SignificanceFull-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two limitations: suboptimal utilization of functional information about the sample and complicated optical design with several moving parts for phase contrast.AimWe report an OCM setup capable of generating dynamic intensity, phase, and pseudo-spectroscopic contrast with single-shot full-field video-rate imaging called bichromatic tetraphasic (BiTe) full-field OCM with no moving parts.ApproachBiTe OCM resourcefully uses the phase-shifting properties of anti-reflection (AR) coatings outside the rated bandwidths to create four unique phase shifts, which are detected with two emission filters for spectroscopic contrast.ResultsBiTe OCM overcomes the disadvantages of previous FF-OCM setup techniques by capturing both the intensity and phase profiles without any artifacts or speckle noise for imaging scattering samples in three-dimensional (3D). BiTe OCM also utilizes the raw data effectively to generate three complementary contrasts: intensity, phase, and color. We demonstrate BiTe OCM to observe cellular dynamics, image live, and moving micro-animals in 3D, capture the spectroscopic hemodynamics of scattering tissues along with dynamic intensity and phase profiles, and image the microstructure of fall foliage with two different colors.ConclusionsBiTe OCM can maximize the information efficiency of FF-OCM while maintaining overall simplicity in design for quantitative, dynamic, and spectroscopic characterization of biological samples.
We present a novel optical coherence microscopy (OCM) setup capable of generating dynamic intensity, phase, and pseudo-spectroscopic contrast with single-shot full-field video-rate imaging called BiTe (Bichromatic Tetraphasic) full-field OCM. BiTe OCM uniquely uses the phase-shifting properties of AR coatings outside their rated bandwidths to create four distinct phase shifts between the sample and reference. BiTe OCM utilizes the raw data effectively to generate three complementary contrasts. We demonstrate BiTe OCM to observe cellular dynamics, capture the spectroscopic hemodynamics of tumors, and image the microstructure of fall foliage in two different color channels.
Simultaneous Label-free Autofluorescence Multiharmonic (SLAM) microscopy is a nonlinear multimodal optical imaging technique with sub-micron spatial resolution, enabling 3-D visualization and analysis of live cells, complex in vitro models, and tissues. SLAM microscopy detects NAD(P)H and FAD autofluorescence as well as second and third harmonic generation signals simultaneously from biological samples. It can be used for a wide range of applications in cell-to-clinic pharmaceutical research. To run proof-of-concept, longitudinal, and clinical studies of interest to GSK project teams, the GSK Center for Optical Molecular Imaging (COMI) was established in 2015. Based on promising results from these studies, GSK contracted with spin-out start-up, LiveBx, to design and develop the first portable SLAM microscope, and is currently being used for studies on-site at GSK. In this presentation, major milestones and challenges in translating the SLAM technology from academia to industry and key learnings from this process will be shared from multiple perspectives.
The anomalous diffusion characteristics of neuronal dynamics are analyzed by label-free, phase-sensitive optical coherence microscopy. The technique provides low-noise images, enabling cellular dynamic characteristics to be measurable. The phase variance is a conventional dynamic parameter that cannot elucidate the ballistic components of neuronal dynamics. Determining the dynamics by phase variance alone omits the ballistic information that can occur from the ion exchange across cellular membranes. The probability density function of phase displacements exerted by cellular dynamics was acquired and the shape of the power-law tail was analyzed. The development of the power-law tail provides a more sensitive dynamic feature.
Label-free imaging has re-emerged as a premier approach for imaging living cells and tissues within their natural environment. Label-free imaging provides a non-destructive, high throughput platform for understanding and defining the biophysical states of cells. We anticipate that the real-time spatially-resolved information about the biophysical states of cells will be useful for evaluating and quantifying the changes of these states in response to a variety of external interventions. Here we present a portable high-resolution microscopy system which combines five imaging channels, including four simultaneously excited multiphoton imaging channels (3-photon, 2-photon, third and second harmonic) and wide-field near-infrared imaging.
Characterizing the performance of fluorescence microscopy and nonlinear imaging systems is an essential step required for imaging system optimization and quality control during longitudinal experiments. Emerging multimodal nonlinear imaging techniques require a new generation of microscopy calibration targets that are not susceptible to bleaching, and can provide a contrast across the multiple modalities. Here, we present a nanodiamond-based calibration target for microscopy, designed for facilitating reproducible measurements at the object plane. Since fluorescent nanodiamonds are not prone to bleaching shelf-stable sample can provide a rapid reference measurements for ensuring consistent performance of microscopy systems in microscopy laboratories and imaging facilities.
Recent advances in tissue engineering and microfabrication have led to development of novel Complex In Vitro models (CIVMs) that more closely mimic pathophysiological functions of human tissues and organs. CIVMs can provide deeper insights into the mechanisms of human disease and pharmacological properties of new drug candidates during early stages of development. In this study, a multimodal optical imaging platform was used for characterizing the structural and functional features of a liver-on-a-chip model (CN Bio Innovations, UK).
Multimodal imaging systems are designed to extract complementary multifaceted information from biological samples; sensorless adaptive optics (AO) seeks to extend their capabilities by estimating the wavefront from image-derived information. Typical sensorless AO techniques need the acquisition of several volumes for optimization. We present a novel single-shot closed-loop sensorless AO technique demonstrated on a label-free multimodal imaging system consisting of optical coherence, two-photon fast fluorescence lifetime imaging, and second harmonic generation microscopy. The wavefront is sensed by performing computational AO on an initial OCM volume that is translated to the deformable mirror to improve the image quality of all modalities in real-time.
The electrical activity of neurons is invariably accompanied by a flux of ions and the motion of the cell membrane. This leads to subtle changes in the refractive index and birefringence of the sample. We present Superfast Polarization-sensitive Off-axis Full-field Optical Coherence Microscopy (SPoOF OCM), a novel setup to capture these variations. Using two orthogonal spatial modulations for each polarization state and a high-speed camera that can operate at up to 4000 Hz, we demonstrate the ability of our setup to observe label-free electrical activity in both the short (1-10 ms) and long (50-500 ms) term.
We present a novel optical coherence microscopy (OCM) implementation which permits accurate measurements of intensity and phase of light returned from the sample. We demonstrate the capacity to acquire stable phase recordings with a phase sensitivity of 0.1 nm rms at 500 Hz frame rates with a field-of-view of 250x250 µm and 1 µm lateral resolution. Instantaneous, coherence gated, quantitative phase imaging at high frame rates enables observations of the dynamics in cells and tissues, providing instant feedback on metabolic activity and tissue states. We demonstrate applications of the technology for ultrasensitive measurement of intracellular activity and cellular dynamics in cell cultures.
Fluorescence Lifetime Imaging Microscopy (FLIM), providing unique quantitative functional information, has gained popularity in various biomedical and molecular biology studies. Here we present an open-source Python package, FlimTK, a toolkit that enables state-of-the-art functions for FLIM image analysis and visualization. It contains comprehensive functionalities for reading FLIM raw files, fluorescence lifetime estimation, heterogeneity analysis, and spatial distribution analysis. FlimTK package is optimized for high performance and ease of use for integration into custom Python-based analysis workflows. FlimTK source code, demo analysis workflows, and tutorial documentation are available for download from GitHub.
One limitation of phase shifting optical coherence microscopy (OCM) is that any changes in imaging conditions, caused by either system-level instability or movement of dynamic samples, will inevitably lead to reduction in image quality. Here we present a novel implementation of OCM which permits accurate measurements of phase and intensity, by simultaneously capturing four phase-shifted images on a single camera sensor. Uniquely, our approach enables probing the sample with unpolarized light, producing four phase-shifted interferograms, separated by π2 in two orthogonally polarized detection channel pairs. The proposed set-up is compatible with spatially incoherent illumination sources, enabling high resolution imaging due to its inherently reduced susceptibility to speckle noise. By simultaneously capturing the four phase-shifted images on a single CCD camera chip we demonstrate the capacity to acquire stable phase recordings with a phase sensitivity of 0.1 nm rms error at 500 Hz frame rates, a field-of-view of 250x250 μm, and 1μm lateral resolution. Instantaneous, coherence-gated, phase-sensitive imaging at high frame rates enables observations of the dynamics in cells and tissues, providing instant feedback on metabolic activity and tissue states. We present the novel design and methodology that enables ultrasensitive label-free coherence-gated measurements of intracellular dynamics in cells and cellular networks.
Significance: Recent advances in nonlinear optics in neuroscience have focused on using two ultrafast lasers for activity imaging and optogenetic stimulation. Broadband femtosecond light sources can obviate the need for multiple lasers by spectral separation for chromatically targeted excitation.
Aim: We present a photonic crystal fiber (PCF)-based supercontinuum source for spectrally resolved two-photon (2P) imaging and excitation of GCaMP6s and C1V1-mCherry, respectively.
Approach: A PCF is pumped using a 20-MHz repetition rate femtosecond laser to generate a supercontinuum of light, which is spectrally separated, compressed, and recombined to image GCaMP6s (930 nm excitation) and stimulate the optogenetic protein, C1V1-mCherry (1060 nm excitation). Galvanometric spiral scanning is employed on a single-cell level for multiphoton excitation and high-speed resonant scanning is employed for imaging of calcium activity.
Results: Continuous wave lasers were used to verify functionality of optogenetic activation followed by directed 2P excitation. Results from these experiments demonstrate the utility of a supercontinuum light source for simultaneous, single-cell excitation and calcium imaging.
Conclusions: A PCF-based supercontinuum light source was employed for simultaneous imaging and excitation of calcium dynamics in brain tissue. Pumped PCFs can serve as powerful light sources for imaging and activation of neural activity, and overcome the limited spectra and space associated with multilaser approaches.
We present a deformable mirror based remote focusing method for three-dimensional imaging in high-resolution microscopy systems. The method relies on predefined mirror mode arrays that are obtained during initial mirror training step with a low complexity wavefront-sensing module. The imaging plane can be refocused over distances over a hundred times greater than the original depth of field of the objective lens along the optical axis at millisecond rates. We will demonstrate the combination of the remote focusing method with spatiotemporally focused two-photon excitation applied to three-dimensional imaging of biological samples.
We present a novel swept source optical coherence tomography configuration, equipped with acousto-optic deflectors
that can be used to simultaneously acquire multiple B-scans OCT images originating from different depths. The
sensitivity range of the configuration is evaluated while acquiring five simultaneous B-scans. Then the configuration is
employed to demonstrate long range B-scan imaging by combining two simultaneous B-scans from a mouse head
sample.
We present a time-domain polarization-sensitive (PS) optical coherence tomography configuration operating at 830 nm, equipped with multichannel acousto-optic deflectors and single photodetectors. The system is used to simultaneously acquire interference information from multiple PS channels and to enable measurement and imaging of backscattered intensity to create both PS and polarization insensitive images. Our approach enables multiple channel imaging without need to divide the object signal. Here, we employ our system in order to demonstrate PS imaging of a thermally damaged muscle tissue.
In this paper we present a frequency multiplexed optical coherence tomography imaging method in which simultaneous
data acquisition is enabled by a multiplexer. The multiplexer is based on a pair of Bragg cells, simultaneously driven at
several frequencies that enable multiple paths in the reference arm. Multiple beams are created in the multiplexer and
then recombined again so that the resulting beam multiplex can be inserted into a single mode fiber. In this paper we
discuss the design principles for a multiplexer that supports the reflectivity measurements as well as polarization
sensitive imaging at several different depths. We show how to design a system with a low footprint, while allowing
broad optical bandwidth to be used for imaging in multiple channels with little crosstalk between adjacent channels.
We present a novel time-domain polarization sensitive optical coherence tomography configuration operating at 830 nm,
equipped with multichannel acousto-optic deflectors (AOD)s. The system can be used to simultaneously acquire
interference information from multiple polarization-sensitive channels and to enable measurement and imaging of
backscattered intensity, birefringence, and fast optic axis orientation. The system is employed here to demonstrate
polarization sensitive imaging of a thermally damaged muscle tissue.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.