Simplified Instrument Calibration for Wide-Field Fluorescence Resonance Energy Transfer (FRET) Measured by the Sensitized Emission Method.

FÓ§rster (or fluorescence) resonance energy transfer (FRET) is a quantifiable energy transfer in which a donor fluorophore nonradiatively transfers its excitation energy to an acceptor fluorophore. A change in FRET efficiency indicates a change of proximity and environment of these fluorophores, which enables the study of intermolecular interactions. Measurement of FRET efficiency using the sensitized emission method requires a donor-acceptor calibrated system. One of these calibration factors named the G factor, which depends on instrument parameters related to the donor and acceptor measurement channels and on the fluorophores quantum efficiencies, can be determined in several different ways and allows for conversion of the raw donor and acceptor emission signals to FRET efficiency. However, the calculated value of the G factor from experimental data can fluctuate significantly depending on the chosen experimental method and the size of the sample. In this technical note, we extend the results of Gates et al. (Cytometry Part A 95A (2018) 201-213) by refining the calibration method used for calibration of FRET from image pixel data. Instead of using the pixel histograms of two constructs with high and low FRET efficiency to determine the G factor, we use pixel histogram data from one construct of known efficiency. We validate this method by determining the G factor with the same constructs developed and used by Gates et al. and comparing the results from the two approaches. While the two approaches are equivalent theoretically, we demonstrate that the use of a single construct with known efficiency provides a more precise experimental measurement of the G factor that can be attained by collecting a smaller number of images. © 2020 International Society for Advancement of Cytometry.

Microfluidic platforms for the study of neuronal injury in vitro.

Traumatic brain injury (TBI) affects 5.3 million people in the United States, and there are 12,500 new cases of spinal cord injury (SCI) every year. There is yet a significant need for in vitro models of TBI and SCI in order to understand the biological mechanisms underlying central nervous system (CNS) injury and to identify and test therapeutics to aid in recovery from neuronal injuries. While TBI or SCI studies have been aided with traditional in vivo and in vitro models, the innate limitations in specificity of injury, isolation of neuronal regions, and reproducibility of these models can decrease their usefulness in examining the neurobiology of injury. Microfluidic devices provide several advantages over traditional methods by allowing researchers to (1) examine the effect of injury on specific neural components, (2) fluidically isolate neuronal regions to examine specific effects on subcellular components, and (3) reproducibly create a variety of injuries to model TBI and SCI. These microfluidic devices are adaptable for modeling a wide range of injuries, and in this review, we will examine different methodologies and models recently utilized to examine neuronal injury. Specifically, we will examine vacuum-assisted axotomy, physical injury, chemical injury, and laser-based axotomy. Finally, we will discuss the benefits and downsides to each type of injury model and discuss how researchers can use these parameters to pick a particular microfluidic device to model CNS injury.

Special Section Guest Editorial: Seeing Inside Tissue with Optical Molecular Probes.

The editorial introduces the Special Section on Seeing Inside Tissue with Optical Molecular Probes.

Förster resonance energy transfer efficiency measurements on vinculin tension sensors at focal adhesions using a simple and cost-effective setup.

Significance: Forces inside cells play a fundamental role in tissue growth, affecting important processes such as cancer cell migration or tissue repair after injury. Förster resonance energy transfer (FRET)-based tension sensors are a remarkable tool for studying these forces and should be made easier to use. Aim: We prove that absolute FRET efficiency can be measured on a simple setup, an order of magnitude more cost-effective than a standard FRET microscopy setup, by applying it to vinculin tension sensors (VinTS) at the focal adhesions of live CHO-K1 cells. Approach: Our setup located at Université Paris-Saclay acquires donor and acceptor fluorescence in parallel on two low-cost CMOS cameras and uses two LEDs for rapid switching of the excitation wavelength at a reduced cost. The calibration required to extract FRET efficiency was achieved using a single construct (TSMod). FRET efficiencies were measured for VinTS and the tail-less control VinTL, lacking the actin-binding domain of vinculin. Measurements were confirmed on the same cell type using a more standard intensity-based setup located at Rutgers University. Results: The average FRET efficiency of VinTS (22.0%±4%) over more than 10,000 focal adhesions is significantly lower (p<10-6) than that of VinTL (30.4%±5%), our control that is insensitive to force, in agreement with the force exerted on vinculin at focal adhesions. Attachment of the CHO-K1 cells on fibronectin decreases FRET efficiency, thus increasing the force, compared with poly-lysine. FRET efficiency for the VinTL control is consistent with all measurements currently available in the literature, confirming the validity of our measurements and hence of our simpler setup. Conclusions: Force measurements, resolved spatially inside a cell, can be achieved using FRET-based tension sensors with a cost effective intensity-based setup. This will facilitate combining FRET with techniques for applying controlled forces such as optical tweezers.

Measurement of object structure from size-encoded images generated by optically-implemented Gabor filters.

We use optical Fourier processing based on two dimensional (2D) Gabor filters to obtain size-encoded images which depict with 20nm sensitivity to size while preserving a 0.36µm spatial resolution, the spatial distribution of structural features within transparent objects. The size of the object feature measured at each pixel in the encoded image is determined by the optimal Gabor filter period, S(max), that maximizes the scattering signal from that location in the object. We show that S(max) (in µm) depends linearly on feature size (also in µm) over a size range from 0.11µm to 2µm. This linear response remains largely unchanged when the refractive index ratio is varied and can be predicted from numerical simulations of Gabor-filtered light scattering. Pixel histograms of the size-encoded images of isolated spheres and diatoms were used to generate highly resolved size distributions ("size spectra") exhibiting sharp peaks characterizing the known major structural features within the studied objects. Dynamic signal associated with changes in selected feature sizes within living cells is also demonstrated. Taken together, our data suggest that a label-free, direct and objective measurement of sample structure is enabled by the size-encoded images and associated pixel histograms generated from a calibrated optical processing microscope based on Gabor filtering.

Glutamate Receptors Mediate Changes to Dendritic Mitochondria in Neurons Grown on Stiff Substrates.

The stiffness of brain tissue changes during development and disease. These changes can affect neuronal morphology, specifically dendritic arborization. We previously reported that N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors regulate dendrite number and branching in a manner that is dependent on substrate stiffness. Since mitochondria affect the shape of dendrites, in this study, we determined whether the stiffness of substrates on which rat hippocampal neurons are grown affects mitochondrial characteristics and if glutamate receptors mediate the effects of substrate stiffness. Dendritic mitochondria are small, short, simple, and scarce in neurons cultured on substrates of 0.5 kPa stiffness. In contrast, dendritic mitochondria are large, long, complex, and low in number in neurons grown on substrates of 4 kPa stiffness. Dendritic mitochondria of neurons cultured on glass are high in number and small with complex shapes. Treatment of neurons grown on the stiffer gels or glass with the NMDA and AMPA receptor antagonists, 2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitroquinoxaline-2,3-dione, respectively, results in mitochondrial characteristics of neurons grown on the softer substrate. These results suggest that glutamate receptors play important roles in regulating both mitochondrial morphology and dendritic arborization in response to substrate stiffness.

Förster resonance energy transfer efficiency of the vinculin tension sensor in cultured primary cortical neuronal growth cones.

Significance: Interaction of neurons with their extracellular environment and the mechanical forces at focal adhesions and synaptic junctions play important roles in neuronal development. Aim: To advance studies of mechanotransduction, we demonstrate the use of the vinculin tension sensor (VinTS) in primary cultures of cortical neurons. VinTS consists of TS module (TSMod), a Förster resonance energy transfer (FRET)-based tension sensor, inserted between vinculin's head and tail. FRET efficiency decreases with increased tension across vinculin. Approach: Primary cortical neurons cultured on glass coverslips coated with poly-d-lysine and laminin were transfected with plasmids encoding untargeted TSMod, VinTS, or tail-less vinculinTS (VinTL) lacking the actin-binding domain. The neurons were imaged between day in vitro (DIV) 5 to 8. We detail the image processing steps for calculation of FRET efficiency and use this system to investigate the expression and FRET efficiency of VinTS in growth cones. Results: The distribution of fluorescent constructs was similar within growth cones at DIV 5 to 8. The mean FRET efficiency of TSMod ( 28.5 ± 3.6 % ) in growth cones was higher than the mean FRET efficiency of VinTS ( 24.6 ± 2 % ) and VinTL ( 25.8 ± 1.8 % ) ( p < 10 - 6 ). While small, the difference between the FRET efficiency of VinTS and VinTL was statistically significant ( p < 10 - 3 ), suggesting that vinculin is under low tension in growth cones. Two-hour treatment with the Rho-associated kinase inhibitor Y-27632 did not affect the mean FRET efficiency. Growth cones exhibited dynamic changes in morphology as observed by time-lapse imaging. VinTS FRET efficiency showed greater variance than TSMod FRET efficiency as a function of time, suggesting a greater dependence of VinTS FRET efficiency on growth cone dynamics compared with TSMod. Conclusions: The results demonstrate the feasibility of using VinTS to probe the function of vinculin in neuronal growth cones and provide a foundation for studies of mechanotransduction in neurons using this tension probe.

Microscopic imaging and spectroscopy with scattered light.

Optical contrast based on elastic scattering interactions between light and matter can be used to probe cellular structure, cellular dynamics, and image tissue architecture. The quantitative nature and high sensitivity of light scattering signals to subtle alterations in tissue morphology, as well as the ability to visualize unstained tissue in vivo, has recently generated significant interest in optical-scatter-based biosensing and imaging. Here we review the fundamental methodologies used to acquire and interpret optical scatter data. We report on recent findings in this field and present current advances in optical scatter techniques and computational methods. Cellular and tissue data enabled by current advances in optical scatter spectroscopy and imaging stand to impact a variety of biomedical applications including clinical tissue diagnosis, in vivo imaging, drug discovery, and basic cell biology.

Detection of mitochondrial fission with orientation-dependent optical Fourier filters.

We utilize a recently developed optical imaging method based on Fourier processing with Gabor-like filters to detect changes in light scattering resulting from alterations in mitochondrial structure in endothelial cells undergoing apoptosis. Imaging based on Gabor filters shows a significant decrease in the orientation of subcellular organelles at 60 to 100 minutes following apoptosis induction and concomitant with mitochondrial fragmentation observed by fluorescence. The optical scatter changes can be detected at low resolution at the whole cell level. At high resolution, we combine fluorescence imaging of the mitochondria with optical Fourier-based imaging to demonstrate that the dynamic decrease in organelle orientation measured by optical Gabor filtering is spatially associated with fluorescent mitochondria and remains largely absent from nonfluorescent subcellular regions. These results provide strong evidence that the optical Gabor responses track mitochondrial fission during apoptosis and can be used to provide label-free, rapid monitoring of this morphological process within single cells.

Optical Scatter Imaging with a digital micromirror device.

We had developed Optical Scatter Imaging (OSI) as a method which combines light scattering spectroscopy with microscopic imaging to probe local particle size in situ. Using a variable diameter iris as a Fourier spatial filter, the technique consisted of collecting images that encoded the intensity ratio of wide-to-narrow angle scatter at each pixel in the full field of view. In this paper, we replace the variable diameter Fourier filter with a digital micromirror device (DMD) to extend our assessment of morphology to the characterization of particle shape and orientation. We describe our setup in detail and demonstrate how to eliminate aberrations associated with the placement of the DMD in a conjugate Fourier plane of our microscopic imaging system. Using bacteria and polystyrene spheres, we show how this system can be used to assess particle aspect ratio even when imaged at low resolution. We also show the feasibility of detecting alterations in organelle aspect ratio in situ within living cells. This improved OSI system could be further developed to automate morphological quantification and sorting of non-spherical particles in situ.

Highly sensitive size discrimination of sub-micron objects using optical Fourier processing based on two-dimensional Gabor filters.

We use optical Gabor-like filtering implemented with a digital micromirror device to achieve nanoscale sensitivity to changes in the size of finite and periodic objects imaged at low resolution. The method consists of applying an optical Fourier filter bank consisting of Gabor-like filters of varying periods and extracting the optimum filter period that maximizes the filtered object signal. Using this optimum filter period as a measure of object size, we show sensitivity to a 7.5 nm change in the period of a chirped phase mask with period around 1 microm. We also show 30 nm sensitivity to change in the size of polystyrene spheres with diameters around 500 nm. Unlike digital post-processing our optical processing method retains its sensitivity when implemented at low magnification in undersampled images. Furthermore, the optimum Gabor filter period found experimentally is linearly related to sphere diameter over the range 0.46 microm-1 microm and does not rely on a predictive scatter model such as Mie theory. The technique may have applications in high throughput optical analysis of subcellular morphology to study organelle function in living cells.

FRET efficiency measurement in a molecular tension probe with a low-cost frequency-domain fluorescence lifetime imaging microscope.

We demonstrate the possibility of measuring FRET efficiency with a low-cost frequency-domain fluorescence lifetime imaging microscope (FD-FLIM). The system utilizes single-frequency-modulated excitation, which enables the use of cost-effective laser sources and electronics, simplification of data acquisition and analysis, and a dual-channel detection capability. Following calibration with coumarin 6, we measured the apparent donor lifetime in mTFP1-mVenus FRET standards expressed in living cells. We evaluated the system's sensitivity by differentiating the short and long lifetimes of mTFP1 corresponding to the known standards' high and low FRET efficiency, respectively. Furthermore, we show that the lifetime of the vinculin tension sensor, VinTS, at focal adhesions (2.30 ± 0.16 ns) is significantly (p < 10 - 6) longer than the lifetime of the unloaded TSMod probe (2.02 ± 0.16 ns). The pixel dwell time was 6.8 µs for samples expressing the FRET standards, with signal typically an order of magnitude higher than VinTS. The apparent FRET efficiency (EFRETapp) of the standards, calculated from the measured apparent lifetime, was linearly related to their known FRET efficiency by a factor of 0.92 to 0.99 (R2 = 0.98). This relationship serves as a calibration curve to convert apparent FRET to true FRET and circumvent the need to measure multiexponential lifetime decays. This approach yielded a FRET efficiency of 18% to 19.5%, for VinTS, in agreement with published values. Taken together, our results demonstrate a cost-effective, fast, and sensitive FD-FLIM approach with the potential to facilitate applications of FLIM in mechanobiology and FRET-based biosensing.

Dynamin and reverse-mode sodium calcium exchanger blockade confers neuroprotection from diffuse axonal injury.

Mild traumatic brain injury (mTBI) is a frequently overlooked public health concern that is difficult to diagnose and treat. Diffuse axonal injury (DAI) is a common mTBI neuropathology in which axonal shearing and stretching induces breakdown of the cytoskeleton, impaired axonal trafficking, axonal degeneration, and cognitive dysfunction. DAI is becoming recognized as a principal neuropathology of mTBI with supporting evidence from animal model, human pathology, and neuroimaging studies. As mitochondrial dysfunction and calcium overload are critical steps in secondary brain and axonal injury, we investigated changes in protein expression of potential targets following mTBI using an in vivo controlled cortical impact model. We show upregulated expression of sodium calcium exchanger1 (NCX1) in the hippocampus and cortex at distinct time points post-mTBI. Expression of dynamin-related protein1 (Drp1), a GTPase responsible for regulation of mitochondrial fission, also changes differently post-injury in the hippocampus and cortex. Using an in vitro model of DAI previously reported by our group, we tested whether pharmacological inhibition of NCX1 by SN-6 and of dynamin1, dynamin2, and Drp1 by dynasore mitigates secondary damage. Dynasore and SN-6 attenuate stretch injury-induced swelling of axonal varicosities and mitochondrial fragmentation. In addition, we show that dynasore, but not SN-6, protects against H2O2-induced damage in an organotypic oxidative stress model. As there is currently no standard treatment to mitigate cell damage induced by mTBI and DAI, this work highlights two potential therapeutic targets for treatment of DAI in multiple models of mTBI and DAI.

The C-terminal transmembrane domain of Bcl-xL mediates changes in mitochondrial morphology.

We investigate the effect of mitochondrial localization and the Bcl-x(L) C-terminal transmembrane (TM) domain on mitochondrial morphology and subcellular light scattering. CSM 14.1 cell lines stably expressed yellow fluorescent protein (YFP), YFP-Bcl-x(L,) YFP-Bcl-x(L)-DeltaTM, containing the remainder of Bcl-x(L) after deletion of the last 21 amino acids corresponding to the TM domain, or YFP-TM, consisting of YFP fused at its C-terminal to the last 21 amino acids of Bcl-x(L). YFP-Bcl-x(L) and YFP-TM localized to the mitochondria. Their expression decreased the intensity ratio of wide-to-narrow angle forward scatter by subcellular organelles, and correlated with an increase in the proportion of mitochondria with an expanded matrix having greatly reduced intracristal spaces as observed by electron microscopy. Cells expressing YFP-TM also exhibited significant autophagy. In contrast, YFP-Bcl-x(L)-DeltaTM was diffusely distributed in the cells, and its expression did not alter light scattering or mitochondrial morphology compared with parental cells. Expression of YFP-Bcl-x(L) or YFP-Bcl-x(L)-DeltaTM provided significant resistance to staurosporine-induced apoptosis. Surprisingly however, YFP-TM expression also conferred a moderate level of cell death resistance in response to staurosporine. Taken together, our results suggest the existence of a secondary Bcl-x(L) function that is mediated by the transmembrane domain, alters mitochondrial morphology, and is distinct from BH3 domain sequestration.

Label-free dynamic segmentation and morphological analysis of subcellular optical scatterers.

Imaging without fluorescent protein labels or dyes presents significant advantages for studying living cells without confounding staining artifacts and with minimal sample preparation. Here, we combine label-free optical scatter imaging with digital segmentation and processing to create dynamic subcellular masks, which highlight significantly scattering objects within the cells' cytoplasms. The technique is tested by quantifying organelle morphology and redistribution during cell injury induced by calcium overload. Objects within the subcellular mask are first analyzed individually. We show that the objects' aspect ratio and degree of orientation ("orientedness") decrease in response to calcium overload, while they remain unchanged in untreated control cells. These changes are concurrent with mitochondrial fission and rounding observed by fluorescence, and are consistent with our previously published data demonstrating scattering changes associated with mitochondrial rounding during calcium injury. In addition, we show that the magnitude of the textural features associated with the spatial distribution of the masked objects' orientedness values, changes by more than 30% in the calcium-treated cells compared with no change or changes of less than 10% in untreated controls, reflecting dynamic changes in the overall spatial distribution and arrangement of subcellular scatterers in response to injury. Taken together, our results suggest that our method successfully provides label-free morphological signatures associated with cellular injury. Thus, we propose that dynamically segmenting and analyzing the morphology and organizational patterns of subcellular scatterers as a function of time can be utilized to quantify changes in a given cellular condition or state.

Label-Free Classification of Bax/Bak Expressing vs. Double-Knockout Cells.

We combine optical scatter imaging with principal component analysis (PCA) to classify apoptosis-competent Bax/Bak-expressing, and apoptosis resistant Bax/Bak-null immortalized baby mouse kidney cells. We apply PCA to 100 stacks each containing 236 dark-field cell images filtered with an optically implemented Gabor filter with period between 0.3 and 2.9 µm. Each stack yields an "eigencell" image corresponding to the first principal component obtained at one of the 100 Gabor filter periods used. At each filter period, each cell image is multiplied by (projected onto) the eigencell image. A Feature Matrix consisting of 236 × 100 scalar values is thus constructed with significantly reduced dimension compared to the initial dataset. Utilizing this Feature Matrix, we implement a supervised linear discriminant analysis and classify successfully the Bax/Bak-expressing and Bax/Bak-null cells with 94.7% accuracy and an area under the curve (AUC) of 0.993. Applying a feature selection algorithm further reveals that the Gabor filter period ranges most significant for the classification correspond to both large (likely nuclear) features as well as small sized features (likely organelles present in the cytoplasm). Our results suggest that cells with a genetic defect in their apoptosis pathway can be differentiated from their normal counterparts by label-free multi-parametric optical scatter data.

Alterations in the characteristic size distributions of subcellular scatterers at the onset of apoptosis: effect of Bcl-xL and Bax/Bak.

Optical scatter imaging is used to estimate organelle size distributions in immortalized baby mouse kidney cells treated with 0.4 microM staurosporine to induce apoptosis. The study comprises apoptosis competent iBMK cells (W2) expressing the proapoptotic proteins Bax/Bak, apoptosis resistant Bax/Bak null cells (D3), and W2 and D3 cells expressing yellow fluorescent protein (YFP) or YFP fused to the antiapoptotic protein Bcl-x(L) (YFP-Bcl-x(L)). YFP expression is diffuse within the transfected cells, while YFP-Bcl-x(L) is localized to the mitochondria. Our results show a significant increase in the mean subcellular particle size from approximately 1.1 to 1.4 microm in both Bax/Bak expressing and Bax/Bak null cells after 60 min of STS treatment compared to DMSO-treated control cells. This dynamic is blocked by overexpression of YFP-Bcl-x(L) in Bax/Bak expressing cells, but is less significantly inhibited by YFP-Bcl-x(L) in Bax/Bak null cells. Our data suggest that the increase in subcellular particle size at the onset of apoptosis is modulated by Bcl-x(L) in the presence of Bax/Bak, but it occurs upstream of the final commitment to programmed cell death. Mitochondrial localization of YFP-Bcl-x(L) and the finding that micron-sized particles give rise to the scattering signal further suggest that alterations in mitochondrial morphology may underlie the observed changes in light scattering.

Optical scatter microscopy based on two-dimensional Gabor filters.

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living cells. The proposed instrument extends the sensitivity of label-free optical microscopy to nanoscale changes in organelle size and shape and can be used to accelerate the study of the structure-function relationship pertaining to organelle dynamics underlying fundamental biological processes, such as programmed cell death or cellular differentiation. The microscope can be easily implemented on existing microscopy platforms, and can therefore be disseminated to individual laboratories, where scientists can implement and use the proposed methods with unrestricted access. The proposed technique is able to characterize subcellular structure by observing the cell through two-dimensional optical Gabor filters. These filters can be tuned to sense with nanoscale (10's of nm) sensitivity, specific morphological attributes pertaining to the size and orientation of non-spherical subcellular organelles. While based on contrast generated by elastic scattering, the technique does not rely on a detailed inverse scattering model or on Mie theory to extract morphometric measurements. This technique is therefore applicable to non-spherical organelles for which a precise theoretical scatter description is not easily given, and provides distinctive morphometric parameters that can be obtained within unstained living cells to assess their function. The technique is advantageous compared with digital image processing in that it operates directly on the object's field transform rather than the discretized object's intensity. It does not rely on high image sampling rates and can therefore be used to rapidly screen morphological activity within hundreds of cells at a time, thus greatly facilitating the study of organelle structure beyond individual organelle segmentation and reconstruction by fluorescence confocal microscopy of highly magnified digital images of limited fields of view. In this demonstration we show data from a marine diatom to illustrate the methodology. We also show preliminary data collected from living cells to give an idea of how the method may be applied in a relevant biological context.

Optical scatter changes at the onset of apoptosis are spatially associated with mitochondria.

We combine optical scatter imaging (OSI) with fluorescence imaging of mitochondria to investigate the spatial relationship between the optical scatter signal and the location and structure of mitochondria within endothelial cells undergoing apoptosis. The OSI data corroborate our previous results showing a decrease in the intensity ratio of wide-to-narrow angle scatter [optical scatter image ratio (OSIR)] during the first 60 min of apoptosis. In addition, we find here that this is followed by an increase in OSIR concurrent with mitochondrial fragmentation. We demonstrate that the dynamic change in light scattering is spatially associated with subcellular regions containing fluorescently labeled mitochondria, and remains absent from adjacent nonfluorescent regions dominated by other organelles. These results lend strong support to the hypothesis that mitochondria act as the source of the optical scatter changes measured at the onset of apoptosis.

Quantifying subcellular dynamics in apoptotic cells with two-dimensional Gabor filters.

We demonstrate an optical Fourier filtering method which can be used to characterize subcellular morphology during dynamic cellular function. In this paper, our Fourier filters were based on two-dimensional Gabor elementary functions, which can be tuned to sense directly object size and orientation. We utilize this method to quantify changes in mitochondrial and nuclear structure during the first three hours of apoptosis. We find that the technique is sensitive to a decrease in particle orientation consistent with apoptosis-induced mitochondrial fragmentation. The scattering signal changes were less pronounced in the nucleus and the remainder of the cytoplasm. Particles in these regions were less oriented than mitochondria and did not change orientation significantly.