive imaging modality, which is used to image tissue microstructure with very high
resolution (less than 20 microns) in real-time. With a view toward needle tip OCT visualization of the spinal
neuroforamen, we conducted animal studies to explore OCT imaging of paraspinal neurovascular structures. With
institutional animal care committee approval, we performed ex-vivo and in situ OCT studies in a euthanized dog, pig,
and rabbit. Image data was gathered on spinal nerve roots, dura, and brachial plexus. Two systems were used: frequency
domain OCT imaging system developed at California Institute of Technology, and time domain Imalux NIRIS system
with a 2.7 mm diameter probe. In a euthanized pig, excised dura was punctured with a 17-gauge Tuohy needle. FDOCT
dural images of the puncture showed a subsurface cone-shaped defect. In a rabbit in situ study, puncture of the dura with
a 26-gauge needle is imaged as a discontinuity. FDOCT imaging of both small artery and large arteries will be
presented, along with H&E and OCT images of the brachial plexus.
We report a new method for obtaining non-trivial phase difference between the output ports of an interferometer
through the use of shallow diffraction gratings. We show that as opposed to a single shallow diffraction grating-based
interferometer (which provides only trivial phase shifts, i.e., 0° or 180°), a pair of harmonically-related shallow
diffraction gratings can be used to design interferometers with non-trivial phase shifts between different output ports.
More importantly, the phase shifts can be adjusted by simply shearing one grating with respect to the other. This
approach does not change the path length relationships of the different interference beams within the interferometer,
which is an advantage for metrology and low coherence interferometry applications.
In this article, we will present a novel differential interference contrast (DIC) phase imaging device based
on Young's interference. It is mainly based on either two or four nano apertures defined in an optically
opaque aluminum film on a CMOS imaging sensor chip. It provides linear and disentangled differential
phase and intensity images simultaneously. Furthermore, it's simple, free of bulky optical elements and
compatible to the planar micro fabrication process. All of these features make it a promising device for the
on-chip high resolution DIC phase imaging and beam profiling. The fabrication and operation of the device
is explained in details. The performance is evaluated theoretically and is verified experimentally by
examining the phase and intensity profile of a Gaussian beam and an optical vortex. The 2D quantitive
differential phase distribution of an optical vortex has been recorded directly by our device with 1μm
resolution.
In this paper, we will report our recent development of a new type of
OptoFluidic Microscope (OFM) that is capable of delivering resolution beyond the
diffraction limit of light. Accurate control of the sample translation is accomplished
by adopting an optical tweezer scanner into the system. During the image acquisition,
a two-dimensional nanoaperture array defined on a thin aluminum film acts as an
array of ultra-fine illumination sources. The imaging system is tested and
demonstrated by using polystyrene beads and green algae (Chlamydomonas).
Properties of the system are reported and discussed.
In this paper, we report the use of holographic gratings, which act as the free-space equivalent of the 3x3 fiber-optic
coupler, to perform full field phase imaging. By recording two harmonically-related gratings in the same holographic
plate, we are able to obtain nontrivial phase shift between different output ports of the gratings-based Mach-Zehnder
interferometer. The phase difference can be adjusted by changing the relative phase of the recording beams when
recording the hologram. We have built a Mach-Zehnder interferometer using harmonically-related holographic gratings
with 600 and 1200 lines/mm spacing. Two CCD cameras at the output ports of the gratings-based Mach-Zehnder
interferometer are used to record the full-field quadrature interferograms, which are subsequently processed to
reconstruct the phase image. The imaging system has ~12X magnification with ~420μmx315μm field-of-view. To
demonstrate the capability of our system, we have successfully performed phase imaging of a pure phase object and a
paramecium caudatum.
A variation to the analysis of phase data achieved with spectral domain optical coherence tomography (SDOCT) is presented. By using the variance of the phase changes observed in the OCT images, scatterer motion has been imaged which is not readily observable with conventional Doppler OCT techniques. Dynamic motion contrast has been demonstrated for imaging Brownian motion of a sample system as well as imaging vasculature of in vivo 3dpf zebrafish.
Vitrectomy (removal of the vitreous humor) is an ophthalmic surgery required as a precursor to several posterior chamber procedures. Vitrectomy is commonly performed using an endoscopic vitreous cutter and fiber based light delivery for observation through a surgical microscope. Cross-sectional visualization of the retina and remnant vitreous layers during surgery using an external optical coherence tomography (OCT) scanner is impractical due to deformation in the shape of the eye and the cornea. We present a forward imaging probe with 820 &mgr;m outer diameter (21 gauge needle) for cross-sectional endoscopic OCT imaging during ophthalmic surgeries. The Paired-Angle-Rotating Scanner (PARS) OCT probe is based on angle polished gradient index (GRIN) lenses which are rotated about the optical axis. The scan pattern is determined by the angle between the GRIN lenses and the relative angular velocity. Endoscopic placement of the PARS-OCT probe tip near the retinal surface permits use of a longer wavelength light, in particular 1310 nm, which would otherwise suffer significant attenuation traversing the vitreous humor. The prototype endoscopic PARS-OCT probe is coupled to a commercially available 1310 nm swept laser source, and uses commercial software for data acquisition, processing, and display of retinal images in real time at an A-scan rate of 16 kHz. We present an analysis of aberrations due to off axis use of GRIN lenses and measure the scan pattern of the PARS probe. Images acquired on an ex vivo porcine retina are presented, motivating development of the endoscopic PARS-OCT probe for clinical evaluation.
We review the current state of research in endoscopic optical coherence tomography (OCT). We first survey the range of available endoscopic optical imaging techniques. We then discuss the various OCT-based endoscopic methods that have thus far been developed. We compare the different endoscopic OCT methods in terms of their scan performance. Next, we examine the application range of endoscopic OCT methods. In particular, we look at the reported utility of the methods in digestive, intravascular, respiratory, urinary and reproductive systems. We highlight two additional applications—biopsy procedures and neurosurgery—where sufficiently compact OCT-based endoscopes can have significant clinical impacts.
KEYWORDS: Microfluidics, Imaging systems, Microscopes, Charge-coupled devices, Signal to noise ratio, Near field scanning optical microscopy, Imaging devices, Animal model studies, Lab on a chip, Microscopy
We demonstrate a novel optical imaging device that can be directly integrated into a microfluidic network, and therefore enables on-chip imaging in a microfluidic system. This micro imaging device, termed optofluidic microscope (OFM) is free of bulk optics and is based on a nanohole array defined in a non-transmissive metallic layer that is patterned onto the floor of the microfluidic channel. The operation of the optofluidic microscope will be explained in details and its performance is examined by using a popular animal model, Caenorhabditis elegans (C. elegans). Images from a large population of nematode worms are efficiently acquired within a short time frame. The quality of the OFM images of C. elegans and the morphological characteristics revealed therein are evaluated. Two groups of early-stage C. elegans larvae, wild-type and dpy-24 are successfully separated even though their morphological difference at the larval stage is subtle. The experimental results support our claim that the methodology described therein can be effectively used to develop a powerful tool for fulfilling high-resolution, high-throughput imaging task in microfluidics-based systems.
The use of indocyanine green (ICG), a U.S. Food and Drug Administration approved dye, in a pump-probe scheme for molecular contrast optical coherence tomography (MCOCT) is proposed and demonstrated for the first time. In the proposed pump-probe scheme, an optical coherence tomography (OCT) scan of the sample containing ICG is first acquired. High fluence illumination (~190 kJ/cm2) is then used to permanently photobleach the ICG molecules—resulting in a permanent alteration of the overall absorption of the ICG. A second OCT scan is next acquired. The difference of the two OCT scans is used to determine the depth resolved distribution of ICG within a sample. To characterize the extent of photobleaching in different ICG solutions, we determine the cumulative probability of photobleaching, B,cum, defined as the ratio of the total photobleached ICG molecules to the total photons absorbed by the ground state molecules. An empirical study of ICG photobleaching dynamics shows that B,cum decreases with fluence as well as with increasing dye concentration. The quantity B,cum is useful for estimating the extent of photobleaching in an ICG sample (MCOCT contrast) for a given fluence of the pump illumination. The paper also demonstrates ICG-based MCOCT imaging in tissue phantoms as well as within stage 54 Xenopus laevis.
In this paper, we present a novel high resolution optical imaging device on chip. It is based on a line of nano holes defined in an optically opaque aluminum film on a CMOS imaging sensor chip. Because it's free of bulky optical elements and compatible to the planar micro fabrication process, it is very promising to become an important component for the on-chip high resolution imaging in the future. The fabrication and operation of this novel on-chip microscope is explained in details. The performance is evaluated
theoretically and is verified experimentally by examining the profile of a laser spot formed by a 10X objective lens.
KEYWORDS: Microfluidics, Microscopes, Imaging systems, Signal to noise ratio, Image resolution, Charge-coupled devices, Biology, Imaging devices, Animal model studies, Lab on a chip
In this article, we will demonstrate a novel optical imaging device that can be directly integrated into a microfluidic network, and therefore enables on-chip imaging in a microfluidic system. This micro imaging device, termed optofluidic microscope (OFM) is potentially free of bulk optics and is based on a nanohole array defined in a nontransmissive metallic layer that is patterned onto the floor of the microfluidic channel. The operation of the optofluidic microscope will be explained in details and its performance is examined by using a popular animal model, Caenorhabditis elegans (C. elegans). Images from a large population of nematode worms are efficiently acquired within a short time frame. The quality of the OFM images of C. elegans and the morphological characteristics revealed therein are evaluated. Two groups of early-stage C elegans larvae, wild-type and dpy-24 are successfully separated even though their morphological difference at the larval stage is subtle. The experimental results support our claim that the methodology described therein can be effectively used to develop a powerful tool for fulfilling high-resolution, highthroughput imaging task in microfluidics-based systems.
Use of indocyanine green (ICG), an FDA-approved dye, in a pump-probe scheme for optical coherence tomography (OCT) is reported. Aqueous solutions of ICG are not stable, i.e., the dye degrades over time especially in the presence of light. Addition of protein such as bovine serum albumin (BSA) stabilizes the ICG; however, when exposed to high intensity illumination, the dye still degrades. Moreover, the photodegradation is permanent and occurs swiftly if the illumination band corresponds to the ICG absorption peak. The permanence of the photobleached state illustrates that ICG photobleaching phenomenon has great potential to achieve contrast in OCT. ICG solutions with 50 micromolar concentration were prepared in water, 1% BSA, and 0.8% agarose to study the dynamics of the dye for different illumination intensity levels. In addition, different molar concentrations of ICG in water were studied for fixed illumination intensity. In each case, probability of photobleaching, defined as the ratio of the total photobleached ICG molecules to the total photons absorbed by the ground-state molecules, is evaluated to characterize the photobleaching phenomenon in ICG. We also demonstrate ICG-based pump-probe MCOCT imaging by mapping the distribution of ICG in a stage 54 Xenopus laevis.
We propose a novel forward-imaging OCT needle probe. The probe is based on the use of two angled GRIN lenses that can freely rotate with respect to each other. The probe is capable of scanning a forward cone volume ahead of the probe tip. Different scanning modes, such as the conventional OCT B-scan mode, spiral mode and starburst B-scan mode, can be obtained by adjusting the angular scan velocities of the two GRIN lenses. We develop a prototype probe and demonstrate its capability to acquire OCT images. In this paper we give the characteristics of the prototype probe and display images of different part of tadpole acquired by the probe. The longitudinal resolution, lateral resolution and the signal-to-noise ratio of the system are 10 μm, 10 μm and 93 dB, respectively.
A variation on the standard time domain optical coherence tomography (TDOCT) system is presented. Using an inexpensive piezoelectric stack to modulate the reference mirror position, the amplitude and phase of the sample reflection is determined without scanning. With the primary scan in the transverse direction, en face and B-scan OCT images can be readily produced with phase information. This project plans to use the dynamic phase information to add an extra level of contrast to the images, based on the motion of the scatterers.
KEYWORDS: Microfluidics, Microscopes, Imaging systems, Charge-coupled devices, Signal to noise ratio, Microscopy, Imaging devices, Aluminum, Gold, Near field optics
In this work, we present a novel optical imaging device that can be directly integrated into a microfluidic network, and can therefore enable on-chip imaging in a microfluidic system. This micro imaging device, termed optofluidic microscope (OFM) is free of bulk optics and is based on a nanohole array defined in a non-transmissive metallic layer that is patterned onto the floor of the microfluidic channel. The operation of the optofluidic microscope is explained in details and its performance is examined with Caenorhabditis elegans (C. elegans) of various genotypes. Images from a large population of worms have been efficiently acquired within a short time frame. The quality of the OFM images of C elegans and the morphological characteristics revealed by the images are evaluated. The experimental results support our claim that the methodology described therein promises to create a powerful tool for fulfilling high- resolution, high-throughput imaging task of the microscopic biological samples.
"Optofluidics" is the marriage of optics, optoelectronics and nanophotonics with fluidics. Such integration represents a new approach for dynamic manipulation of optical properties at length scales both greater than and smaller than the wavelength of light with applications ranging from reconfigurable photonic circuits to fluidically adaptable optics to high sensitivity bio-detection currently under development. The capabilities in terms of fluidic control, mixing, miniaturization and optical property tuning afforded by micro-, nano- and electro-fluidics combined with soft lithography based fabrication provides an ideal platform upon which to build such devices. In this paper we provide a general overview of some of the important issues related to the fabrication, integration and operation of optofluidic devices and present three comprehensive application examples: nanofluidically tunable photonic crystals, optofluidic microscopy and DFB dye lasers.
Molecular contrast OCT (MCOCT) is an extension of OCT in which specific molecular species are imaged based on their spectroscopic characteristics. In order to improve the sensitivity and specificity of MCOCT, several techniques have recently been introduced which depend upon coherent detection of inelastically scattered light from molecules of interest in a sample. These techniques include harmonic generation, coherent anti-Stokes Raman scattering, and several different forms of pump-probe spectroscopy. We have developed a theoretical framework to facilitate the comparison of different inelastic scattering-based contrast mechanisms for molecular contrast OCT. This framework is based upon the observation that since the noise floor is defined by the reference arm power in a shot-noise limited heterodyne detection system, the relevant comparison among the techniques is isolated to the available molecular-specific signal power. We have derived the value of the molecular contrast signal power for second harmonic generation OCT (SHOCT) and three different pump-probe OCT (PPOCT) techniques. Motivated by this analysis, we have constructed a preliminary ground state recovery pump-probe OCT system, and demonstrated its performance using rhodamine 6G as the MCOCT contrast agent.
We report that the complex conjugate ambiguity in spectral domain OCT approaches (including swept source OCT and Fourier-domain OCT) may be removed by the use of novel interferometer designs based on NxN couplers. An interferometer based on a 3x3 truly fused fiber coupler with equal splitting ratios provides simultaneous access to components of the complex interferometric signal separated by 120o. These phase components may be converted to quadrature components by use of a simple trigonometric operation, and then inverse Fourier transformed to obtain A-scans and images free of complex conjugate artifact. We demonstrate instantaneous complex spectral-domain OCT using a novel Fourier-domain OCT system employing photodiode arrays, and will also report on a similar system design for instantaneous complex swept-source OCT.
We describe a novel technique for contrast enhancement in optical coherence tomography (OCT) which uses optically switchable protein based chromophores. Photosensitive proteins, such as bacteriorhodopsin and phytochrome, are promising OCT molecular contrast agents by reason of their remarkably low transition activation intensities compatible with in vivo imaging, and their potential for use as genetically expressible markers for molecular imaging. This study details the use of a novel optical switch suppression scheme which uses the absorption change between the two state groups of phytochrome to extract concentration and distribution information of the contrast agent within a target sample.
We report on the use of two matched linearly chirped fiber Bragg grating (FBG) in the reference arm of a Michelson interferometer as a means to achieve variable optical delay. We demonstrate that the properties of a linearly chirped FBG can be exploited to achieve millimeters of optical delay with physical stretches of the FBG on the order of tens of microns; this allows for optical delay line configurations that are easily driven by piezo-electric actuators.
We describe two novel techniques for contrast enhancement in optical coherence tomography (OCT) which enables molecular specific imaging. The first, a pump-probe technique, is employed in which a pulsed pump laser is tuned to ground-state absorption in a molecule of interest. The location of the target molecule population is derived from the resulting transient absorption of OCT sample arm light acting as probe light. Preliminary results exhibiting contrast enhancement in cross-sectional OCT images using methylene blue dye are presented. The second method is an optical switch suppression technique based on the use of a transmembrane protein called bacteriorhodopsin. Initial experiments indicate that biochemical optical switches, such as bacteriorhodopsin, are excellent contrast agent candidates for molecular contrast OCT.
We present a fast scanning transmission-mode confocal scanning laser microscope system based on the use of a second harmonic generation (SHG) crystal for signal detection. The quadratic intensity dependence of SHG is exploited to preferentially reveal unscattered signal light and reject out-of-focus scattered background. The SHG crystal plays the role of a virtual pinhole that remains self-aligned without a need for de-scanning. We demonstrate that this new microscope method produces images with higher contrast and less speckle than transmission scanning microscopy with linear detection.
We report the use of a highly sensitive phase based motion measurement technique to study the correlation of cellular metabolic rate with cellular motions. The technique is based on a modified Michelson interferometer with a composite laser beam of 1550 nm low coherence light and 775 nm CW light. In this system, motional artifacts from vibrations in the interferometer are completely eliminated. We demonstrate that the system is sensitive to motions as small as 3.6 nm and velocities as small as 1 nm/s. Using the system, we show that the cellular motions are strongly dependent on the ambient temperature. We observe that the dependency does not conform to Brownian motion predictions but instead appears to correlate with the optical ambient temperature that the cells have evolved to operate in.
We describe our phase-sensitive interferometry technique implemented as phase dispersion microscopy (PDM)/optical tomography (PDOT). The technique is based on measuring the phase difference between fundamental and second harmonic low coherence light in a novel interferometer. We attain high sensitivity to subtle refractive index differences due to dispersion with a differential optical path sensitivity of 5 nm. Using PDM, we show that ballistic light in a turbid medium undergoes a phase velocity change that is dependent on scatterer size. We demonstrate that the microscopy technique performs better than a conventional phase contrast microscope in imaging dispersive and weakly scattering samples. The tomographic implementation of the technique (PDOT) can complement Optical Coherence Tomography (OCT) by providing phase information about the scanned object.
A modified Michelson interferometer is used to measure path- length resolved angular distributions of light backscattered by turbid media. The path length resolution is obtained by exploiting the coherence properties of a broadband source. The angular distribution is mapped out using a simple optical system to scan the angle at which the reference field intersects the detector plane. Angular scattering distributions can be compared to Mie theory to determine the size and refractive index of spherical scatterers. Initial studies utilizing this system demonstrate the potential of low coherence interferometry for obtaining structural information using angular distributions.
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