Weak magnetic fields affect a multitude of biological processes including cell metabolism and are hypothesized to be a result of magnetic field-sensitive spin-selective radical-pair reactions. To provide much needed visualization of this process, we demonstrate the use of a custom-built multimodal nonlinear optical imaging system capable of measuring the redox state of cells through multi-photon-excited autofluorescence and autofluorescence lifetime of metabolic cofactors. We demonstrate a custom multi-axis Helmholtz coil system to apply time-varying magnetic fields across the sample during imaging. This imaging platform allows for characterization and optimization of the effects of magnetic fields on live cells and tissues.
Nonlinear microscopy encompasses a range of imaging techniques that leverage laser technology to reveal the chemical composition and structure of a sample. Nonlinear microscopes exploit femtosecond laser pulses to target intrinsic biomolecules of cells and tissues. Fiber lasers have limited bandwidth and reduced wavelength tunability, leading to long pulse durations and limited molecular applications. Supercontinuum generation solves this problem, enabling <50 fs pulses and a larger range of molecular excitation. This paper presents nonlinear microscopy with supercontinuum generation from a Yttrium Aluminum Garnet (YAG) crystal enabling simultaneous label-free autofluorescence multi-harmonic (SLAM) microscopy with high resolution and specificity in biological tissues.
Quality control in molecular optical sectioning microscopy is indispensable for transforming acquired digital images from qualitative descriptions to quantitative data. Although numerous tools, metrics, and phantoms have been developed, accurate quantitative comparisons of data from different microscopy systems with diverse acquisition conditions remains a challenge. Here, we develop a simple tool based on an absolute measurement of bulk fluorophore solutions with related Poisson photon statistics, to overcome this obstacle. Demonstrated in a prototypical multiphoton microscope, our tool unifies the unit of pixelated measurement to enable objective comparison of imaging performance across different modalities, microscopes, components/settings, and molecular targets. The application of this tool in live specimens identifies an attractive methodology for quantitative imaging, which rapidly acquires low signal-to-noise frames with either gentle illumination or low-concentration fluorescence labeling.
Melanopsin, a tri-stable photopigment found in intrinsically-photosensitive retinal ganglion cells (ipRGCs), drives circadian rhythms and other non-image forming functions in the nervous system. Despite increased understanding of the biomolecular and spectroscopic properties of melanopsin, its multiphoton and ultrafast optical absorption properties remain underexplored. We demonstrate the effects of two-photon absorption of melanopsin using 900-1160 nm optical stimulation. Excitation in this bandwidth causes consistent increases in calcium levels in transfected HEK293T cells. Our results demonstrate the first reported nonlinear optical properties and corresponding functional responses of two-photon excitation of melanopsin in vitro, along with the effects of spectral-phase modulation on activation.
We propose a versatile pulse shaper using a 2D spatial light modulator to perform simultaneous and independent control of the spectral amplitude and phase of a fiber-based supercontinuum source. Different spectral optimizations will be demonstrated in a multimodal optical imaging system, combining optical coherence tomography (OCT), multiphoton microscopy (MPM), and nonlinear Raman imaging. This custom pulse shaper enables fast wavelength-tunable excitation for Raman spectroscopy with high spectral resolution. Single beam simultaneous MPM and OCT can also be enabled by compressing a selected multiphoton excitation band while chirping the whole spectrum for ultra-high-resolution OCT.
This research presents our discovery of two new nonlinear optical biomarkers of Alzheimer’s disease (AD), namely 3-photon autofluorescence (3PAF), and Third Harmonic Generation (THG). A hallmark of AD is the aggregation of the Amyloid-Beta (Aβ) protein and Tau protein. Identification of these plaques and analysis of the surrounding cells and tissue is most often done using immunohistochemistry, often with inconsistent results. Using label-free nonlinear optical microscopy, new optical biomarkers were found for identifying a plaque. We present longitudinal imaging of AD progression in mice ranging from 8 to 52 weeks in age, in the hippocampal and cortical regions.
One of the challenges in indirect measurements of electrical activity is its representation as biologically-relevant features. Non-invasive techniques for controlling neural activity, such as optogenetics with simultaneous optical imaging, have emerged as powerful and versatile tools. We demonstrated Superfast Polarization-sensitive Off-axis Full-field (SPoOF) OCM to image changes to both the optical phase and birefringence from the electrical activity of neurons at cellular resolution for an entire network at a millisecond scale. Here, we demonstrate all-optical neurophysiology with SPoOF OCM and optical excitation as a non-invasive versatile technique for studying neural circuits at high throughput, and a method to convert optical metrics to biologically relevant electrical features.
Active neurons experience rapid changes in their metabolic states since they have dynamic energy requirements. In this presentation, we demonstrate fast dual-channel label-free fluorescence lifetime imaging microscopy (FLIM) of NAD(P)H and FAD as a method for neurophysiology by performing computational photon counting in the onboard FPGA of the digitizer. The data throughput is reduced by 4x for each channel by compressing the photocurrents (16 bits) to photon counts (4 bits); the parallel processing on the FPGA ensures no lag. The setup was demonstrated for mammalian stem-cell-derived neurons under chemical stimulation, ion-channel blockers, and optical excitation. Fast FLIM on the FPGA enables dual-channel label-free metabolic optophysiology of neural activity in real time.
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.
We present Superfast Polarization-sensitive Off-axis Full-field Optical Coherence Microscopy (SPoOF OCM) as a novel all-optical technique for neurophysiology. Both the optical path length and birefringence induced by the millisecond-scale electrical activity of neurons are captured by SPoOF OCM at 4000 frames per second and with a field-of-view of 200×200 µm sq., 1 µm transverse resolution, 4.5 µm axial resolution, and 300 pm phase sensitivity. With an ability to capture responses spanning three orders of magnitude in both space and time, SPoOF OCM meets the exacting needs of a comprehensive neurophysiology tool and overcomes the existing limitations of traditional electrophysiology and fluorescence microscopy.
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.
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.
It is still unclear how neuronal metabolism is affected by opioids. We developed a multimodal nonlinear optical imaging platform that can resolve single neurons while monitoring endogenous metabolic molecules in a label-free manner. These capabilities enable investigation of morphine-induced metabolic changes in neurons. Additionally, mouse brain slices were used to study the effects of morphine on metabolism in the brain. We found that after morphine exposure, the nucleus accumbens area showed increased metabolic activity compared to the cerebral cortex. Further studies will reveal how opioids alter the metabolism of the brain and individual neurons at the sub-cellular level.
Multicore fiber bundles for imaging and stimulating optogenetically modified neurons have been largely adopted in neurophotonics research. They allow for directed, single-cell stimulation and imaging of neuronal activity. An inherent limitation of these bundles is the presence and detection of the empty space between individual fibers, resulting in a loss of significant amounts of data, and reduced image quality due to pixilation effects. We propose a novel approach and algorithm to depixelation and image reconstruction from fiber bundles that utilizes multiple image frames collected during on-axis fiber bundle rotation. The approach involves first acquiring the Fourier transform of a stationary, unrotated image, followed by its rotated counterparts. The phase information from each image is then acquired, cross-correlated, and the angle of rotation determined from this correlation. Rotated images are then weighed and summed to generate a final reconstructed, depixelated image. Simulations were initially performed using Matlab demo images. Experimentation was done with a resolution chart, and thereafter with a cell culture. 488 nm and 561 nm continuous wave laser sources (Coherent, Inc.) were used for imaging GCaMP6s and C1V1-mCherry, respectively, in hippocampal neuronal cultures. The light sources were coupled to a multicore fiber bundle (Schott, 1534702) containing 4,200, 7.5 µm fibers. Cell cultures were prepared from 2 day old transgenic mice (GCaMP6s, Jackson Labs) transfected with C1V1(E122T/E162T)-TS-p2A-mCherry (Karl Deisseroth, Stanford). The results demonstrate this as an effective technique alongside fiber bundle imaging, serving as a useful and powerful tool for removing undesired artifacts associated with these fibers.
All-optical systems for stimulating and imaging neuronal activity have served as powerful tools for understanding the underlying circuitry of the brain. Experiments using these setups, however, tend to choose stimulation locations based solely on what brain regions are of interest, and take for granted that stimulation effects may vary even within localized brain regions. We thus have developed an algorithm for acquiring neuronal activity via calcium imaging data to assess network connectivity. These parameters include the signal rise time, decay time, inter-event intervals, and the timing and amplitude of signal peaks. These parameters are then compared between cell clusters for similarities, and used as a basis for establishing interconnectivity. Additionally, we have incorporated both temporal and spatial correlation functions to assess inter-neuronal connectivity based on these parameters. This data is then run through a genetic algorithm, applying weights to cells with similar parameters to learn which are interconnected in a given field-of-view. For this study, hippocampal neurons extracted from 2 day old transgenic mice (GCaMP6s, Jackson Labs), - cultured for 2 weeks and imaged under single and two-photon conditions. Single-photon imaging was performed under a commercial Zeiss microscope, whereas two-photon imaging was performed with an in-house imaging system. Results demonstrate a strong correlation between these parameters and cellular connectivity, making them noteworthy markers for targeted stimulation. This study demonstrates an efficient method of assessing network connectivity for various imaging techniques, and hence directed targeting for optogenetic stimulation.
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