Name: high-resolution volume imaging of neurons by the use of fluorescence exclusion method and dedicated microfluidic devices
Uploaded: 2018-03-26T12:30:00.000+00:00
Duration: 9 min 11 s
Description: Watch this Scientific Journal Video about High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices at JoVE.com

The authors want to acknowledge ChiLab, Materials and Microsystems Laboratory - Politecnico di Torino - DISAT, in the person of Prof. C F Pirri, Dr. M Cocuzza and Dr. S L Marasso, for their precious support in the process development and device fabrication. We thank Victor Racine from Quantacell for discussion and support in image processing. We are grateful to Isabelle Grandjean and Manon Chartier from the Animal Facility of the Institut Curie for their support for mice, and Pablo Vargas and Ana-Maria Lennon (Institut Curie) for providing us with the GFP LifeAct mice. We are grateful to Olivier Thouvenin from the Institut Langevin and Clotilde Cadart, Larisa Venkova and Matthieu Piel from the Institut Curie - UMR 144, for their help in the understanding of the Fluorescence eXclusion Method. Finally, we thank the Technological platform of Institut Pierre-Gilles de Gennes (CNRS UMS 3750) for their support in microfabrication. This work was supported in part by the European Research Council Advanced Grant No. 321107 &#34;CellO,&#34; PSL Universit&#233; (SwithNeuroTrails project), ANR Investissement d&#39;Avenir, and the IPGG Labex and Equipex.

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The authors declare no conflict of interest.

Volume imaging of neurons constitutes a challenge for the FXm technique due to the long and thin extensions of these cells. This protocol describes variants of the same type of microfluidic device dedicated to neuron imaging.
Beside the aspects of microfluidic design, the choice of the objective is fundamental for fluorescence exclusion imaging and implies a trade-off between lateral resolution and the image clarity. It has been shown in 13 that a high NA leading to a depth of focus smaller than the chamber height was not detrimental for the precision of volume measurement if imaging was performed at focus and if a sufficient margin is left between the contour of the object of interest and the boundaries of the integration surface. However, the use of a chamber much higher than the depth of focus impairs the image clarity due to photon diffusion, which smoothes the edges of the objects of interest. The fabrication of a 3 &#181;m high chamber reduced this lateral blurring and provided exceptionally well defined fluorescent exclusion images even using high NA (0.8) 40X objectives to visualize neuronal branches with high lateral resolution.
Chip assembling is a critical step, in particular in the case of 3 &#181;m high chambers, but careful manipulation as described in 4.1.2 avoids the collapse of the roof. The high surface to volume ratio associated to these thin chambers raised also the issue of the stability of Dextran concentration over time. We have checked that the surface absorption of the Dextran after one night of incubation was negligible: after replacing Dextran by PBS, the difference of intensity between the pillar and the background was about 1 per 1000 of the initial intensity contrast between these two regions in the presence of Dextran. Note that neurons may adhere both on the bottom coverslip and on the PDMS roof. This effect disappears when using patterned coverslips (i.e. when we do not incubate adhesive molecules within the PDMS chamber), as the coating is therefore strictly localized on the bottom of the chamber.
Apart from their challenging morphology, neurons are rather suited to FXm due to the fact that one of the major limitation of the method, i.e. dextran endocytosis, is very limited in these cells. We choose a 10 kDa formulation to suppress in the long range (hours) any visible endocytosis phenomena.
In conclusion, the conceptual simplicity of FXm is balanced by a set of experimental issues which have been solved by the present protocol, such as nanometric PDMS roughness and micrometric chamber height, or background correction to correct for the unevenness of the PDMS ceiling between pillars. However, the use of a close microfluidic chamber to confine the fluorescent medium yields a few specific constraints like the need of support pillars, which lowers the effective surface available for cell adhesion, or the necessity to exclude soma from the central chamber to observe neuronal extensions with the highest clarity, which restricts the regions of the cell accessible to high resolution observation. One possible evolution of this method would be to get rid of this physical confinement, to be replaced by an optical one. The new development of light sheet microscopy might be combined advantageously with FXm in the future.

The precise knowledge of cellular volume has attracted increased attention in the last years, driven by the issue of cell size homeostasis in single-celled microorganisms 1 and more generally in mitotic cells 2. However, the question of cell volume is pertinent also for post-mitotic cells, for which neurons constitute a paradigmatic example.
Volume is indeed an important signature of physiological and pathological events at different scales and time-points in neuronal life, from transient axonal deformation associated to electrical activity (millisecond scale) 3 to the irreversible neuronal swelling occurring during the asymptomatic phase of neurodegenerative diseases (over years in humans) 4. However, the largest volume change occurs in an intermediate time scale of days or weeks (depending on the considered organism) during neuronal growth. The extended and complex morphology of neurons raises multiples issues, among which the regulation of cell size. Axonal length and diameter are indeed tightly regulated in vivo, with values specific to each neuronal type 5,6.
These issues, complex to address in vivo, can also be addressed in a simplified way in vitro. In that aim, a method dedicated to volume measurement fast enough to follow growth dynamics (i.e. in a time scale of minutes) and compatible with observation over hours or days is required. Several methods have been developed over the years to provide a direct or indirect access to cellular volume in vitro. Cell reconstruction from confocal imaging is one of them, but this method implies labeling and repeated exposures to light while showing a limited axial resolution of about 500 nm 7. Note that these two last drawbacks are partially overcome by a more sophisticated and recent method named lattice light-sheet microscopy 8. Atomic Force Microscopy has been used 9 but this scanning method is by essence slow and tedious. Moreover, the physical contact it requires with the cell might interfere with the measurement considering the extreme softness of neurons 10. Indirect method using impedance or resonance have been employed for different cell types 11, but are inadequate for extended adhesive cells like neurons.
One of the most promising methods is based on the measure of the excluded volume of cells in a close chamber filled with a fluorescent dye. The Fluorescence eXclusion method (FXm) is simple in its principle as it requires no labeling, and is suitable for fast, long term optical imaging of cell populations with a potentially sub-optical axial resolution. More precisely, the resolution in z depends on the maximum fluorescence intensity in the culture chamber (i.e. in region devoid of cells) divided by the dynamic range of the camera, although several sources of noise limit this ultimate resolution. This method has been very powerful to follow the volume of migrating adherent cells 12 or to study volume change during mitosis of mammalian cells, as thoroughly described in 13. However, neurons constitute a methodological challenge for FXm considering their extensive ramification into sub-micrometric processes.
We present here a method leading to the fabrication of smooth FXm chambers to access with high precision the volume and height of neuronal branches and dynamic structures involved in neuronal growth like growth cones.
Chambers should have similar heights than the object to measure in order to optimize axial resolution. Therefore, we designed different FXm devices characterized by central measurement chambers of three different heights. The thinnest (3 &#181;m in height) is dedicated to neurite measurement: this low height excludes soma, which remain in the near 15 &#181;m high intermediate chamber. Thicker central chambers (10 and 12 &#181;m) are sufficiently high to follow the whole cell growth. The device also includes two reservoirs located on either side of the central chamber. Four injection holes (IH) are thus implemented and are designated as follows: the inlet and outlet serve to introduce the cellular suspensions into the chip, whereas the two others feed the reservoirs.
We have first fabricated calibration coverslips for height measurements using photoresist structures of known geometry. We have then imaged free growing neurons, but also morphologically constrained neurons into micropatterns of adhesion.

<table><tbody><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plasmalab System 100</td><td>Oxford Instruments</td><td></td><td>To perform DRIE</td></tr><tr><td>MJB4 mask aligner</td><td>SUSS MicroTec</td><td></td><td>SU-8 photolithography</td></tr><tr><td>NXQ 4006 Mask aligner</td><td>Neutronix Quintel</td><td></td><td>Photolithography associated to DRIE</td></tr><tr><td>Plasma cleaner</td><td>Diener Electronic</td><td>Pico PCCE</td><td></td></tr><tr><td>Cell culture hood</td><td>ADS Laminaire</td><td>Optimale 12</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Fisher Scientific</td><td>Heraeus Multifuge X1R</td><td></td></tr><tr><td>Incubator 37 &amp;deg;C 5% CO2</td><td>Panasonic</td><td>MCO-170AICUVH-PE IncuSafe CO2 Incubator</td><td></td></tr><tr><td>Epifluorescence microscope</td><td>Leica</td><td>DMi8</td><td></td></tr><tr><td>PDMS Oven between 65 and 80 &amp;deg;C</td><td>Memmert</td><td></td><td></td></tr><tr><td>Mechanical profilometer</td><td>Veeco</td><td>Dektak 6M Stylus Profiler</td><td></td></tr><tr><td>Optical profilometer</td><td>Veeco</td><td>Wyko NT9100</td><td></td></tr><tr><td>Vacuum desiccator</td><td>Verrerie Villeurbanaise (Kartel Labware)</td><td>230KAR</td><td>Diameter 200 mm</td></tr><tr><td>Ultrasonic bath sonicator</td><td>Labo Moderne</td><td>SHE1000</td><td>Volume of the bath: 0.8 liter</td></tr><tr><td>Hotplate</td><td>Stuart SD162</td><td>SD162</td><td></td></tr><tr><td>&lt;strong&gt;Masks&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DRIE</td><td></td><td></td><td>Supplementary data</td></tr><tr><td>Su8 Mask 1</td><td></td><td></td><td>Supplementary data</td></tr><tr><td>Su8 Mask 2</td><td></td><td></td><td>Supplementary data</td></tr><tr><td>Su8 Mask 3</td><td></td><td></td><td>Supplementary data</td></tr><tr><td>S1805 calibration stripes</td><td></td><td></td><td>Supplementary data</td></tr><tr><td>&lt;strong&gt;Small laboratory equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.5 mm hole puncher</td><td>Sigma-Aldrich</td><td>29002519 (US reference)</td><td></td></tr><tr><td>Scalpel or razor blade</td><td></td><td></td><td></td></tr><tr><td>9&quot; Stainless Steel Flat Spatula with Spoon</td><td>VWR International</td><td>82027-532</td><td>To demold PDMS</td></tr><tr><td>Top Lip Wafer Handling</td><td>VWR International</td><td>63042-096</td><td></td></tr><tr><td>Curved tweezer</td><td>FST</td><td>Dumont #7 Forceps - Standard / Dumoxel</td><td>To manipulate glass coverslips</td></tr><tr><td>&lt;strong&gt;Substrates&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Silicon wafer</td><td>Prolog Semicor Ltd</td><td></td><td></td></tr><tr><td>24x24 mm glass coverslips</td><td>VWR</td><td>631-0127</td><td></td></tr><tr><td>&lt;strong&gt;Photoresists and developpers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AZ 1518 positive photoresist</td><td>Microchemicals GmbH</td><td></td><td>Before DRIE process, thicknes 1.8 &amp;micro;m</td></tr><tr><td>AZ 351B developer</td><td>Microchemicals GmbH</td><td></td><td>To develop positive AZ 1518 photoresist</td></tr><tr><td>SU-8 2007</td><td>MicroChem</td><td></td><td></td></tr><tr><td>SU-8 2025</td><td>MicroChem</td><td></td><td></td></tr><tr><td>SU-8 2050</td><td>MicroChem</td><td></td><td></td></tr><tr><td>PGMA developer</td><td>Technic</td><td></td><td>To develop SU-8 negative photoresist</td></tr><tr><td>Microposit S1805 resist</td><td>Chimie Tech Services</td><td></td><td>Positive photoresist used to obtain 0.5&amp;micro;m high structures</td></tr><tr><td>MF 26A developer</td><td>Chimie Tech Services</td><td></td><td>To develop positive S1805 photoresist</td></tr><tr><td>&lt;strong&gt;Laboratory consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Disposable plastic pipette 3 mL</td><td>LifeTechnologies - ThermoFisher</td><td></td><td></td></tr><tr><td>P100 Petri dishes</td><td>TPP</td><td>93100</td><td></td></tr><tr><td>20 mL syringe</td><td>Terumo</td><td>SS+20ES1</td><td></td></tr><tr><td>Transparent scotch tape</td><td></td><td></td><td></td></tr><tr><td>Square wipes</td><td>VWR</td><td>115-2148</td><td></td></tr><tr><td>Parafilm</td><td>DUTSCHER</td><td>90260</td><td>Plastic paraffin film</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>(3-Methacryloxypropyl)trichlorosilane</td><td>abcr</td><td>AB 109004</td><td></td></tr><tr><td>PDMS and curing agent Sylgard 184</td><td>Sigma-Aldrich</td><td>761036</td><td></td></tr><tr><td>isopropanol</td><td></td><td>W292907</td><td></td></tr><tr><td>poly-ornithine</td><td>Sigma-Aldrich</td><td>P4957 - 50 mL</td><td></td></tr><tr><td>Ethanol absolute</td><td>Sigma-aldrich</td><td>02865</td><td>99.8%</td></tr><tr><td>3-methacryloxypropyl-trimethoxysilane</td><td>Sigma-aldrich</td><td>M6514-25ML</td><td>(C4H5O2)-(CH2)3- Si(OCH3)3</td></tr><tr><td>acetic acid</td><td>Sigma-aldrich</td><td>71251-5ML-F</td><td></td></tr><tr><td>Dextran 10kW conjugated with Alexa488</td><td>LifeTechnologies - ThermoFisher</td><td>D22910</td><td>Absoprtion at 488 nm</td></tr><tr><td>Dextran 10kW conjugated with Alexa647</td><td>LifeTechnologies - ThermoFisher</td><td>D22914</td><td>Absoprtion at 647 nm</td></tr><tr><td>&lt;strong&gt;Culture medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MEM</td><td>LifeTechnologies - ThermoFisher</td><td>21090-022</td><td></td></tr><tr><td>Horse Serum</td><td>LifeTechnologies - ThermoFisher</td><td>26050088</td><td></td></tr><tr><td>B27</td><td>LifeTechnologies - ThermoFisher</td><td>12587-010</td><td></td></tr><tr><td>Glutamax 200 mM</td><td>LifeTechnologies - ThermoFisher</td><td>35050-061</td><td></td></tr><tr><td>Sodium Pyruvate GIBCO 100 mM</td><td>LifeTechnologies - ThermoFisher</td><td>11360-070</td><td></td></tr><tr><td>Gentamicin</td><td>LifeTechnologies - ThermoFisher</td><td>15710-049</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537-500ML</td><td></td></tr><tr><td>HBSS 10x</td><td>LifeTechnologies - ThermoFisher</td><td>14180-046</td><td></td></tr><tr><td>Hepes 1M</td><td>LifeTechnologies - ThermoFisher</td><td>15630-056</td><td></td></tr><tr><td>trypsin-EDTA</td><td>Sigma-Aldrich</td><td>59418C-100ML</td><td></td></tr><tr><td>Neurobasal</td><td>LifeTechnologies - ThermoFisher</td><td>21103-049</td><td></td></tr><tr><td>Neurobasal without phenol red</td><td>LifeTechnologies - ThermoFisher</td><td>12348-017</td><td></td></tr><tr><td>&lt;strong&gt;Softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Routine in Matlab for background normalization</td><td>Quantacell</td><td>Contact Victor Racine: victor.racine@quantacell.com</td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td>To select specific ROI for image analysis</td></tr><tr><td>Routine</td><td>ImageJ</td><td></td><td>Supplementary data</td></tr><tr><td>Routine</td><td>Matlab</td><td></td><td>Supplementary data</td></tr></tbody></table>

high-resolution volume imaging of neurons by the use of fluorescence exclusion method and dedicated microfluidic devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

The result of the process of fabrication described in sections 1 and 2 is illustrated by the images of Figure 1A-1B and the curve of Figure 1C. The table of Figure 1D displays the roughness values of two different representative areas of the PDMS chip, i.e. in the central and the 20 &#181;m high next intermediate chamber. A decrease in roughness by a factor of about 7 has been obtained by using etched Si wafer instead of SU-8 photoresist. Then, FXm was first applied on a photoresist stripe of known geometry (Figure 2A) within a 10 &#181;m high chamber. After image processing and intensity to height conversion (see the graph of Figure 2B), FXm profiles performed on cross-sections along this stripe (Figure 2C) provide the desired height profiles (Figure 2D). Figure 2D shows the comparison between profiles obtained using mechanical profilometry and FXm methods. These profiles, including edge and plateau value, are very similar, validating the method. Note that the scattering of FXm data is not representative of the ultimate resolution of the method, as further assessed in Figure 3 and Figure 4, but results from the low intensity employed to avoid a possible effect of the very weak auto-fluorescence of the photoresist in the GFP channel.
Then, we observed neurites in 3 &#181;m and 10 &#181;m high chambers (Figure 3). The standard deviation of the background noise is about 18 nm after intensity to height conversion and background correction. This value is slightly higher than the physical roughness of PDMS surfaces casted on silicon surfaces (12 nm, see the table of Figure 1D) but much lower than the roughness measured on PDMS obtained from SU-8 molds. These results highlight the added value of drilling wells into silicon wafers rather than opening holes into SU-8 photoresist to cast pillars. Such a low value allows a high signal to noise ratio and very clear images in volume such as the one displayed in Figure 3A. As an example of the data that can be retrieved from such images, we computed the volume of 1.6 &#181;m (i.e. 10 pixels) wide neurite slice (see the graph of Figure 3B). Using in a first approximation a linear fit of these data gives a mean neurite height value of about 400 nm, to be compared with e.g. the 500-nm axonal diameter found in 10 days old pups within the corpus callosum 5. We also combined FXm with micropatterns of adhesion consisting of serially abutted 2 &#181;m and 6 &#181;m wide stripes of 30 &#181;m in length. Our aim was to study the influence of the neurite width on its 3D shape. Figure 3C shows a 3D representation in false color of a whole neuron image obtained in a 10 &#181;m high chamber. Neurites are spreading on 2 &#181;m and 6 &#181;m wide stripes, whereas the soma is located on the extremity of the largest stripe. Height profiles were drawn in three different cross sections. In coherence with the graph displayed in Figure 3A, the surface integrated over the cross-sections increase with the neurite width (Figure 3D).
We also focused on growth cone (GC) 3D structures. Figure 4A-B displays two different GC profiles obtained in a 3 &#181;m high chamber, which highlight their branched sub-structure. In addition, we performed time-lapse experiments to follow the dynamics of the volume of GCs in a 12 &#181;m high chamber. Figure 4C displays a cycle of shrinking and reactivation of a given GC within a time scale of a few tens of minutes. Thanks to the use of GFP-lifeact mice, growth cones were localized in the GFP emission wavelength (510 nm) from their high actin concentration. The surface identified at the wavelength was used to integrate over the dextran emission wavelength at 647 nm to compute GC volume. Figure 4D shows finally the distribution of GC volume at different time points and location on three different neurons, centered on a value of about 6 &#181;m3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56923/56923fig1v2.jpg" /> 
Figure 1: FXm PDMS chambers. (A) Schemes of the four different main steps of microfabrication leading to the final mold. The location of the inlet, outlet and reservoirs are indicated. Scale bars: 1 mm. (B) Image of the PDMS FXm chamber obtained using an optical profilometer. This image shows the central chamber containing 3 rows of 10 &#181;m high pillars and the intermediate chambers of 20, 50 and 90 &#181;m in height. Scale bar: 500 &#181;m. (C) Cross-sectional view of the chip along the two dashed lines drawn in (B). Yellow/gold: cross-section along pillars, blue: cross-section between pillars. (D) Mean values of the PDMS roughness measured on 50 &#215; 50 &#181;m2 areas molded on silicon and on the 20 &#181;m high SU-8 intermediate chamber (see arrows for the location of these areas). Mean values were obtained from the measurements of three different areas. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56923/56923fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56923/56923fig2.jpg" /> 
Figure 2: Calibration of FXm method using a photoresist stripe as the object of interest. (A) GFP-fluorescence image taken in a 10 &#181;m high chamber filled with 10,000 MW dextran absorbing at 488 nm at 1 mg/mL. (B: background, P: pillar). Observation with a dry 40X NA 0.8 objective. Scale bar: 50 &#181;m. (B) Linear calibration law obtained from the mean intensity of the two colored rectangles shown in A. (C) Fluorescence intensity profile obtained at the level of the blue dashed line displayed in (A), crossing the photoresist stripe (0.45 &#181;m high positive photoresist). (D) Comparison of the profiles obtained from mechanical profilometer (black dots) and FXm after intensity to height conversion of the data of (B) (blue dots). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56923/56923fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56923/56923fig3.jpg" /> 
Figure 3: Neurite volume imaging. (A) Neurite extending into the central 3 &#181;m high chamber from soma located in the next 15 &#181;m intermediate chamber. Imaging performed using 10,000 MW dextran absorbing at 488 nm and a 40X, NA 0.8 dry objective. The inset obtained after the use of the background reduction routine highlights the two neurites and chosen to plot the graph on the right. Scale bars: 30 &#181;m. (B) Neurite slice volume as a function of neurite width obtained from the 22 profiles (average on 10 pixels, i.e. on a 1.6 &#181;m &#34;neurite slice&#34;) shown in (A). The solid line represents a linear fit of slope 0.4 &#181;m passing through the origin. (C) False color image of a patterned neuron on an adhesive stripe made of successive 2 &#181;m and 6 &#181;m wide stumps (represented in white). Measurements were made in a 10 &#181;m high chamber filled with 10,000 MW dextran absorbing at 647 nm and using a 40x NA 0.8 dry objective. (D) Height profiles corresponding to the colored dashed lines shown in (C), keeping the same color code. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56923/56923fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56923/56923fig4.jpg" /> 
Figure 4: Static and dynamic growth cone imaging. (A-B) Growth cone height profiles obtained in a 3 &#181;m high chamber after intensity to height conversion along the yellow lines displayed in associated images. Observation performed using a fill with 10,000 MW dextran absorbing at 488 nm and a 40X, NA 0.8 dry objective. (C) Whole neuron imaging in a 12 &#181;m high chamber filled with 10,000 MW dextran absorbing at 647 nm. Observations have been made in two fluorescent channels: GFP for growth cone localization (dashed yellow lines), and CY5 to compute GC volume from fluorescence exclusion. The surface included by dashed yellow lines was used to compute GC volume. The graph shows the variation of GC volume over time, and associated morphologies in both GFP and CY5 channels at two representative different time points. All data were acquired using a 40x NA 0.8 dry objective every 3 min. Scale bars: 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56923/56923fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Mask 1:8 &#181;m layer</td>
			<td>Mask 2:30 &#181;m layer</td>
			<td>Mask 3:40 &#181;m layer</td>
		</tr>
		<tr>
			<td>SU-8 type</td>
			<td>2007</td>
			<td>2025</td>
			<td>2050</td>
		</tr>
		<tr>
			<td>Spincoating</td>
			<td>30 s @ 2000 rpm</td>
			<td>30 s @ 3050 rpm</td>
			<td>30 s @ 3250 rpm</td>
		</tr>
		<tr>
			<td>Soft bake</td>
			<td>3 min @ 95 &#176;C</td>
			<td>2 min @ 65 &#176;C + 6 min @ 95 &#176;C</td>
			<td>3 min @ 65 &#176;C + 7 min @ 95 &#176;C</td>
		</tr>
		<tr>
			<td>Exposure energy</td>
			<td>110 mJ/cm2</td>
			<td>155 mJ/cm2</td>
			<td>170 mJ/cm2</td>
		</tr>
		<tr>
			<td>Post-exposure bake</td>
			<td>4 min @ 95 &#176;C</td>
			<td>1 min @ 65 &#176;C + 6 min @ 95 &#176;C</td>
			<td>2 min @ 65 &#176;C + 7 min @ 95 &#176;C</td>
		</tr>
		<tr>
			<td>Development</td>
			<td>2 min 30 s</td>
			<td>5 min</td>
			<td>6 min</td>
		</tr>
		<tr>
			<td>Hard bake (optional)</td>
			<td>3-5 min @ 200 &#176;C</td>
			<td>3-5 min @ 200 &#176;C</td>
			<td>3-5 min @ 200 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 1: Photolithography steps performed to build a device containing a central chamber of 12 &#956;m in height. Heights of the intermediate chambers: 20, 50 and 90 &#181;m.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Mask 1:10 &#181;m layer</td>
			<td>Mask 2:30 &#181;m layer</td>
			<td>Mask 3:40 &#181;m layer</td>
		</tr>
		<tr>
			<td>SU-8 type</td>
			<td>2007</td>
			<td>2025</td>
			<td>2050</td>
		</tr>
		<tr>
			<td>Spincoating</td>
			<td>30 s @ 1500 rpm</td>
			<td>30 s @ 3050 rpm</td>
			<td>30 s @ 3250 rpm</td>
		</tr>
		<tr>
			<td>Soft bake</td>
			<td>3 min @ 95 &#176;C</td>
			<td>2 min @ 65 &#176;C + 6 min @ 95 &#176;C</td>
			<td>3 min @ 65 &#176;C + 7 min @ 95 &#176;C</td>
		</tr>
		<tr>
			<td>Exposure energy</td>
			<td>125 mJ/cm2</td>
			<td>155 mJ/cm2</td>
			<td>170 mJ/cm2</td>
		</tr>
		<tr>
			<td>Post-exposure bake</td>
			<td>4 min @ 95 &#176;C</td>
			<td>1 min @ 65 &#176;C + 6 min @ 95 &#176;C</td>
			<td>2 min @ 65 &#176;C + 7 min @ 95 &#176;C</td>
		</tr>
		<tr>
			<td>Development</td>
			<td>2 min 30 s</td>
			<td>5 min</td>
			<td>6 min</td>
		</tr>
		<tr>
			<td>Hard bake (optional)</td>
			<td>3-5 min @ 200 &#176;C</td>
			<td>3-5 min @ 200 &#176;C</td>
			<td>3-5 min @ 200 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 2: Photolithography steps performed to build a device containing a central chamber of 10 &#956;m in height. Heights of the intermediate chambers: 20, 50 and 90 &#181;m.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Mask 1:12 &#181;m layer</td>
			<td>Mask 2:32 &#181;m layer</td>
			<td>Mask 3:40 &#181;m layer</td>
		</tr>
		<tr>
			<td>SU-8 type</td>
			<td>2015</td>
			<td>2025</td>
			<td>2050</td>
		</tr>
		<tr>
			<td>Spincoating</td>
			<td>30 s @ 3250 rpm</td>
			<td>30 s @ 2500 rpm</td>
			<td>30 s @ 3250 rpm</td>
		</tr>
		<tr>
			<td>Soft bake</td>
			<td>3 min @ 95 &#176;C</td>
			<td>2 min @ 65 &#176;C + 5 min @ 95 &#176;C</td>
			<td>3 min @ 65 &#176;C + 7 min @ 95 &#176;C</td>
		</tr>
		<tr>
			<td>Exposure time</td>
			<td>140 mJ/cm2</td>
			<td>157 mJ/cm2</td>
			<td>170 mJ/cm2</td>
		</tr>
		<tr>
			<td>Post-exposure bake</td>
			<td>4 min @ 95 &#176;C</td>
			<td>1 min @ 65 &#176;C + 5 min @ 95 &#176;C</td>
			<td>2 min @ 65 &#176;C + 7 min @ 95 &#176;C</td>
		</tr>
		<tr>
			<td>Development</td>
			<td>3 min</td>
			<td>5 min</td>
			<td>6 min</td>
		</tr>
		<tr>
			<td>Hard bake (optional)</td>
			<td>3-5 min @ 200 &#176;C</td>
			<td>3-5 min @ 200 &#176;C</td>
			<td>3-5 min @ 200 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 3: Photolithography steps performed to build a device containing a central chamber of 3 &#956;m in height. Heights of the intermediate chambers: 18, 50 and 90 &#181;m.
Supplementary data 1: masks_neuron_volume_chips.tiff. Schematic view of the masks used to fabricate the PDMS device (DRIE mask and masks 1-3). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56923/masks_neuron_volume_chips.tiff">Please click here to download this file.</a>
Supplementary data 2: file &#34;masks_neuron_volume_chips.dxf&#34;. Electronic files allowing to fabricate the DRIE mask and masks 1-3. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56923/masks_neuron_volume_chips.dxf">Please click here to download this file.</a>
Supplementary data 3: &#34;Mask_Photoresist-stripes.dxf&#34;. Electronic files allowing to fabricate the mask used for the photolithography of photoresist stripes. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56923/Mask_Photoresist-stripes.dxf">Please click here to download this file.</a>
Supplementary data 4: file conversion_mattotiff.m <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56923/conversion_mattotiff.m">Please click here to download this file.</a>
Supplementary data 5: file importfilevol.m <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56923/importfilevol.m">Please click here to download this file.</a>

Watch this Scientific Journal Video about High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices at JoVE.com

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique ...

high-resolution-volume-imaging-neurons-use-fluorescence-exclusion

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.0K Views.  Université PSL, CNRS. Volume is een belangrijke parameter over fysiologische en pathologische eigenschappen van cellen. Beschrijven we een fluorescerende uitsluiting methode waardoor volledige-veld meting van in vitro neuronale volume met sub Micrometrische axiale resolutie die nodig zijn voor de analyse van neurites en dynamische structuren geïmpliceerd in neuronale groei.

Video: High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Biology

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

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 how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

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

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a commercially available pre-assembled plastic chip for compartmentalizing cultured primary rat hippocampal neurons. These plastic chips, contained within the footprint of a standard microscope slide, are compatible with high-resolution, live, and fluorescence imaging. This protocol demonstrates how to retrograde label neurons via isolated axons using a modified rabies virus encoding a fluorescent protein, create isolated microenvironments within one compartment, and perform axotomy and immunocytochemistry on-chip. Neurons are cultured for &gt;3 weeks within the plastic chips, illustrating the compatibility of these chips for long-term neuronal cultures.

This protocol describes the use of plastic chips to culture and compartmentalize primary murine neurons. These chips are preassembled, user-friendly, and compatible with high-resolution, live, and fluorescence imaging. This protocol describes how to plate rat hippocampal neurons within these chips and perform fluidic isolation, axotomy and immunostaining.

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a ...

Multilayer Imaging with Microprism in Head-Fixed and Freely Moving Animals

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

Author Spotlight: Comparative Imaging of Neural Activity in Awake and Freely Moving States

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

Source: Tsuruta, F., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55488">Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes.</a> J. Vis. Exp. (2017).
This video demonstrates how neuronal vesicles are labeled with specific fluorescent proteins to visualize their movement. First, a plasmid coding for a fluorescent protein is incubated with a transfection agent and then introduced into a culture of neurons. Subsequently, images of the neurons are acquired and analyzed to track the vesicles&#39; movement.

1. Imaging Vesicle Motility

	Prepare the plasmids that will label each type of vesicle. For instance, use EGFP-Rab5 for early endosomes, EGFP-Rab7 for ...

JoVE Encyclopedia of Experiments

Neuropathology

Developmental and Pediatric Disorders

Simultaneous Optical and Electrophysiological Monitoring of Neuronal Cells in Brain Slices

Source:&#160; Murphy, C. A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61333">Optogenetic activation of afferent pathways in brain slices and modulation of responses by volatile anesthetics.</a> J. Vis. Exp. (2020)
This video outlines a method for simultaneously imaging and recording electrical activity from transgenic brain slices that contain neuron-specific fluorescent proteins. The procedure involves maintaining the brain slice in a perfusion chamber with aerated artificial cerebrospinal fluid, using a light source to excite fluorescent-labeled cells, and recording the electrical signals with a multichannel probe and patch clamp.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;&#160;&#160;&#160;&#160;
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High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

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