Name: spectral reflectometric microscopy on myelinated axons in situ
Uploaded: 2018-07-02T15:00:00
Description: Watch this Scientific Journal Video about Spectral Reflectometric Microscopy on Myelinated Axons In Situ at JoVE.com

This work was supported by the Institute of Basic Science (IBS-R015-D1) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1A6A1A03015642).

All surgical procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University.
1. Sample Preparation
Note: Autoclave all surgical instruments before animal handling. Conduct all surgical performance in a room dedicated to surgical procedures. Sterile surgical gowns and gloves should be worn by all personnel in the surgical room at all times.
<ol>
	<li>Tissue Fixation
	<ol>
		<li>Prepare two 10 mL syrin...

<ol>
	<li>Fernandez-Moran, H., Finean, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscope+and+low-angle+x-ray+diffraction+studies+of+the+nerve+myelin+sheath.">Electron microscope and low-angle x-ray diffraction studies of the nerve myelin sheath.</a> Journal of Cell Biology. 3, (1957).</li><li>Blaurock, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spaces+between+membrane+bilayers+within+PNS+myelin+as+characterized+by+X-ray+diffraction.">The spaces between membrane bilayers within PNS myelin as characterized by X-ray diffraction.</a> Brain Research. 210, 383-387 (1981).</li><li>Shim, S. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+fluorescence+imaging+of+organelles+in+live+cells+with+photoswitchable+membrane+probes.">Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes.</a> Proceedings of the National Academy of Sciences. , 13978-13983 (2012).</li><li>Urban, N. T., Willig, K. I., Hell, S. W., N&#228;gerl, U. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+nanoscopy+of+actin+dynamics+in+synapses+deep+inside+living+brain+slices.">STED nanoscopy of actin dynamics in synapses deep inside living brain slices.</a> Biophysics Journal. 101, 1277-1284 (2011).</li><li>Manley, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density+mapping+of+single-molecule+trajectories+with+photoactivated+localization+microscopy.">High-density mapping of single-molecule trajectories with photoactivated localization microscopy.</a> Nature Methods. 5, 155-157 (2008).</li><li>Peters, A., Sethares, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+there+remyelination+during+aging+of+the+primate+central+nervous+system?.">Is there remyelination during aging of the primate central nervous system?.</a> Journal of Comparative Neurology. 460, 238-254 (2003).</li><li>De Campos Vidal, B., Silveira Mello, M. L., Caseiro-Filho, A. C., Godo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+properties+of+the+myelin+sheath.">Anisotropic properties of the myelin sheath.</a> Acta Histochemica. 66, 32-39 (1980).</li><li>Schain, A. J., Hill, R. A., Grutzendler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+in+vivo+imaging+of+myelinated+axons+in+health+and+disease+with+spectral+confocal+reflectance+microscopy.">Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy.</a> Nature Medicine. 20, 443-449 (2014).</li><li>Kwon, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+nanoscale+optical+metrology+on+myelinated+axons+in+vivo.">Label-free nanoscale optical metrology on myelinated axons in vivo.</a> Nature Communication. 8, 1832 (2017).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Animal+Perfusion+Fixation+for+Rodents.">Whole Animal Perfusion Fixation for Rodents.</a> JoVE. (65), e3564 (2012).</li><li>Segev, A., Garcia-Oscos, F., Kourrich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+Patch-clamp+Recordings+in+Brain+Slices.">Whole-cell Patch-clamp Recordings in Brain Slices.</a> JoVE. (112), e54024 (2016).</li><li>Waldchen, S., Lehmann, J., Klein, T., Van De Linde, S., Sauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+cell+damage+in+live-cell+super-resolution+microscopy.">Light-induced cell damage in live-cell super-resolution microscopy.</a> Scientific Reports. 5, 15348 (2015).</li><li>Chang, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iterative+expansion+microscopy.">Iterative expansion microscopy.</a> Nature Methods. 14, 593-599 (2017).</li><li>Polishchuk, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+light-electron+microscopy+reveals+the+tubular-saccular+ultrastructure+of+carriers+operating+between+Golgi+apparatus+and+plasma+membrane.">Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane.</a> Journal of Cell Biology. 148, 45-58 (2000).</li></ol>

SpeRe is a new label-free imaging modality based on spectral interferometry, which for the first time, offers the nanoscale information in live myelinated axons. In the current acquisition protocol, the spatial resolution for the axon diameter is of the order of 10 nm. Moreover, SpeRe utilizes orders-of-magnitude lower light dose compared to other super-resolution microscopies; thus, it is free from phototoxicity and photobleaching. SpeRe would provide a new avenue for studying nanoscale dynamics of the myelinated axons....

In the mammalian nervous system, myelin provides rapid nerve conduction and axonal integrity by enwrapping the axon fibers with multilayered membranous sheaths. Its multilayered structure is composed of alternating nanoscale thin-films composed of plasma membranes (~5 nm), cytosol (~3 nm), and extracellular spaces (~7 nm)1,2. Optical microscopy, including the recent super-resolution microscopy, are not suitable for observing the nanoscale myelin dynamics due to their insufficient resolution due to optical diffraction3,4,5. Although electron microscopy can provide fine details of the myelin nanostructure, it is not compatible with the living biological systems due to highly invasive sample preparations involving chemical fixation and ultrasectioning6,7. Until recently, there has been no technique applicable to observe nanoscale dynamics of myelinated axons in situ.
Schain et al. previously reported that myelinated axons exhibit colorful light reflectance8. By adopting the spectroscopic analysis on the reflected light, we have devised a new imaging modality for nanoscale imaging of myelinated axons, named spectral reflectometry (SpeRe)9. SpeRe is based on the thin-film interference occurring in the multi-layered structure of the myelin sheath (Figure 1). By optic simulation on various axons, we have revealed that the reflectance spectrum is a periodic function of wavenumber and its periodicity (<img alt="Equation 1" src="/files/ftp_upload/57965/57965eq1.jpg" />) is inversely proportional to the axon diameter (d). This simple relationship (<img alt="Equation 2" src="/files/ftp_upload/57965/57965eq2.jpg" />) offers facile quantification of axon diameter from the SpeRe data. Utilizing this, we revealed the prevalent axon bulging under mild traumatic brain injury in our prior report.
The SpeRe system is based on confocal microscopy and consists of a specialized laser source and filters (Figure 2). The input source is a white-light laser, providing broadband spectral output visible to infrared regions. For the spectral scan, the system is equipped with two acousto-optic devices: an acousto-optic tunable filter (AOTF) for delivering a selected wavelength from the input broadband source and an acousto-optic beam splitter (AOBS) for guiding the selected reflected wavelength to the detector. The software for hyperspectral confocal microscopy (see Table of Materials) provides a customizable spectral scan option to sequentially acquire the reflectance images at various input wavelengths. In addition, chromatic aberration can critically interfere in the spectral measurement; therefore, use of an apochromat objective lens is recommended.
Of note, white-light lasers produce an uneven spectral output and the optical components also affect the spectral profile. Therefore, the acquired spectra need to be calibrated for the subsequent quantitative analysis. A protected silver mirror is typically used as a reference, which provides a nearly constant reflectance (&#62; 97%) over the full visible region. The acquired spectra are then divided by the reference spectra from the mirror.
The spectral step size for the spectral scan determines the acquisition speed; thus, it needs to be optimized. As a larger axon has a higher spectral period, it requires finer spectral sampling. For example, an axon with a diameter of 10 &#181;m, one of the largest physiologic axons, has a spectral period of ~8 nm. By applying Nyquist sampling criteria, we employed the spectral sampling interval of 4 nm to cover all the physiologic axons in the mouse nervous tissues. This approach typically takes over several seconds for a full spectral scan and thus is not suitable for in vivo applications, where physiologic motion (e.g. respiration and heartbeat) interferes stable spectral acquisition. We previously solved this issue by instrumenting a customized upright microscope, designed to acquire the full spectrum for each point using an array spectrometer (acquisition speed &#8776; 30 ms per pixel).
In this report, we describe a detailed protocol on the SpeRe imaging on a fixed brain slice, which can be performed in a commercial hyperspectral microscope (see Table of Materials). Thus, the protocol can be completed by experimenters without expertise in optical instrumentation. We also cover the potential issues and troubleshooting for acquisition and analysis of SpeRe data.

<table>
	<tbody>
		<tr>
			<td>Glass cutter</td>
			<td>-</td>
			<td>-</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>-</td>
			<td>-</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>Apochromat objective 40&#215;, NA 1.1</td>
			<td>Leica Microsystems</td>
			<td>15506357</td>
			<td>Water-immersion type</td>
		</tr>
		<tr>
			<td>Fluoromyelin Green</td>
			<td>Thermo Fisher</td>
			<td>F34651</td>
			<td>Alternatively, Fluoromyelin Red (F34652) can be used.</td>
		</tr>
		<tr>
			<td>Leica SP8 TCS microscope</td>
			<td>Leica Microsystems</td>
			<td>SP8</td>
			<td>Refer to the &#34;Configuration of microscope&#34; in Introduction Section for details.</td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Leica Microsystems</td>
			<td>LAS-X</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Mirror</td>
			<td>Thorlabs</td>
			<td>PF10-03-P01</td>
			<td>Coated with protected silver.</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Life technologies</td>
			<td>14190-136</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Biosolution</td>
			<td>BP031a</td>
			<td>4% v/v in PBS</td>
		</tr>
		<tr>
			<td>Cover slip</td>
			<td>Thermo Fisher</td>
			<td>3306</td>
			<td>Thickness: #1 (0.13 to 0.17 mm)</td>
		</tr>
		<tr>
			<td>Slide glass</td>
			<td>Muto Pure Chemicals</td>
			<td>5116-20F</td>
			<td>Thickness: ~1 mm</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Henkel</td>
			<td>Loctite 406</td>
			<td>Use a dispensing equipment to avoid skin or eye contact.</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Brainetree Scientific</td>
			<td>BS-8000 DUAL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica Biosystems</td>
			<td>VT1200S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>White-light laser</td>
			<td>NKT photonics</td>
			<td>EXB-6</td>
			<td>EXB-6 was discontinued and replaced by EXU-6.</td>
		</tr>
	</tbody>
</table>

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.

According to the protocol, a fixed brain slice was prepared with exogenous staining, a myelin-targeting fluorophore (see Table of Materials). SpeRe imaging was performed on the brain slice using a commercial hyperspectral confocal microscope in conjunction with confocal fluorescence imaging (Figure 4a). For SpeRe, input optical intensity was set to 5 &#181;W/&#181;m2 with a pixel dwell time of ~1 &#181;s. This light dose is over an...

Watch this Scientific Journal Video about Spectral Reflectometric Microscopy on Myelinated Axons In Situ at JoVE.com

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.

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 ...

Research

JoVE Journal

Neuroscience

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...