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>Equipments</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 &#176;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 &#176;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>Masks</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>Small laboratory equipment</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&#34; 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>Substrates</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>Photoresists and developpers</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 &#181;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&#181;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>Laboratory consumables</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>Chemicals</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>Culture medium</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>Softwares</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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/files/ftp_upload/56923/conversion_mattotiff.m">Please click here to download this file.</a>
Supplementary data 5: file importfilevol.m <a href="http://cloudflare.jove.com/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

Research

JoVE Journal

Neuroscience

7.0K Views.  Université PSL, CNRS. 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.

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

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

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 ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...