This work was supported by Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACYT) through the following grants: infraestructura 226450 to VP-CIO, infraestructura 255277 to V.P., and FORDECYT-PRONACES/194171/2020 to V.H. We acknowledge the support of Juvenal Hern&#225;ndez Guevara at CIO in the video-making.

All procedures described were done in compliance with the laws and codes approved in the seventh title of the Regulation of the General Health Law Regarding Health Research of the Mexican Government (NOM-062-ZOO-1999) and in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Experimental Animals and were approved by the institutional committee of bioethics in research of the Universidad de Guanajuato and Benem&#233;rita Universidad Aut&#243;noma de Puebla.
1. Microscope settings
<ol>
	<li>Switch on the microscopy system.</li>
	<li>Turn on the pulsed laser to guarantee that it will be ready to lase at an optimal and steady power level after the sample extraction and preparation.</li>
	<li>Tune the laser to 810 nm. To study tissular microtubules, 10%-20% of the available laser power is used, which, in the described system, corresponds to 13-26 mW measured at the back focal plane of the objective.</li>
	<li>Make sure the microscope is K&#246;hler-aligned (Supplementary File 1), with the objective that will be used for the SHG imaging. 
	NOTE: This is important since, in the commercial setup used in this work, the detection is performed in transmission mode and through the condenser.</li>
	<li>Remove unwanted filters from the optical detection path (Figure 1A).</li>
	<li>Make sure the condenser diaphragm is fully opened to ensure that no light is unnecessarily stopped at this level.</li>
	<li>Prepare a 25x/0.8 numerical aperture (NA) oil-immersion objective with a small drop of immersion oil in the lens. 
	&#8203;NOTE: This objective was used to best match the condenser NA, which was 0.55 (ideally, NAcond&#8805; NAobj).</li>
</ol>
2. Microscope preliminary controls
NOTE: Perform the preliminary microscope controls once, unless the setup is modified.
<ol>
	<li>Image dry corn starch sandwiched between glass slides with SHG parameters to generate SHG images that reveal the laser polarization direction. Follow the steps reported in Supplementary File 2, except for the laser power, which is less than 5% for corn starch.</li>
	<li>Take one image of sparse corn starch grains, and mark the orientation of the SHG lobules in the x-y plane. This orientation corresponds to the laser orientation (Figure 2A).</li>
	<li>As a control, take another image of the same sample, inserting into the optical path a half-wave plate (position shown in Figure 1A) to alter the oscillation direction of the laser. The resulting image displays rotated SHG signal lobules (Figure 2B, C).</li>
	<li>If using a different type of bandpass filter for the SHG, make sure it has optimal transmission properties by comparing the images (Figure 2D, E) and the signal intensities pixel by pixel along a line-scan (Figure 2F).</li>
</ol>
3. Tissue extraction
NOTE: Always use clean tools to perform the surgical procedures.
<ol>
	<li>Use 6-12 month old taiep rats and age-matched Sprague-Dawley&#160;controls (WT).</li>
	<li>Anesthetize the animals with an i.p. injection of a mixture of ketamine-xylazine (0.125 mg/Kg and 5 mg/Kg) diluted in a 0.9% sterile NaCl solution.
	<ol>
		<li>Check the pain reflexes, and proceed only if they are absent.</li>
	</ol>
	</li>
	<li>Sacrifice the animal via decapitation.</li>
	<li>Once the head is isolated, open the bones of the cranial vault carefully along the midline, starting from the most caudal point at the top toward the nose, from the orbital cavities toward the midline, and then from the most caudal point to below the brain and cerebellum. 
	NOTE: Bone cutters and Rongeurs allow for proper removal of the head bones without damaging the brain structures.</li>
	<li>Release the brain and cerebellum from the bone. The two hemispheres could be separated. If the hemispheres are divided, use one half for SHG imaging, and fix the other half in 3.7% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min inside a chemical hood for subsequent sectioning and staining.</li>
</ol>
4. Vibratome sectioning
<ol>
	<li>Prepare a vibratome buffer tray filled with warm (37 &#176;C) Hank&#39;s Balanced Salt Solution (HBSS).</li>
	<li>Fix the brain to the specimen plate using cyanoacrylate glue via a piece of masking tape. The most caudal portion/region contacts the glue so that the usable coronal sections from the opposite, rostral portion can be cut.
	<ol>
		<li>Allow ~10 s for the glue to polymerize to obtain effective attachment of the sample. 
		NOTE: The best results are obtained when the brain is oriented so that the blade cuts &#34;from top to bottom&#34; rather than &#34;from side to side&#34; (Figure 1B).</li>
	</ol>
	</li>
	<li>Transfer the specimen plate onto its magnetic support inside the buffer tray.</li>
	<li>Start cutting 300-500 &#181;m sections until the slices produced encompass the entire surface of the brain.</li>
	<li>At this point, reduce the thickness of the section to 160 &#181;m.</li>
	<li>Once a complete 160 &#181;m section is cut at the level of the corpus callosum, recover it with a modified glass Pasteur pipette with a large, flamed orifice (Figure 1C).</li>
	<li>Transfer it to a clean Petri dish with new warm HBSS or directly onto the glass support for microscopy (coverslip or glass-bottom dish). 
	&#8203;NOTE: For the described experimental conditions, adding a coverslip above the sample is detrimental to the imaging.</li>
</ol>
5. Transfer to the microscope
<ol>
	<li>If the microscope is in a different room, prepare an insulated box with warmed gel packs to transfer the tissue sections while maintaining the temperature. The box also serves to keep the sections warm until they are imaged.</li>
	<li>Put the sample under the microscope, and make sure it is positioned properly under the objective by direct observation through the oculars with transmitted light. 
	NOTE: The goal is to align the predicted emitting structures with the oscillation direction of the laser to maximize the emitted (and, therefore, detected) SHG signal.</li>
	<li>Remove the excess HBSS so that a thin liquid film covers the entire sample. Visually check the liquid film every few minutes to avoid excessive evaporation and drying of the sample. 
	NOTE: In the conditions described, a section is used for no more than 20 min. Drying of the sample causes drastic artifactual effects (Supplementary Figure 1A). Rather than adding more HBSS, periodic soaking of the section in warm, fresh medium is recommended if longer imaging times are needed. The use of perfusion chambers and glue or low-melting agarose to immobilize the tissue is also recommended when longer experiments are to be done.</li>
	<li>Prepare the microscope stage for non-descanned imaging, which might include closing all the doors of the dark incubation chamber or covering the incubation chamber with a black nylon polyurethane-coated fabric.</li>
</ol>
6. Imaging
<ol>
	<li>Select the &#34;non-descanned&#34; imaging mode along the transmission path. This way, the capture of the weak SH signal of tubulin will be optimized.</li>
	<li>Select the LCI Plan-Neofluar 25x/0.8 NA objective.</li>
	<li>Set a laser power between 13 mW and 26 mW, with a pixel dwell time of 12.6 &#181;s. Take images no bigger than 512 pixels x 512 pixels, with speed 5 and averaging 2, for an average acquisition time of about 15 s. 
	NOTE: Using higher laser powers and/or longer dwell times may damage the sample (Supplementary Figure 1B).</li>
	<li>Take images first using a 485 nm short-pass filter (SP485), and, in a second step, add a sharp 405 nm bandpass filter (BP405; Figure 3). Follow the steps reported in Supplementary File 2. 
	NOTE: Pseudo-brightfield images can be taken through the same detector using the residual laser light of a visible line (405 nm or 488 nm lasers work best).</li>
</ol>
7. Processing of the cerebellum
<ol>
	<li>First, cut the cerebellum with a scalpel into two hemispheres, and then glue them to the vibratome support by the middle portion (the flat part obtained after cutting).</li>
	<li>Section and image the cerebellum in the same way as was described for the brain (160 &#181;m sections, same vibratome settings, same blade, same microscope settings, etc.). 
	NOTE: Due to the anatomy of the cerebellum, its orientation at the moment of sectioning is not critical; in most of the slices generated away from the surface, there will be folia sectioned well into the white matter.</li>
</ol>

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The authors have no competing financial interests.

SHG microscopy is part of a group of non-linear optics techniques, which include two-photon excitation microscopy, third harmonic generation microscopy, and coherent anti-Stokes Raman scattering microscopy, that have contributed to expanding the range of applications of conventional optical microscopy to the life sciences20.
Specifically, the major strength and weakness of SHG microscopy relate to the same condition: the signal generator is non-centrosymmetric<sup class...

The optical imaging of cytoskeletal structures in tissues and organ preparations is not an easy task. Cytoskeletal filaments are ubiquitous, so if generic staining is performed, for example, against alpha-tubulin or beta-actin or potentially keratin in an epithelial sample, the signal will likely be distributed rather homogeneously all over the sample. To restrict the staining to a more meaningful subset of cellular components, one can either use transgenic mice with targeted expression1 or plan to use isoform-specific antibodies. While very few of the latter are on the market (and very few exist at all2,3,4), a transgenic animal model might be available. However, it needs to be acquired by the lab and properly housed, with all the expenses involved in the process. Certain antibodies or chemicals, for example, fluorophore-conjugated drugs like phalloidin or paclitaxel, may be partially or fully incompatible with use in living cells or tissues, thus limiting their applicability to only studies of fixed samples.
In the case of tubulin, an additional aspect has to be taken into consideration, which is the sensitivity of the polymer to fixation. Conventional chemical fixation with formaldehyde is known for not being adequate for optimally preserving the integrity of microtubules5. Additionally, a recent report confirms that formaldehyde crosslinking induces subtle changes in the ultrastructure of the microtubule, similar to what happens with the binding of some drugs or physiological molecules such as GTP6.
The direct visualization of microtubules in unstained, unfixed samples is, therefore, often desirable. To achieve this, one technical solution is second harmonic generation (SHG) microscopy7, which is based on the ability of bundles of parallel microtubules to act as harmonophores and to emit frequency-doubled light when properly illuminated with an intense, pulsed infrared laser. Although a stronger and more stable second harmonic signal can be generated from collagen and myosin, which are the only other two biological materials known to be capable of frequency-doubling, the signal from tubulin has been used so far mostly to study mitotic spindle rearrangements8,9,10 and axonal microtubule morphology11,12,13.
In this work, we introduce a novel use of SHG microscopy as a diagnostic tool to distinguish central nervous system (CNS) tissues affected by tubulin beta 4 A (TUBB4A) tubulinopathy from their healthy counterparts14. Some of the mutations occurring in this predominantly neural isoform of tubulin, like those causing hypomyelination and atrophy of the basal ganglia and cerebellum (H-ABC), induce microtubule overfilling in the oligodendrocytes15,16; the cytoskeletal alterations, in turn, are associated with downstream effects like dysmyelination, with profound impairment of the motor and sensory pathways16,17,18,19. The taiep murine model used in this work displays abnormal microtubule content in the oligodendrocytes and recapitulates most of the sensory-motor symptoms of H-ABC patients17. The protocol explains how to image structures as the corpus callosum and the cerebellum, which are usually highly myelinated and which are severely affected in human patients as well as in the taiep rat19, to highlight the differences in SH signals between healthy and mutant tissues.

<table><tbody><tr><td>405/10 nm BrightLine(R) single-band bandpass filter&amp;nbsp;</td><td>Semrock</td><td>FF01-405/10-25</td><td>32 mm diameter, with housing ring</td></tr><tr><td>Black Nylon, Polyurethane-Coated Fabric</td><td>Thorlabs</td><td>BK5</td><td>5&#039; x 9&#039; (1.5 m x 2.7 m) x 0.005&quot; (0.12 mm) Thick&amp;nbsp;</td></tr><tr><td>Blades for vibratome</td><td>any commercial; e.g. Wilkinson Sword</td><td></td><td>&amp;nbsp;Classic stainless steel double edge razor blades</td></tr><tr><td>Cell culture dishes, 35 mm</td><td>any commercial; e.g. Falcon</td><td>351008</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM710NLO AxioObserver Z1</td><td>Inverted microscope, objective used is LCI Plan-Neofluar 25x/0.8 NA&amp;nbsp;</td></tr><tr><td>Cooler</td><td>any commercial</td><td></td><td>Any insulated, polystyrene box could work, to mantain the sample at about 37 &amp;deg;C</td></tr><tr><td>Corn stach</td><td>e.g. Maizena</td><td></td><td>From the supermarket</td></tr><tr><td>Coverslips #1.5</td><td>any commercial</td><td></td><td>Rectangular</td></tr><tr><td>Cyanoacrylate glue</td><td>e.g. Loctite</td><td></td><td>To glue the brain to the masking tape</td></tr><tr><td>Fine forceps</td><td>fine science tools</td><td>11412-11</td><td>To manipulate tissue sections by handling from the meninges</td></tr><tr><td>Fine scissors</td><td>fine science tools</td><td>14370-22</td><td>To cut the skin&amp;nbsp;</td></tr><tr><td>Fine scissors curved tip</td><td>fine science tools</td><td>14061-09</td><td>To cut along the midline</td></tr><tr><td>Formaldehyde 37%</td><td>Sigma-Aldrich</td><td>252549</td><td>To dilute 1:10 in PBS</td></tr><tr><td>Friedman Rongeur</td><td>fine science tools</td><td>16000-14</td><td>To cut the bone</td></tr><tr><td>Gel packs</td><td>any commercial</td><td></td><td>Prewarmed to 37 &amp;deg;C, to help mantaining the temperature inside the cooler</td></tr><tr><td>Glass Pasteur pipette, modified</td><td>any commercial</td><td></td><td>To transfer the tissue section</td></tr><tr><td>Hanks&amp;prime; Balanced Salt solution (HBSS)</td><td>Gibco</td><td>14025-076</td><td>Could be prepared from powders</td></tr><tr><td>Kelly hemostats</td><td>fine science tools</td><td>13018-14</td><td>To separate the bone&amp;nbsp;</td></tr><tr><td>Masking tape</td><td>any commercial</td><td></td><td>To protect th surface of the specimen plate</td></tr><tr><td>NDD module, type C</td><td>Zeiss</td><td>000000-1410-101</td><td>To detect the signal, reducing light loss. Housing the 000000-1935-163 filter set with the SP485</td></tr><tr><td>Offset bone nippers</td><td>fine science tools</td><td>16101-10</td><td>To cut the bone</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Gibco</td><td>10010-031</td><td>Could be prepared from powders or tabs</td></tr><tr><td>Pulsed laser</td><td>Coherent</td><td>Chameleon Vision II</td><td>680&amp;ndash;1080 nm tunable laser</td></tr><tr><td>Scalpel</td><td>any commercial</td><td></td><td>Straight blade with sharp point</td></tr><tr><td>Standard pattern forceps</td><td>fine science tools</td><td>11000-18</td><td></td></tr><tr><td>Vannas spring scissors</td><td>fine science tools</td><td>15018-10</td><td>To cut meninges that remain joined to both the slice obtained from vibratome cutting and the section glued to the specimen plate.</td></tr><tr><td>Vibratome</td><td>any commercial; e.g. Leica</td><td>VT1200</td><td></td></tr></tbody></table>

label-free non-linear optics for the study of tubulin-dependent defects in central myelin

The satisfactory visualization of cytoskeletal components in the brain is challenging. The ubiquitous distribution of the networks of microtubules, microfilaments, and intermediate filaments in all the neural tissues, together with the variability in the outcomes of fluorescent protein fusion strategies and their limited applicability to dynamic studies of antibodies and drugs as chromophore vehicles, make classical optical approaches not as effective as for other proteins. When tubulin needs to be studied, the label-free generation of second harmonics is a very suitable option due to the non-centrosymmetric organization of the molecule. This technique, when conjugated to microscopy, can qualitatively describe the volumetric distribution of parallel bundles of microtubules in biological samples, with the additional advantage of working with fresh tissues that are unfixed and unpermeabilized. This work describes how to image tubulin with a commercial second harmonic generation microscopy setup to highlight microtubules in the tubulin-enriched structures of the oligodendrocytes, as&#160;in hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) tubulinopathy, a recently described myelin disorder.

The images obtained with this methodology have an intrinsic low background level due to the very limited number of harmonophores present in biological tissues, which is one of the significant advantages of the method.
When the fibers of the corpus callosum are imaged, fiber-like short structures and rounded elements can be consistently found in the taiep brain (Figure 3B), while the corpus callosum of the control brain shows a much more heterogeneous and ...

Watch this Scientific Journal Video about Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin at JoVE.com

Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin

In this article, we present a protocol to detect microtubule-loaded oligodendrocytes in a model of tubulinopathy through a simple, innovative second harmonic generation microscopy approach.

The satisfactory visualization of cytoskeletal components in the brain is challenging. The ubiquitous distribution of the networks of microtubules, ...

label-free-non-linear-optics-for-study-tubulin-dependent-defects

Research

JoVE Journal

Neuroscience

1.8K Views.  Centro de Investigaciones en Óptica A.C. (CIO). Second-Harmonic Generation imaging van biologische monsters is een optische techniek die de visualisatie van niet-centrosymmetrische moleculen en hun assemblages mogelijk maakt zonder de noodzaak van een tag of een kleurstof. De beelden die met deze methode worden gegenereerd, hebben een lage achtergrond, omdat zeer weinig biologische moleculen als een harmonale kracht kunnen fungeren. Dit protocol beschrijft de toepassing van de techniek voor diagnostische beeldvorming van Tubulin beta-4A in...

Video: Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

Developmental Biology

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Self-Assembly of Microtubule Tactoids

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is especially the case during mitosis or meiosis, where the microtubules form the spindle during cell division. The spindle is the machinery used to segregate genetic material during cell division. Toward creating self-organized spindles in vitro, we recently developed a technique to reconstitute microtubules into spindle-like assemblies with a minimal set of microtubule-associated proteins and crowding agents. Specifically, MAP65 was used, which is an antiparallel microtubule crosslinker from plants, a homolog of Ase1 from yeast and PRC1 from mammalian organisms. This crosslinker self-organizes microtubules into long, thin, spindle-like microtubule self-organized assemblies. These assemblies are also similar to liquid crystal tactoids, and microtubules could be used as mesoscale mesogens. Here, protocols are presented for creating these microtubule tactoids, as well as for characterizing the shape of the assemblies using fluorescence microscopy and the mobility of the constituents using fluorescence recovery after photobleaching.

This article presents a protocol for the formation of microtubule assemblies in the shape of tactoids using MAP65, a plant-based microtubule crosslinker, and PEG as a crowding agent.

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is ...

Biochemistry

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and&#160;wide-field&#160;illumination&#160;microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.&#160;

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...

Bioengineering

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the dynamic properties of the cytoskeleton. This protocol offers a technique for human primary fibroblast transfection, which can be difficult because of the specifics of primary cell cultivation conditions. Additionally, cytoskeleton dynamic property maintenance requires a low level of transfection to obtain a good signal-to-noise ratio without causing microtubule stabilization. It is important to take measures to protect the cells from light-induced stress and fluorescent dye fading. In the course of our work, we tested different transfection methods and protocols as well as different vectors to select the best combination of conditions suitable for human primary fibroblast studies. We analyzed the resulting time-lapse videos and calculated microtubule dynamics using ImageJ. The dynamics of microtubules' plus-ends in the different cell parts are not similar, so we divided the analysis into subgroups - the centrosome region, the lamella, and the tail of fibroblasts. Notably, this protocol can be used for in vitro analysis of cytoskeleton dynamics in patient samples, enabling the next step towards understanding the dynamics of the various disease development.

This protocol is dedicated to the microtubule plus-end visualization by EB3 protein transfection to study their dynamic properties in primary cell culture. The protocol was implemented on human primary skin fibroblasts obtained from Huntington's disease patients.

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Biology

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin

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