We study the control of coherent light propagation through multiple-scattering media in the presence of measurement noise. In our experiments, we use a two-step optimization procedure to find the optimal incident wavefront that generates a bright focal spot behind the medium. We conclude that the control of coherent light propagation through a multiple-scattering medium is only determined by the number of photoelectrons detected per optimized segment. The prediction of our model agrees well with the experimental results. Our results offer opportunities for imaging applications through scattering media such as biological tissue in the shot noise limit.
(030.6600) Statistical optics; (110.7050) Turbid media; (290.4210) Multiple scattering
Optical coherence tomography (OCT) allows for non-invasive 3D visualization of biological tissue at cellular level resolution. Often hindered by speckle noise, the visualization of important biological tissue details in OCT that can aid disease diagnosis can be improved by speckle noise compensation. A challenge with handling speckle noise is its inherent non-stationary nature, where the underlying noise characteristics vary with the spatial location. In this study, an innovative speckle noise compensation method is presented for handling the non-stationary traits of speckle noise in OCT imagery. The proposed approach centers on a non-stationary spline-based speckle noise modeling strategy to characterize the speckle noise. The novel method was applied to ultra high-resolution OCT (UHROCT) images of the human retina and corneo-scleral limbus acquired in-vivo that vary in tissue structure and optical properties. Test results showed improved performance of the proposed novel algorithm compared to a number of previously published speckle noise compensation approaches in terms of higher signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR) and better overall visual assessment.
(110.4500) Optical coherence tomography; (030.6140) Speckle; (100.2980) Image enhancement; (100.3010) Image reconstruction techniques; (170.4460) Ophthalmic optics and devices
The rapid growth of microfluidic cell culturing in biological and biomedical research and industry calls for fast, non-invasive and reliable methods of evaluating conditions such as pH inside a microfluidic system. We show that by careful calibration it is possible to measure pH within microfluidic chambers with high accuracy and precision, using a direct single-pass measurement of light absorption in a commercially available phenol-red-containing cell culture medium. The measurement is carried out using a standard laboratory microscope and, contrary to previously reported methods, requires no modification of the microfluidic device design. We demonstrate the validity of this method by measuring absorption of light transmitted through 30-micrometer thick microfluidic chambers, using an inverted microscope fitted with a scientific-grade digital camera and two bandpass filters. In the pH range of 7–8, our measurements have a standard deviation and absolute error below 0.05 for a measurement volume smaller than 4 nL.
(120.0120) Instrumentation, measurement, and metrology; (120.3940) Metrology; (120.7000) Transmission; (300.1030) Absorption; (170.1420) Biology
This work reports a multimodal system for label-free tissue diagnosis combining fluorescence lifetime imaging (FLIm), ultrasound backscatter microscopy (UBM), and photoacoustic imaging (PAI). This system provides complementary biochemical, structural and functional features allowing for enhanced in vivo detection of oral carcinoma. Results from a hamster oral carcinoma model (normal, precancer and carcinoma) are presented demonstrating the ability of FLIm to delineate biochemical composition at the tissue surface, UBM and related radiofrequency parameters to identify disruptions in the tissue microarchitecture and PAI to map optical absorption associated with specific tissue morphology and physiology.
(300.6500) Spectroscopy, time-resolved; (170.7180) Ultrasound diagnostics; (170.5120) Photoacoustic imaging; (170.3880) Medical and biological imaging
Angle-resolved low coherence interferometry (a/LCI) is an approach for assessing tissue structure based on light scattering data. Recent advances in a/LCI have extended the analysis to study scattering distributions in two dimensions. In order to provide suitable scattering phantoms for 2D a/LCI, we have developed phantoms based on soft lithography which can provide a range of structures including long range order. Here we characterize these phantoms and demonstrate their utility for providing standardized multi-scale structural information for light scattering measurements.
(290.0290) Scattering; (290.3200) Inverse scattering; (290.5820) Scattering measurements; (120.3180) Interferometry
Non-invasive reflectance imaging of the human RPE cell mosaic is demonstrated using a modified
confocal adaptive optics scanning light ophthalmoscope (AOSLO). The confocal circular aperture in
front of the imaging detector was replaced with a combination of a circular aperture 4 to 16 Airy
disks in diameter and an opaque filament, 1 or 3 Airy disks thick. This arrangement reveals the RPE
cell mosaic by dramatically attenuating the light backscattered by the photoreceptors. The RPE cell
mosaic was visualized in all 7 recruited subjects at multiple retinal locations with varying degrees
of contrast and cross-talk from the photoreceptors. Various experimental settings were explored for
improving the visualization of the RPE cell boundaries including: pinhole diameter, filament
thickness, illumination and imaging pupil apodization, unmatched imaging and illumination focus,
wavelength and polarization. None of these offered an obvious path for enhancing image contrast. The
demonstrated implementation of dark-field AOSLO imaging using 790 nm light requires low light
exposures relative to light safety standards and it is more comfortable for the subject than the
traditional autofluorescence RPE imaging with visible light. Both these factors make RPE dark-field
imaging appealing for studying mechanisms of eye disease, as well as a clinical tool for screening
and monitoring disease progression.
(170.4460) Ophthalmic optics and devices; (170.4470) Ophthalmology; (290.4210) Multiple scattering; (110.1080) Active or adaptive optics
Superoxide anion is the key radical that causes intracellular oxidative stress. The lack of a method to directly monitor superoxide concentration in vivo in real time has severely hindered our understanding on its pathophysiology. We made transgenic zebrafish to specifically express yellow fluorescent proteins, a reversible superoxide-specific indicator, in the liver and used a fiber-optic fluorescent probe to noninvasively monitor the superoxide concentration in real time. Several superoxide-inducing and scavenging reagents were administrated onto the fish to alter superoxide concentrations. The distinct biochemical pathways of the reagents can be discerned from the transient behaviors of fluorescence time courses. These results demonstrate the feasibility of this method for analyzing superoxide dynamics and its potential as an in vivo pharmaceutical screening platform.
(060.2370) Fiber optics sensors; (170.1470) Blood or tissue constituent monitoring; (170.2655) Functional monitoring and imaging; (170.6280) Spectroscopy, fluorescence and luminescence
We propose and demonstrate a dark-field imaging technique capable of automated identification of individual bacteria. An 87-channel multispectral system capable of angular and spectral resolution was used to measure the scattering spectrum of various bacteria in culture smears. Spectra were compared between various species and between various preparations of the same species. A 15-channel system was then used to prove the viability of bacterial identification with a relatively simple microscope system. A simple classifier was able to identify four of six bacterial species with greater than 90% accuracy in bacteria-by-bacteria testing.
(170.0180) Microscopy; (170.4580) Optical diagnostics for medicine; (290.5820) Scattering measurements
High resolution microscopy is essential for advanced study of biological structures and accurate diagnosis of medical diseases. The spatial resolution of conventional microscopes is light diffraction limited. Structured illumination has been extensively explored to break the diffraction limit in wide field light microscopy. However, deployable application of the structured illumination in scanning laser microscopy is challenging due to the complexity of the illumination system and possible phase errors in sequential illumination patterns required for super-resolution reconstruction. We report here a super-resolution scanning laser imaging system which employs virtually structured detection (VSD) to break the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost and phase-artifact free strategy to achieve super-resolution in scanning laser microscopy.
(100.6640) Superresolution; (110.3080) Infrared imaging; (170.3880) Medical and biological imaging; (180.5810) Scanning microscopy
Optical coherence microscopy (OCM) is a widely used structural imaging modality. To extend its application in molecular imaging, gold nanorods are widely used as contrast agents for OCM. However, they very often offer limited sensitivity as a result of poor signal to background ratio. Here we experimentally demonstrate that a novel OCM implementation based on dark-field circular depolarization detection can efficiently detect circularly depolarized signal from gold nanorods and at the same time efficiently suppress the background signals. This results into a significant improvement in signal to background ratio.
(110.4500) Optical coherence tomography; (120.5820) Scattering measurements; (180.3170) Interference microscopy; (290.5850) Scattering, particles; (290.5855) Scattering, polarization
Fluorescence correlation spectroscopy (FCS) is one of the most sensitive methods for enumerating low concentration nanoparticles in a suspension. However, biological nanoparticles such as viruses often exist at a concentration much lower than the FCS detection limit. While optically generated trapping potentials are shown to effectively enhance the concentration of nanoparticles, feasibility of FCS for enumerating field-enriched nanoparticles requires understanding of the nanoparticle behavior in the external field. This paper reports an experimental study that combines optical trapping and FCS to examine existing theoretical predictions of particle concentration. Colloidal suspensions of polystyrene (PS) nanospheres and HIV-1 virus-like particles are used as model systems. Optical trapping energies and statistical analysis are used to discuss the applicability of FCS for enumerating nanoparticles in a potential well produced by a force field.
(350.4855) Optical tweezers or optical manipulation; (140.7010) Laser trapping; (290.1990) Diffusion; (180.2520) Fluorescence microscopy; (170.1790) Confocal microscopy
Light sheet microscopy allows rapid imaging of three-dimensional fluorescent samples, using illumination and detection axes that are orthogonal. For imaging large samples, this often forces the objective to be tilted relative to the sample’s surface; for samples that are not precisely matched to the immersion medium index, this tilt introduces aberrations. Here we calculate the nature of these aberrations for a simple tissue model, and show that a low-dimensional parametrization of these aberrations facilitates online correction via a deformable mirror without introduction of beads or other fiducial markers. We use this approach to demonstrate improved image quality in living tissue.
(110.1080) Active or adaptive optics; (180.2520) Fluorescence microscopy
In a blood-lipid liquid phantom the prototype near-infrared spectroscopy oximeter OxyPrem was calibrated against the INVOS® 5100c adult sensor in respect to values of regional tissue oxygen haemoglobin saturation (rStO2) for possible inclusion in the randomised clinical trial - SafeBoosC. In addition different commercial NIRS oximeters were compared on changing haemoglobin oxygen saturation and compared against co-oximetry. The best calibration was achieved with a simple offset and a linear scaling of the OxyPrem rStO2 values. The INVOS adult and pediatric sensor gave systematically different values, while the difference between the NIRO® 300 and the two INVOS sensors were magnitude dependent. The co-oximetry proved unreliable on such low haemoglobin and high Intralipid levels.
(170.1470) Blood or tissue constituent monitoring; (300.6190) Spectrometers
Optical pacing has been demonstrated to be a viable alternative to electrical pacing in embryonic hearts. In this study, the feasibility of optically pacing an adult rabbit heart was explored. Hearts from adult New Zealand White rabbits (n = 9) were excised, cannulated and perfused on a modified Langendorff apparatus. Pulsed laser light (λ = 1851 nm) was directed to either the left or right atrium through a multimode optical fiber. An ECG signal from the left ventricle and a trigger pulse from the laser were recorded simultaneously to determine when capture was achieved. Successful optical pacing was demonstrated by obtaining pacing capture, stopping, then recapturing as well as by varying the pacing frequency. Stimulation thresholds measured at various pulse durations suggested that longer pulses (8 ms) had a lower energy capture threshold. To determine whether optical pacing caused damage, two hearts were perfused with 30 µM of propidium iodide and analyzed histologically. A small number of cells near the stimulation site had compromised cell membranes, which probably limited the time duration over which pacing was maintained. Here, short-term optical pacing (few minutes duration) is demonstrated in the adult rabbit heart for the first time. Future studies will be directed to optimize optical pacing parameters to decrease stimulation thresholds and may enable longer-term pacing.
(140.3460) Lasers; (170.3890) Medical optics instrumentation
Spectrally encoded confocal microscopy (SECM) is a reflectance confocal microscopy technology that uses a diffraction grating to illuminate different locations on the sample with distinct wavelengths. SECM can obtain line images without any beam scanning devices, which opens up the possibility of high-speed imaging with relatively simple probe optics. This feature makes SECM a promising technology for rapid endoscopic imaging of internal organs, such as the esophagus, at microscopic resolution. SECM imaging of the esophagus has been previously demonstrated at relatively low line rates (5 kHz). In this paper, we demonstrate SECM imaging of large regions of esophageal tissues at a high line imaging rate of 100 kHz. The SECM system comprises a wavelength-swept source with a fast sweep rate (100 kHz), high output power (80 mW), and a detector unit with a large bandwidth (100 MHz). The sensitivity of the 100-kHz SECM system was measured to be 60 dB and the transverse resolution was 1.6 µm. Excised swine and human esophageal tissues were imaged with the 100-kHz SECM system at a rate of 6.6 mm2/sec. Architectural and cellular features of esophageal tissues could be clearly visualized in the SECM images, including papillae, glands, and nuclei. These results demonstrate that large-area SECM imaging of esophageal tissues can be successfully conducted at a high line imaging rate of 100 kHz, which will enable whole-organ SECM imaging in vivo.
(170.1790) Confocal microscopy; (170.2680) Gastrointestinal; (170.4730) Optical pathology
Flow cytometry is a powerful tool for cell counting and biomarker detection in biotechnology and medicine especially with regards to blood analysis. Standard flow cytometers perform cell type classification both by estimating size and granularity of cells using forward- and side-scattered light signals and through the collection of emission spectra of fluorescently-labeled cells. However, cell surface labeling as a means of marking cells is often undesirable as many reagents negatively impact cellular viability or provide activating/inhibitory signals, which can alter the behavior of the desired cellular subtypes for downstream applications or analysis. To eliminate the need for labeling, we introduce a label-free imaging-based flow cytometer that measures size and cell protein concentration simultaneously either as a stand-alone instrument or as an add-on to conventional flow cytometers. Cell protein concentration adds a parameter to cell classification, which improves the specificity and sensitivity of flow cytometers without the requirement of cell labeling. This system uses coherent dispersive Fourier transform to perform phase imaging at flow speeds as high as a few meters per second.
(170.0180) Microscopy; (170.1530) Cell analysis; (170.7160) Ultrafast technology; (100.5070) Phase retrieval; (170.3890) Medical optics instrumentation
Acoustical and optical non-diffracting beams are potentially useful for manipulating particles
and larger objects. An extended optical theorem for a non-diffracting beam was given recently in the
context of acoustics. The theorem relates the extinction by an object to the scattering at the
forward direction of the beam’s plane wave components. Here we use this theorem to examine
the extinction cross section of a sphere centered on the axis of the beam, with a non-diffracting
Bessel beam as an example. The results are applied to recover the axial radiation force and torque
on the sphere by the Bessel beam.
(350.4855) Optical tweezers or optical manipulation; (290.2200) Extinction; (290.4020) Mie theory; (290.5850) Scattering, particles
As contrast agents, microbubbles have been playing significant roles in ultrasound imaging. Investigation of microbubble oscillation is crucial for microbubble characterization and detection. Unfortunately, 3-dimensional (3D) observation of microbubble oscillation is challenging and costly because of the bubble size—a few microns in diameter—and the high-speed dynamics under MHz ultrasound pressure waves. In this study, a cost-efficient optical confocal microscopic system combined with a gated and intensified charge-coupled device (ICCD) camera were developed to detect 3D microbubble oscillation. The capability of imaging microbubble high-speed oscillation with much lower costs than with an ultra-fast framing or streak camera system was demonstrated. In addition, microbubble oscillations along both lateral (x and y) and axial (z) directions were demonstrated. Accordingly, this system is an excellent alternative for 3D investigation of microbubble high-speed oscillation, especially when budgets are limited.
(170.0110) Imaging systems; (170.1790) Confocal microscopy; (170.7160) Ultrafast technology; (170.7170) Ultrasound
We report the use of a twisted nematic liquid-crystal spatial light modulator (TNLC-SLM) for quantitative phase imaging. The experimental setup is a new implementation of the SLIM principle, which is a phase shifting, white light method for quantitative phase imaging. The approach is based on switching between the phase and amplitude modulation modes of the SLM. Our system is able to deliver a 0.99 nm spatial and 1.33 nm temporal pathlength sensitivity while retaining the optical transverse resolution. The system is implemented as an additional module mounted to a conventional microscope, which makes the system very easy to deploy and integrate with other imaging modalities.
(180.0180) Microscopy; (180.3170) Interference microscopy; (070.6120) Spatial light modulators; (110.0110) Imaging systems; (060.5060) Phase modulation
Multi-scale multimodal microscopy is a very useful technique by providing multiple imaging contrasts with adjustable field of views and spatial resolutions. Here, we present a tri-modal microscope combining multiphoton microscopy (MPM), optical coherence microscopy (OCM) and optical coherence tomography (OCT) for subsurface visualization of biological tissues. The advantages of the tri-modal system are demonstrated on various biological samples. It enables the visualization of multiple intrinsic contrasts including scattering, two-photon excitation fluorescence (TPEF), and second harmonic generation (SHG). It also enables a rapid scanning over a large tissue area and a high resolution zoom-in for cellular-level structures on regions of interest. The tri-modal microscope can be important for label-free imaging to obtain a sufficient set of parameters for reliable sample analysis.
(180.4315) Nonlinear microscopy; (140.7090) Ultrafast lasers; (110.4500) Optical coherence tomography; (170.3880) Medical and biological imaging
Custom high-resolution high-speed anterior segment spectral domain Optical Coherence Tomography (OCT) provided with automatic quantification and distortion correction algorithms was used to characterize three-dimensionally (3-D) the human crystalline lens in vivo in four subjects, for accommodative demands between 0 to 6 D in 1 D steps. Anterior and posterior lens radii of curvature decreased with accommodative demand at rates of 0.73 and 0.20 mm/D, resulting in an increase of the estimated optical power of the eye of 0.62 D per diopter of accommodative demand. Dynamic fluctuations in crystalline lens radii of curvature, anterior chamber depth and lens thickness were also estimated from dynamic 2-D OCT images (14 Hz), acquired during 5-s of steady fixation, for different accommodative demands. Estimates of the eye power from dynamical geometrical measurements revealed an increase of the fluctuations of the accommodative response from 0.07 D to 0.47 D between 0 and 6 D (0.044 D per D of accommodative demand). A sensitivity analysis showed that the fluctuations of accommodation were driven by dynamic changes in the lens surfaces, particularly in the posterior lens surface.
(110.4500) Optical coherence tomography; (330.7322) Visual optics, accommodation; (120.6650) Surface measurements, figure; (120.4640) Optical instruments; (110.6880) Three-dimensional image acquisition; (330.7327) Visual optics, ophthalmic instrumentation
In this paper, we demonstrate a new single-cell optoporation and transfection technique using a femtosecond Gaussian laser beam and optical tweezers. Tightly focused near-infrared (NIR) femtosecond laser pulse was employed to transiently perforate the cellular membrane at a single point in MCF-7 cancer cells. A distinct technique was developed by trapping the microparticle using optical tweezers to focus the femtosecond laser precisely on the cell membrane to puncture it. Subsequently, an external gene was introduced in the cell by trapping and inserting the same plasmid-coated microparticle into the optoporated cell using optical tweezers. Various experimental parameters such as femtosecond laser exposure power, exposure time, puncture hole size, exact focusing of the femtosecond laser on the cell membrane, and cell healing time were closely analyzed to create the optimal conditions for cell viability. Following the insertion of plasmid-coated microparticles in the cell, the targeted cells exhibited green fluorescent protein (GFP) under the fluorescent microscope, hence confirming successful transfection into the cell. This new optoporation and transfection technique maximizes the level of selectivity and control over the targeted cell, and this may be a breakthrough method through which to induce controllable genetic changes in the cell.
(140.7090) Ultrafast lasers; (320.2250) Femtosecond phenomena; (020.7010) Laser trapping; (000.1430) Biology and medicine; (140.3538) Lasers, pulsed; (020.4180) Multiphoton processes
Imaging of simultaneous two-photon absorption and stimulated Raman scattering is accomplished by detecting the intensity changes of the two-color pulses simultaneously and the mathematical operations of addition and subtraction. The stimulated Raman scattering is quantitatively separated from the two-photon absorption, generated in a mixed solution in which a glycerin solution is miscible in various proportions with a quantum dot solution. Our technique is applied to simultaneous two-photon absorption and stimulated Raman scattering imaging.
(180.4315) Nonlinear microscopy; (180.5655) Raman microscopy; (190.4180) Multiphoton processes
One of the most promising applications of the X-ray phase-contrast imaging is the three dimensional tomographic reconstruction of the index of refraction. However, results reported so far are limited to relatively small samples. We present here the tomographic reconstruction of the index of refraction distribution of a large biomedical sample (> 10 cm diameter). A quantitative study comparing the absorption and phase contrast (analyzer-based) tomography images shows that the distribution of the index of refraction obtained with the phase contrast method provides a more accurate depiction (3–10 times larger signal to noise ratio values) of the sample internal structure. Thanks to the higher sensitivity of this method, the improved precision was obtained using an incoming photon fluence on the sample several times smaller than in the case of absorption imaging.
(110.6955) Tomographic imaging; (110.7440) X-ray imaging; (120.5710) Refraction
We propose a novel compressive sensing (CS) method on spectral domain optical coherence tomography (SDOCT). By replacing the widely used uniform discrete Fourier transform (UDFT) matrix with a new sensing matrix which is a modification of the non-uniform discrete Fourier transform (NUDFT) matrix, it is shown that undersampled non-linear wavenumber spectral data can be used directly in the CS reconstruction. Thus k-space grid filling and k-linear mask calibration which were proposed to obtain linear wavenumber sampling from the non-linear wavenumber interferometric spectra in previous studies of CS in SDOCT (CS-SDOCT) are no longer needed. The NUDFT matrix is modified to promote the sparsity of reconstructed A-scans by making them symmetric while preserving the value of the desired half. In addition, we show that dispersion compensation can be implemented by multiplying the frequency-dependent correcting phase directly to the real spectra, eliminating the need for constructing complex component of the real spectra. This enables the incorporation of dispersion compensation into the CS reconstruction by adding the correcting term to the modified NUDFT matrix. With this new sensing matrix, A-scan with dispersion compensation can be reconstructed from undersampled non-linear wavenumber spectral data by CS reconstruction. Experimental results show that proposed method can achieve high quality imaging with dispersion compensation.
(170.4500) Optical coherence tomography; (100.3010) Image reconstruction techniques