Name: high-resolution volume imaging of neurons by the use of fluorescence exclusion method and dedicated microfluidic devices
Uploaded: 2018-03-26T12:30:00
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>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 ...

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