Engineered fluidic device to achieve multiplexed monitoring of cell cultures with digital holographic microscopy.

We present a low-cost, 3D-printed, and biocompatible fluidic device, engineered to produce laminar and homogeneous flow over a large field-of-view. Such a fluidic device allows us to perform multiplexed temporal monitoring of cell cultures compatible with the use of various pharmacological protocols. Therefore, specific properties of each of the observed cell cultures can be discriminated simultaneously during the same experiment. This was illustrated by monitoring the agonists-mediated cellular responses, with digital holographic microscopy, of four different cell culture models of cystic fibrosis. Quantitatively speaking, this multiplexed approach provides a time saving factor of around four to reveal specific cellular features.

Simultaneous measurements of a specimen quantitative-phase signal and its surrounding medium refractive index using quantitative-phase imaging.

This Letter demonstrates a method to simultaneously measure the quantitative-phase signal (QPS) of the observed specimen and the refractive index of its surrounding medium (nm) in a time-resolved manner using a micro-structured coverslip. Such coverslips, easily integrated into perfused live-cell imaging chambers, allow to use various quantitative-phase imaging techniques to achieve this dual measurement. Since QPS is crucially dependent on nm, the measurement of the latter paves the way for its manipulation in a controlled manner leading to a QPS contrast modulation for appealing applications, including visualizing the interior of cells.

Sample and substrate preparation for exploring living neurons in culture with quantitative-phase imaging.

Quantitative-phase imaging (QPI) has recently emerged as a powerful new quantitative microscopy technique suitable for the noninvasive exploration of the structure and dynamics of transparent specimens, including living cells in culture. Indeed, the quantitative-phase signal (QPS), induced by transparent living cells, can be detected with a nanometric axial sensitivity, and contains a wealth of information about both cell morphology and content. However, QPS is also sensitive to various sources of experimental noise. In this chapter, we emphasize how to properly and specifically measure the cell-mediated QPS in a wet-lab environment, when measuring with a digital holographic microscope (DHM). First, we present the substrate-requisite characteristics for properly achieving such cell-mediated QPS measurements at single-cell level. Then, we describe how quantitative-phase digital holographic microscopy (QP-DHM) can be used to numerically process holograms and subsequently reshape wavefronts in association with post-processing algorithms, thereby allowing for highly stable QPS obtainable over extended periods of time. Such stable QPS is a prerequisite for exploring the dynamics of specific cellular processes. We also describe experimental procedures that make it possible to extract important biophysical cellular parameters from QPS including absolute cell volume, transmembrane water permeability, and the movements of water in and out of the cell. To illustrate how QP-DHM can reveal the dynamics of specific cellular processes, we show how the monitoring of transmembrane water movements can be used to resolve the neuronal network dynamics at single-cell level. This is possible because QPS can measure the activity of electroneutral cotransports, including NKCC1 and KCC2, during a neuronal firing mediated by glutamate, the main excitatory neurotransmitter in the brain. Finally, we added a supplemental section, with more technical details, for readers who are interested in troubleshooting live-cell QP-DHM.

Comparative study of quantitative phase imaging techniques for refractometry of optical waveguides.

A comparative study of quantitative phase imaging techniques for refractometry of optical waveguides is presented. Three techniques were examined: a method based on the transport-of-intensity equation, quadri-wave lateral shearing interferometry and digital holographic microscopy. The refractive index profile of a SMF-28 optical fiber was thoroughly characterized and served as a gold standard to assess the accuracy and precision of the phase imaging methods. Optical waveguides were inscribed in an Eagle2000 glass chip using a femtosecond laser and used to evaluate the sensitivity limit of these phase imaging approaches. It is shown that all three techniques provide accurate, repeatable and sensitive refractive index measurements. Using these phase imaging methods, we report a comprehensive map of the photosensitivity to femtosecond pulses of Eagle2000 glass. Finally, the reported data suggests that the phase imaging techniques are suited to be used as precise and non-destructive refractive index shift measuring tools to study and control the inscription process of optical waveguides.

Betacellulin regulates schwann cell proliferation and myelin formation in the injured mouse peripheral nerve.

When a nerve fiber is cut or crushed, the axon segment that is separated from the soma degenerates distal from the injury in a process termed Wallerian degeneration (WD). C57BL/6OlaHsd-WldS (WldS ) mutant mice exhibit significant delays in WD. This results in considerably delayed Schwann cell and macrophage responses and thus in impaired nerve regenerations. In our previous work, thousands of genes were screened by DNA microarrays and over 700 transcripts were found to be differentially expressed in the injured sciatic nerve of WldS compared with wild-type (WT) mice. One of these transcripts, betacellulin (Btc), was selected for further analysis since it has yet to be characterized in the nervous system, despite being known as a ligand of the ErbB receptor family. We show that Btc mRNA is strongly upregulated in immature and dedifferentiated Sox2+ Schwann cells located in the sciatic nerve distal stump of WT mice, but not WldS mutants. Transgenic mice ubiquitously overexpressing Btc (Tg-Btc) have increased numbers of Schmidt-Lantermann incisures compared with WT mice, as revealed by Coherent anti-Stokes Raman scattering (CARS). Tg-Btc mice also have faster nerve conduction velocity. Finally, we found that deficiency in Btc reduces the proliferation of myelinating Schwann cells after sciatic nerve injury, while Btc overexpression induces Schwann cell proliferation and improves recovery of locomotor function. Taken together, these results suggest a novel regulatory role of Btc in axon-Schwann cell interactions involved in myelin formation and nerve repair. GLIA 2017 GLIA 2017;65:657-669.

Understanding the nervous system: lessons from Frontiers in Neurophotonics.

The Frontiers in Neurophotonics Symposium is a biennial event that brings together neurobiologists and physicists/engineers who share interest in the development of leading-edge photonics-based approaches to understand and manipulate the nervous system, from its individual molecular components to complex networks in the intact brain. In this Community paper, we highlight several topics that have been featured at the symposium that took place in October 2022 in Québec City, Canada.

A robust and reliable methodology to perform GECI-based multi-time point neuronal calcium imaging within mixed cultures of human iPSC-derived cortical neurons.

Introduction: Human induced pluripotent stem cells (iPSCs), with their ability to generate human neural cells (astrocytes and neurons) from patients, hold great promise for understanding the pathophysiology of major neuropsychiatric diseases such as schizophrenia and bipolar disorders, which includes alterations in cerebral development. Indeed, the in vitro neurodifferentiation of iPSCs, while recapitulating certain major stages of neurodevelopment in vivo, makes it possible to obtain networks of living human neurons. The culture model presented is particularly attractive within this framework since it involves iPSC-derived neural cells, which more specifically differentiate into cortical neurons of diverse types (in particular glutamatergic and GABAergic) and astrocytes. However, these in vitro neuronal networks, which may be heterogeneous in their degree of differentiation, remain challenging to bring to an appropriate level of maturation. It is therefore necessary to develop tools capable of analyzing a large number of cells to assess this maturation process. Calcium (Ca2+) imaging, which has been extensively developed, undoubtedly offers an incredibly good approach, particularly in its versions using genetically encoded calcium indicators. However, in the context of these iPSC-derived neural cell cultures, there is a lack of studies that propose Ca2+ imaging methods that can finely characterize the evolution of neuronal maturation during the neurodifferentiation process. Methods: In this study, we propose a robust and reliable method for specifically measuring neuronal activity at two different time points of the neurodifferentiation process in such human neural cultures. To this end, we have developed a specific Ca2+ signal analysis procedure and tested a series of different AAV serotypes to obtain expression levels of GCaMP6f under the control of the neuron-specific human synapsin1 (hSyn) promoter. Results: The retro serotype has been found to be the most efficient in driving the expression of the GCaMP6f and is compatible with multi-time point neuronal Ca2+ imaging in our human iPSC-derived neural cultures. An AAV2/retro carrying GCaMP6f under the hSyn promoter (AAV2/retro-hSyn-GCaMP6f) is an efficient vector that we have identified. To establish the method, calcium measurements were carried out at two time points in the neurodifferentiation process with both hSyn and CAG promoters, the latter being known to provide high transient gene expression across various cell types. Discussion: Our results stress that this methodology involving AAV2/retro-hSyn-GCaMP6f is suitable for specifically measuring neuronal calcium activities over multiple time points and is compatible with the neurodifferentiation process in our mixed human neural cultures.

Erratum: Polychromatic digital holographic microscopy: a quasicoherent-noise-free imaging technique to explore the connectivity of living neuronal networks (Publisher's Note).

[This corrects the article DOI: 10.1117/1.NPh.7.4.040501.].

Polychromatic digital holographic microscopy: a quasicoherent-noise-free imaging technique to explore the connectivity of living neuronal networks.

Significance: Over the past decade, laser-based digital holographic microscopy (DHM), an important approach in the field of quantitative-phase imaging techniques, has become a significant label-free modality for live-cell imaging and used particularly in cellular neuroscience. However, coherent noise remains a major drawback for DHM, significantly limiting the possibility to visualize neuronal processes and precluding important studies on neuronal connectivity. Aim: The goal is to develop a DHM technique able to sharply visualize thin neuronal processes. Approach: By combining a wavelength-tunable light source with the advantages of hologram numerical reconstruction of DHM, an approach called polychromatic DHM (P-DHM), providing OPD images with drastically decreased coherent noise, was developed. Results: When applied to cultured neuronal networks with an air microscope objective ( 20 × , 0.8 NA), P-DHM shows a coherent noise level typically corresponding to 1 nm at the single-pixel scale, in agreement with the 1 / N -law, allowing to readily visualize the 1 - µ m -wide thin neuronal processes with a signal-to-noise ratio of ∼ 5 . Conclusions: Therefore, P-DHM represents a very promising label-free technique to study neuronal connectivity and its development, including neurite outgrowth, elongation, and branching.

Sensory Afferents Use Different Coding Strategies for Heat and Cold.

Primary afferents transduce environmental stimuli into electrical activity that is transmitted centrally to be decoded into corresponding sensations. However, it remains unknown how afferent populations encode different somatosensory inputs. To address this, we performed two-photon Ca2+ imaging from thousands of dorsal root ganglion (DRG) neurons in anesthetized mice while applying mechanical and thermal stimuli to hind paws. We found that approximately half of all neurons are polymodal and that heat and cold are encoded very differently. As temperature increases, more heating-sensitive neurons are activated, and most individual neurons respond more strongly, consistent with graded coding at population and single-neuron levels, respectively. In contrast, most cooling-sensitive neurons respond in an ungraded fashion, inconsistent with graded coding and suggesting combinatorial coding, based on which neurons are co-activated. Although individual neurons may respond to multiple stimuli, our results show that different stimuli activate distinct combinations of diversely tuned neurons, enabling rich population-level coding.

Probing pain pathways with light.

We have witnessed an accelerated growth of photonics technologies in recent years to enable not only monitoring the activity of specific neurons, while animals are performing certain types of behavior, but also testing whether specific cells, circuits, and regions are sufficient or necessary for initiating, maintaining, or altering this or that behavior. Compared to other sensory systems, however, such as the visual or olfactory system, photonics applications in pain research are only beginning to emerge. One reason pain studies have lagged behind is that many of the techniques originally developed cannot be directly implemented to study key relay sites within pain pathways, such as the skin, dorsal root ganglia, spinal cord, and brainstem. This is due, in part, to difficulties in accessing these structures with light. Here we review a number of recent advances in design and delivery of light-sensitive molecular probes (sensors and actuators) into pain relay circuits to help decipher their structural and functional organization. We then discuss several challenges that have hampered hardware access to specific structures including light scattering, tissue movement and geometries. We review a number of strategies to circumvent these challenges, by delivering light into, and collecting it from the different key sites to unravel how nociceptive signals are encoded at each level of the neuraxis. We conclude with an outlook on novel imaging modalities for label-free chemical detection and opportunities for multimodal interrogation in vivo. While many challenges remain, these advances offer unprecedented opportunities to bridge cellular approaches with context-relevant behavioral testing, an essential step toward improving translation of basic research findings into clinical applications.

Intravital assessment of myelin molecular order with polarimetric multiphoton microscopy.

Myelin plays an essential role in the nervous system and its disruption in diseases such as multiple sclerosis may lead to neuronal death, thus causing irreversible functional impairments. Understanding myelin biology is therefore of fundamental and clinical importance, but no tools currently exist to describe the fine spatial organization of myelin sheaths in vivo. Here we demonstrate intravital quantification of the myelin molecular structure using a microscopy method based on polarization-resolved coherent Raman scattering. Developmental myelination was imaged noninvasively in live zebrafish. Longitudinal imaging of individual axons revealed changes in myelin organization beyond the diffraction limit. Applied to promyelination drug screening, the method uniquely enabled the identification of focal myelin regions with differential architectures. These observations indicate that the study of myelin biology and the identification of therapeutic compounds will largely benefit from a method to quantify the myelin molecular organization in vivo.

Maintaining polarization in polarimetric multiphoton microscopy.

Polarimetric measurements in multiphoton microscopy can reveal information about the local molecular order of a sample. However, the presence of a dichroic through which the excitation beam propagates will generally scramble its polarization. We propose a simple scheme whereby a second properly-oriented compensation dichroic is used to negate any alteration regardless of the wavelength and the initial polarization. We demonstrate how this robust and rapid approach simplifies polarimetric measurements in second-harmonic generation, two-photon excited fluorescence and coherent anti-Stokes Raman scattering. Illustration of the polarization maintaining strategy with the compensating dichroic oriented such that its s- and p-axes are interchanged with these of the primary dichroic.

Automated method for the segmentation and morphometry of nerve fibers in large-scale CARS images of spinal cord tissue.

A fully automated method for large-scale segmentation of nerve fibers from coherent anti-Stokes Raman scattering (CARS) microscopy images is presented. The method is specifically designed for CARS images of transverse cross sections of nervous tissue but is also suitable for use with standard light microscopy images. After a detailed description of the two-part segmentation algorithm, its accuracy is quantified by comparing the resulting binary images to manually segmented images. We then demonstrate the ability of our method to retrieve morphological data from CARS images of nerve tissue. Finally, we present the segmentation of a large mosaic of CARS images covering more than half the area of a mouse spinal cord cross section and show evidence of clusters of neurons with similar g-ratios throughout the spinal cord.

Local assessment of myelin health in a multiple sclerosis mouse model using a 2D Fourier transform approach.

We present an automated two-dimensional Fourier transform (2D-FT) approach to analyze the local organization of myelinated axons in the spinal cord. Coherent anti-Stokes Raman scattering (CARS) microscopy was used to observe lesions in a commonly used animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). A 2D-FT was applied on the CARS images to find the average orientation and directional anisotropy of the fibers within contiguous image domains. We introduce the corrected correlation parameter (CCP), a measure of the correlation between orientations of adjacent domains. We show that in the EAE animal model of MS, the CCP can be used to quantify the degree of organization/disorganization in the myelin structure. This analysis was applied to a large image dataset from animals at different clinical scores and we show that some descriptors of the CCP probability density function are strongly correlated with the clinical scores. This procedure, compatible with live animal imaging, has been developed to perform local in situ evaluation of myelinated axons afflicted by EAE.

Live animal myelin histomorphometry of the spinal cord with video-rate multimodal nonlinear microendoscopy.

In vivo imaging of cellular dynamics can be dramatically enabling to understand the pathophysiology of nervous system diseases. To fully exploit the power of this approach, the main challenges have been to minimize invasiveness and maximize the number of concurrent optical signals that can be combined to probe the interplay between multiple cellular processes. Label-free coherent anti-Stokes Raman scattering (CARS) microscopy, for example, can be used to follow demyelination in neurodegenerative diseases or after trauma, but myelin imaging alone is not sufficient to understand the complex sequence of events that leads to the appearance of lesions in the white matter. A commercially available microendoscope is used here to achieve minimally invasive, video-rate multimodal nonlinear imaging of cellular processes in live mouse spinal cord. The system allows for simultaneous CARS imaging of myelin sheaths and two-photon excitation fluorescence microendoscopy of microglial cells and axons. Morphometric data extraction at high spatial resolution is also described, with a technique for reducing motion-related imaging artifacts. Despite its small diameter, the microendoscope enables high speed multimodal imaging over wide areas of tissue, yet at resolution sufficient to quantify subtle differences in myelin thickness and microglial motility.

In vivo optical monitoring of tissue pathologies and diseases with vibrational contrast.

Studies of tissue remodeling require in vivo imaging techniques that are as minimally invasive as possible to avoid microenvironment perturbations. To this end, spontaneous Raman techniques have been used but low signals have limited their application mostly to point spectroscopy measurements. Novel Raman-based techniques such as coherent and stimulated Raman scattering can overcome this limitation. This manuscript discusses imaging and spectroscopy applications with Raman-based contrast for in vivo tissue monitoring, and how these can be combined into spectral imaging.

Long-term stable device for tuning fiber Bragg gratings.

It is demonstrated that with a proper choice of embedding material, the composite beam bending method constitutes an effective and reliable approach for tuning fiber Bragg gratings. A long-term stable device is presented with a dynamic range of 80 nm, which exhibits insertion losses smaller than 0.28 dB and small variations of the full width at half-maximum.