This project was funded by the Land Baden-W&#252;rttemberg as well as by the European Union, Europ&#228;ischer Fonds f&#252;r die Regionale Entwicklung. The authors thank Rainer Wittig (ILM Ulm) for providing the U251-MG-L106-Grx1-roGFP2 cell line and Claudia Hintze for skillful technical assistance.

1. Cell Spheroid Growing and Incubation
<ol>
	<li>Experiment 1: Cell spheroids incubated with a chemotherapeutic drug
	<ol>
		<li>Prepare agarose 1.5% in culture medium by adding 0.45 g of agarose to 30 ml culture medium (sufficient for 6 plates).</li>
		<li>Heat the mixture from step 1.1.1 to at least 80 &#176;C while stirring it from time to time.</li>
		<li>Fill 50 &#181;l of the heated mixture from step 1.1.2 into each well of a 96-well plate and let it solidify within 1&#8211;2 hr. Store the plates closed and covered in the refrigerator at 5 &#176;C until use.</li>
		<li>Seed about 150 MCF-7 breast cancer cells in each well of the prepared 96-well plate in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with Ham&#8217;s F-12 culture medium supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin.</li>
		<li>Grow spheroids for 5&#8211;7 days up to a diameter of about 200&#8211;300 &#181;m in an incubator at 5% CO2 and 37 &#176;C.</li>
		<li>Incubate the cell spheroids with the anthracycline antibiotic doxorubicin hydrochloride for 6 hr at a concentration ranging between 2 &#181;M and 8 &#181;M (in culture medium) in an incubator at 5% CO2 and 37 &#176;C.</li>
		<li>Wash the cell spheroids with culture medium or Earle&#39;s Balanced Salt Solution (EBSS) prior to microscopy. </li>
	</ol>
	</li>
	<li>Experiment 2: Oxidation process in spheroids expressing a redox sensor
	<ol>
		<li>Seed about 400 U251-MG-L106 glioblastoma cells permanently transfected with the glutathione sensitive green fluorescent redox sensor Grx1-roGFP2 in agarose gel&#8211;coated wells (procedure described in steps 1.1.1&#8211;1.1.3) of a 96-well plate in DMEM culture medium supplemented with 10% FCS, 1% penicillin/streptomycin and hygromycin B (150 &#181;g/ml).</li>
		<li>Grow spheroids for 5&#8211;7 days up to a diameter of about 200&#8211;300 &#181;m in an incubator at 5% CO2 and 37 &#176;C.</li>
		<li>Add 10 &#181;l of hydrogen peroxide solution (purum p.a., &#8805; 30%) to 990 &#181;l bi-distilled water to create a 100 mM stock solution to be used within 12 hr.</li>
		<li>Dilute the stock solution from step 1.2.3 to 50 &#181;M in EBSS right before the experiment.</li>
		<li>Incubate the cell spheroids via the microfluidic system during measurement with hydrogen peroxide 50 &#181;M in EBSS for oxidation of the redox sensor.</li>
	</ol>
	</li>
	<li>Experiment 3: Initiation and labeling of cellular necrosis within spheroids
	<ol>
		<li>Seed about 400 cells (e.g., U251-MG-L106-Grx1-roGFP2 glioblastoma cells) in agarose gel&#8211;coated wells (procedure described in steps 1.1.1&#8211;1.1.3) of a 96-well plate in DMEM culture medium supplemented with 10% FCS, 1% penicillin/streptomycin and hygromycin B (150 mg/ml).</li>
		<li>Grow spheroids for 5&#8211;7 days up to a diameter of about 200&#8211;300 &#181;m in an incubator at 5% CO2 and 37 &#176;C.</li>
		<li>Incubate the cell spheroids with the mitochondrial electron transport inhibitor rotenone for 3 hr at a concentration of 1 &#181;M in culture medium to induce cellular necrosis. Additionally, co-incubate with a green cytotoxicity dye for 3 hr at a concentration of 1 &#181;l in 1 ml culture medium to label necrotic cells.</li>
		<li>Wash the cell spheroids with culture medium or EBSS prior to microscopy.</li>
	</ol>
	</li>
</ol>
2. Light Sheet Adjustment
NOTE: For achieving best results it has to be ensured that the beam waist of the light sheet is in the focus plane of the objective lens. The position of the beam waist can only be aligned by observing the light sheet in vertical position with respect to the capillary (see steps 2.5 - 2.8).
<ol>
	<li>Use an inverted microscope and mount the SPIM excitation module, as described in Ref. 16, to the baseplate of the positioning table (see Figure 1A).</li>
	<li>Equip the microscope with a 10X/0.3 or 20X/0.5 microscope objective lens and an appropriate long-pass filter (e.g., &#955; &#8805; 515 nm) in the detection path of the microscope.</li>
	<li>Mount an integrating camera to the detection port of the microscope.</li>
	<li>Use a parallel collimated beam of a laser or a laser diode with an excitation wavelength of preferentially 470 nm and apply it to the SPIM module.</li>
	<li>Rotate the cylindrical lens by 90 degrees, which transfers the light sheet into a vertical position for an axial adjustment of the beam waist of the light sheet.</li>
	<li>Place a capillary containing a liquid with a fluorescent dye in the sample holder.</li>
	<li>Attach the sample holder with the capillary to the positioning table of the microscope and align the position of the capillary such that it is centered and the beam waist of the light sheet is in focus.</li>
	<li>Adjust the beam waist by variation of the axial position of the cylindrical lens itself. Turn back the lens after adjustment.</li>
</ol>
3. Cell Spheroid Application and Microscope Feed Synchronization
<ol>
	<li>Place the cell spheroids separately or in groups within rectangular borosilicate glass capillaries16 with an inner cross section of 600 &#181;m x 600 &#181;m and a wall thickness of 120 &#181;m. Two techniques for the application of a cell spheroid have proven to be successful.
	<ol>
		<li>Take the empty capillary upright with your thumb and middle finger and seal the upper opening with your forefinger. Bring the lower opening close to the cell spheroid in its surrounding liquid (EBSS or culture medium). Release the forefinger from the upper opening. Liquid with the cell spheroid in it will be soaked in immediately by capillary forces. Adjust the position of the spheroid within the filled capillary by gravitation in respective upright position of the capillary.</li>
		<li>Alternatively, apply the cell spheroid to the capillary via pipetting. Take care that the opening of the tip of the pipette is, on the one hand, big enough for the spheroid size and, on the other hand, less or equal in size to the inner diameter of the capillary.</li>
	</ol>
	</li>
	<li>Place the capillary with the spheroid in it in a special sample holder for microscopy.</li>
	<li>Attach the sample holder with the capillary to the positioning table of the microscope and align the position of the cell spheroid such that it is centered and focused.</li>
	<li>Set the matching between the light sheet and the focal plane of the microscope objective lens in the detection path by adjusting the position of the reflection mirror and the cylindrical lens (see adjustment screw in Figure 1B).</li>
	<li>Adjust the micrometer screw (see Figure 1B) according to the refraction indices and the numerical aperture of the objective lens to compensate the fishtank effect. 
	NOTE: Different refraction indices of the sample and the media surrounding the objective lens lead to a difference between the shift of the objective lens and the shift of the focal plane. Since the light sheet is moved by the objective turret, the mismatch between the shift of the light sheet (corresponding to the shift of the objective lens) and the shift of the focal plane is compensated by a lever arm (see Figure 1B). The micrometer screw is used to adapt the distance between the upper and the lower pin to adjust the shift of the light sheet according to the numerical aperture of the objective lens and the refraction indices.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51993/51993fig1highres.jpg" width="500" /> 
Figure 1. (A) Picture of the single plane illumination module mounted to the baseplate of the positioning table of an inverted microscope, (B) setup of the single plane illumination module and the microscope feed synchronization to compensate the lifting-mismatch (fishtank effect) between focal plane and illumination plane. &#916;zo indicates the shift of the objective lens and &#916;zf the shift of the focal plane and the light sheet. The inlay shows cross sections of spheroid containing capillaries. Left: rectangular capillary with inner diameter of 600 &#181;m (wall 120 &#181;m). Right: Setup used for rotation. An inner round capillary (inner diameter 400 &#181;m, outer diameter 550 &#181;m) is rotated within the outer rectangular capillary. The space between the two capillaries is filled with an immersion fluid.
4. Measurement in Dynamic Liquid Environment
NOTE: The previous protocol is used for static incubation prior to a measurement where the cell spheroid has previously been incubated and then held in place in the capillary by simple gravitation. There is no need for further fixation. However, for measurements in flowing media where a dynamic incubation and, therefore, a dynamic environment is desirable to measure uptake kinetics, one can accomplish this sub-protocol. Steps 4.5&#8211;4.9 are critical to avoid air bubbles reaching the sample. 
<ol>
	<li>Fill the capillary with FCS for 30 min to support later cellular adhesion of a cell spheroid to the inner glass surface.</li>
	<li>Let the remaining FCS coating in the depleted capillary dry out for at least 12 hr.</li>
	<li>Introduce the cell spheroid to the FCS coated capillary as described in step 3.2.</li>
	<li>Leave the capillary in the incubator at 5% CO2 and 37 &#176;C for additional 2&#8211;4 hr to cause cellular adhesion of the spheroid.</li>
	<li>Set up the afflux part of the microfluidic system (see Figure 2). Use a peristaltic pump to fill the afflux of the tubing (inner diameter: 0.89 mm) with culture medium containing the fluorescent dye, drug or agent.</li>
	<li>Connect the afflux part of the microfluidic system to a bubble trap (see Figure 2).</li>
	<li>First clamp the capillary bubble-free to the afflux tubing and then clamp the other side of the capillary to the drain tubing.</li>
	<li>Tune the liquid temperature of the water bath to the desired value (e.g., 37 &#176;C).</li>
	<li>Adjust the peristaltic pump to the desired pump velocity (e.g., flow rate: 9 &#181;l/min; pump velocity in the capillary: 25 mm/min).</li>
	<li>Collect the pumped liquid either in a recipient (open-loop setup), feed it back to its source (closed-loop setup) or feed it directly into the tubing of the peristaltic pump (tight closed-loop setup).</li>
</ol>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51993/51993fig2highres.jpg" width="500" /> 
Figure 2. Microfluidic setup (open-loop and tight closed-loop); inlay: Illumination of a spheroid within a micro-capillary using light sheet based fluorescence microscopy coupled to an inverted microscope.
5. Data Acquisition and Analysis
<ol>
	<li>Set the laser power and the integration time for the image acquisition.</li>
	<li>Take care that the parameters defined in step 5.1 do not exceed light dose values about 50&#8211;100 J/cm2 for native cells or 10&#8211;20 J/cm2 for cells incubated with a fluorescent dye or transfected with a fluorescent protein encoding plasmid to avoid phototoxicity (for details see Ref. 12).</li>
	<li>Set the increment for the z-stack to &#8710;z = 5&#8211;10 &#181;m which is preferable for 3-dimensional data analysis as the light sheet thickness is about 10 &#181;m.</li>
	<li>Perform measurements of single images or z-stacks by variation of the focal plane within the cell spheroid.</li>
</ol>

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No conflicts of interest declared.

The present manuscript describes a light sheet or single plane illumination microscopy (SPIM) device which is optimized for 3-dimensional cell systems, e.g., multi-cellular tumor spheroids (MCTS). Three exemplary applications include (1) uptake of a cytostatic drug and its partial conversion to a degradation product (whose contribution to chemotherapeutic efficacy still remains to be evaluated), (2) measurements of the redox state by use of a genetically encoded glutathione sensor upon addition of an oxidizing a...

In addition to well established methods (confocal or multi-photon laser scanning microscopy1-4, structured illumination microscopy5,6) light sheet or single plane illumination microscopy (SPIM) has proven to be a valuable method of 3D imaging7,8,9. Of particular interest is its application to 3-dimensional cell cultures, e.g., multi-cellular tumor spheroids (MCTS), which are used increasingly for drug discovery research10,11. Furthermore, SPIM is a preferential method when even upon long-term exposure or repetitive measurements low light doses are required to maintain the sample&#8217;s viability, since for measurement of each plane of the sample only this plane is exposed to light. This is in contrast to other microscopy techniques, where for detection of each focal plane the whole sample is illuminated, so that upon recording of numerous planes the light dose sums up and may damage the sample12.
Light sheet microscopy or SPIM is based on illumination of the sample in perpendicular direction to the observation path either by use of a cylindrical lens or by scanning the exciting laser beam (for a review see Ref. 8). This often requires special sample chambers13,14 or matrices, e.g., agarose7,15, implemented in special high-cost microscopes. As an alternative to those systems a comparably simple illumination device for SPIM has been developed and adapted to a conventional inverted microscope16 (see Figure 1). It consists of a laser beam expanded to a diameter of 8&#160;mm and focused by a cylindrical lens (focal length: 50&#160;mm, numerical aperture: 0.08) to a light sheet of 6&#8211;10&#160;&#181;m thickness over a depth of field of about 100&#160;&#181;m. Samples are located in a rectangular capillary of 600&#8211;900&#160;&#181;m inner diameter placed in front of the microscope objective lens for fluorescence detection. These main features are presently completed and optimized by the use of advanced micro-capillary approaches for holding and for rotating the samples, the synchronous adjustment of the illuminating light sheet (in axial direction) and the objective lens used for fluorescence detection (identical optical path lengths of displacement require a correction of the mechanical feed), and the adaptation of a microfluidic system for application of fluorescent dyes, pharmaceutical agents or drugs, thus minimizing the required quantities and expenses.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:243px;">Name of the Material/Equipment</td>
			<td style="width:201px;">Company</td>
			<td style="width:124px;">&#160;Catalog Number</td>
			<td style="width:376px;">Comments/Description(optional)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">microtiter plate</td>
			<td>Orange Scientific</td>
			<td>4430100</td>
			<td>for cell spheroid growing</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">agarose&#160;</td>
			<td>Carl Roth GmbH</td>
			<td>3810.1</td>
			<td>for cell spheroid growing</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">MCF-7 cell line</td>
			<td>CLS Cell Lines Service GmbH</td>
			<td>300273</td>
			<td>cell line</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">U251-MG-L106 cell line</td>
			<td></td>
			<td></td>
			<td>cell line</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">DMEM</td>
			<td>Biochrom AG (Merck Millipore)</td>
			<td>FG0435</td>
			<td>culture medium</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">DMEM/Ham&#39;s F-12</td>
			<td>Biochrom AG (Merck Millipore)</td>
			<td>FG4815</td>
			<td>culture medium</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">FCS</td>
			<td>Biochrom AG (Merck Millipore)</td>
			<td>S0615</td>
			<td>cell culture supplement</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">penicillin/streptomycin</td>
			<td>Biochrom AG (Merck Millipore)</td>
			<td>A 2213</td>
			<td>antibiotics</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">hygromycin B</td>
			<td style="width:201px;">PAA Laboratories&#160;&#160;</td>
			<td>P02-015</td>
			<td>antibiotic</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">EBSS</td>
			<td>Sigma-Aldrich Inc.</td>
			<td>E3024</td>
			<td>cell culture supplement</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">doxorubicin</td>
			<td>Sigma-Aldrich Inc.</td>
			<td>D1515</td>
			<td>fluorescent dye (CAUTION: acute toxicity)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">green cytotoxicity dye</td>
			<td>Promega GmbH</td>
			<td>G8742</td>
			<td>CellTox - fluorescent cytotoxicity dye</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">rotenone</td>
			<td>Sigma-Aldrich Inc.</td>
			<td>R8875</td>
			<td>cellular inhibitor (CAUTION: acute toxicity)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">hydrogen peroxide (H2O2)</td>
			<td>Sigma-Aldrich Inc.</td>
			<td>95302</td>
			<td>reagent for oxidation (CAUTION: acute toxicity)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">capillary</td>
			<td>VitroCom&#160;</td>
			<td>8260-050</td>
			<td>sample preparation</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">microscope</td>
			<td>Carl Zeiss Jena</td>
			<td>Axiovert 200M</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">AxioCam MRc CCD-camera</td>
			<td>Carl Zeiss MicroImaging GmbH</td>
			<td>426508-9901-000</td>
			<td>CCD-camera</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">AxioVision data aquisition software</td>
			<td>Carl Zeiss MicroImaging GmbH</td>
			<td>version 4.8.2.</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">laser diode</td>
			<td>Pico Quant GmbH</td>
			<td>LDH-P-C-470</td>
			<td>used with driver PDL800-B</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">perestaltic pump</td>
			<td>Ismatec Labortechnik</td>
			<td>MS-1 Reglo</td>
			<td>re-callibrated to reduce the minimum pump speed by 1/10&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">silicone tubes</td>
			<td>IDEX Health &#38; Science GmbH</td>
			<td>TYGON R3607</td>
			<td></td>
		</tr>
	</tbody>
</table>

single plane illumination module and micro-capillary approach for a wide-field microscope

A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures, e.g., multi-cellular tumor spheroids (MCTS). The SPIM excitation module shapes and deflects the light such that the sample is illuminated by a light sheet perpendicular to the detection path of the microscope. The system is characterized by use of a rectangular capillary for holding (and in an advanced version also by a micro-capillary approach for rotating) the samples, by synchronous adjustment of the illuminating light sheet and the objective lens used for fluorescence detection as well as by adaptation of a microfluidic system for application of fluorescent dyes, pharmaceutical agents or drugs in small quantities. A protocol for working with this system is given, and some technical details are reported. Representative results include (1) measurements of the uptake of a cytostatic drug (doxorubicin) and its partial conversion to a degradation product, (2) redox measurements by use of a genetically encoded glutathione sensor upon addition of an oxidizing agent, and (3) initiation and labeling of cell necrosis upon inhibition of the mitochondrial respiratory chain. Differences and advantages of the present SPIM module in comparison with existing systems are discussed.

Experiment 1: Cell spheroids incubated with a chemotherapeutic drug
A z-stack scan of a previously incubated MCF-7 cell spheroid (8&#160;&#181;M doxorubicin, 6&#160;hr) is depicted in Figure 3. It gives detailed information about the cellular uptake and distribution of doxorubicin17 and its degradation product18,19. Within the outer cell layer of the spheroid red fluorescent doxorubicin is mainly localized in the nucleus, whereas in inner...

Watch this Scientific Journal Video about Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope at JoVE.com

Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

A module for single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures. The sample is located within a rectangular capillary, and via a microfluidic system fluorescent dyes, pharmaceutical agents or drugs can be applied in small quantities.

A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and ...

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Research

JoVE Journal

Engineering

9.7K Views.  Aalen University.  Модуль для одной плоскости освещения микроскопии (SPIM) описывается который легко адаптируется к перевернутой широкого поля микроскопа и оптимизирован для культур 3-мерных клеток. Образец находится в пределах прямоугольного капилляра, а также через микрожидк...

Video: Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities

High resolution microscope systems with optical traps allow for precise manipulation of various refractive objects, such as dielectric beads 1 or cellular organelles 2,3, as well as for high spatial and temporal resolution readout of their position relative to the center of the trap. The system described herein has one such "traditional" trap operating at 980 nm. It additionally provides a second optical trapping system that uses a commercially available holographic package to simultaneously create and manipulate complex trapping patterns in the field of view of the microscope 4,5 at a wavelength of 1,064 nm. The combination of the two systems allows for the manipulation of multiple refractive objects at the same time while simultaneously conducting high speed and high resolution measurements of motion and force production at nanometer and piconewton scale.

The system described herein employs a traditional optical trap as well as an independent holographic optical trapping line, capable of creating and manipulating multiple traps. This allows for the creation of complex geometric arrangements of refractive particles while also permitting simultaneous high-speed, high-resolution measurements of the activity of biological enzymes.

High resolution microscope systems with optical traps allow for precise manipulation of various refractive objects, such as dielectric beads 1 or cellular ...

Analysis of Contact Interfaces for Single GaN Nanowire Devices

Single GaN nanowire (NW) devices fabricated on SiO2 can exhibit a strong degradation after annealing due to the occurrence of void formation at the contact/SiO2 interface. This void formation can cause cracking and delamination of the metal film, which can increase the resistance or lead to a complete failure of the NW device. In order to address issues associated with void formation, a technique was developed that removes Ni/Au contact metal films from the substrates to allow for the examination and characterization of the contact/substrate and contact/NW interfaces of single GaN NW devices. This procedure determines the degree of adhesion of the contact films to the substrate and NWs and allows for the characterization of the morphology and composition of the contact interface with the substrate and nanowires. This technique is also useful for assessing the amount of residual contamination that remains from the NW suspension and from photolithographic processes on the NW-SiO2 surface prior to metal deposition. The detailed steps of this procedure are presented for the removal of annealed Ni/Au contacts to Mg-doped GaN NWs on a SiO2 substrate.

A technique was developed that removes Ni/Au contact metal films from their substrate to allow for the examination and characterization of the contact/substrate and contact/NW interfaces of single GaN nanowire devices.

Single GaN nanowire (NW) devices fabricated on SiO2 can exhibit  a strong degradation after annealing due to the  occurrence of void formation at the ...

Measuring Material Microstructure Under Flow Using 1-2 Plane Flow-Small Angle Neutron Scattering

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is presented. The SANS shear cell consists of a concentric cylinder Couette geometry that is sealed and rotating about a horizontal axis so that the vorticity direction of the flow field is aligned with the neutron beam enabling scattering from the 1-2 plane of shear (velocity-velocity gradient, respectively). This approach is an advance over previous shear cell sample environments as there is a strong coupling between the bulk rheology and microstructural features in the 1-2 plane of shear. Flow-instabilities, such as shear banding, can also be studied by spatially resolved measurements. This is accomplished in this sample environment by using a narrow aperture for the neutron beam and scanning along the velocity gradient direction. Time resolved experiments, such as flow start-ups and large amplitude oscillatory shear flow are also possible by synchronization of the shear motion and time-resolved detection of scattered neutrons. Representative results using the methods outlined here demonstrate the useful nature of spatial resolution for measuring the microstructure of a wormlike micelle solution that exhibits shear banding, a phenomenon that can only be investigated by resolving the structure along the velocity gradient direction. Finally, potential improvements to the current design are discussed along with suggestions for supplementary experiments as motivation for future experiments on a broad range of complex fluids in a variety of shear motions.

A shear cell is developed for small-angle neutron scattering measurements in the velocity-velocity gradient plane of shear and is used to characterize complex fluids. Spatially resolved measurements in the velocity gradient direction are possible for studying shear-banding materials. Applications include investigations of colloidal dispersions, polymer solutions, and self-assembled structures.

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is ...

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1&#160;to 24 hr&#160;after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 &#956;m. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response:&#160;elemental, molecular, or cellular.

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle&#039;s Mapping and Quantification in Organ Tissue

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 &#956;m and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) ...

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. The aggressive scaling of feature sizes, both on devices and interconnects, leads to serious challenges to ensure the required product reliability. Standard reliability tests and post-mortem failure analysis provide only limited information about the physics of failure mechanisms and degradation kinetics. Therefore it is necessary to develop new experimental approaches and procedures to study the TDDB failure mechanisms and degradation kinetics in particular. In this paper, an in situ experimental methodology in the transmission electron microscope (TEM) is demonstrated to investigate the TDDB degradation and failure mechanisms in Cu/ULK interconnect stacks. High quality imaging and chemical analysis are used to study the kinetic process. The in situ electrical test is integrated into the TEM to provide an elevated electrical field to the dielectrics. Electron tomography is utilized to characterize the directed Cu diffusion in the insulating dielectrics. This experimental procedure opens a possibility to study the failure mechanism in interconnect stacks of microelectronic products, and it could also be extended to other structures in active devices.

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. This paper demonstrates the procedure of an in situ TDDB experiment in the transmission electron microscope, which opens a possibility to study the failure mechanism in microelectronic products.

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. ...

Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and automotive. However, effective inspection and characterization metrology systems are needed to ensure the functional reliability of MEMS. This study presents a system based on digital holography as a tool for MEMS metrology. Digital holography has gained increasing attention in the past 20 years. With the fast development and decreasing cost of sensor arrays, resolution of such systems has increased broadening potential applications. Thus, it has attracted attention from both research and industry sides as a potential reliable tool for industrial metrology. Indeed, by recording the interference pattern between an object beam (which contains sample height information) and a reference beam on a CCD camera, one can retrieve the quantitative phase information of an object. However, most of digital holographic systems are bulky and thus not easy to implement on industry production lines. The novelty of the system presented is that it is lens-less and thus very compact. In this study, it is shown that the Compact Digital Holographic Microscope (CDHM) can be used to evaluate several characteristics typically consider as criteria in MEMS inspections. The surface profiles of MEMS in both static and dynamic conditions are presented. Comparison with AFM is investigated to validate the accuracy of the CDHM.

We present a compact reflection digital holographic system (CDHM) for inspection and characterization of MEMS devices. A lens-less design using a diverging input wave providing natural geometrical magnification is demonstrated. Both static and dynamic studies are presented.

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and ...

Hand Controlled Manipulation of Single Molecules via a Scanning Probe Microscope with a 3D Virtual Reality Interface

Considering organic molecules as the functional building blocks of future nanoscale technology, the question of how to arrange and assemble such building blocks in a bottom-up approach is still open. The scanning probe microscope (SPM) could be a tool of choice; however, SPM-based manipulation was until recently limited to two dimensions (2D). Binding the SPM tip to a molecule at a well-defined position opens an opportunity of controlled manipulation in 3D space. Unfortunately, 3D manipulation is largely incompatible with the typical 2D-paradigm of viewing and generating SPM data on a computer. For intuitive and efficient manipulation we therefore couple a low-temperature non-contact atomic force/scanning tunneling microscope (LT NC-AFM/STM) to a motion capture system and fully immersive virtual reality goggles. This setup permits &#34;hand controlled manipulation&#34; (HCM), in which the SPM tip is moved according to the motion of the experimenter&#39;s hand, while the tip trajectories as well as the response of the SPM junction are visualized in 3D. HCM paves the way to the development of complex manipulation protocols, potentially leading to a better fundamental understanding of nanoscale interactions acting between molecules on surfaces. Here we describe the setup and the steps needed to achieve successful hand-controlled molecular manipulation within the virtual reality environment.

We demonstrate the precise manipulation of individual organic molecules on a metal surface with the tip of a scanning probe microscope driven in 3D by the experimenter's hand using a motion capture system and fully immersive virtual reality goggles.

Considering organic molecules as the functional building blocks of future nanoscale technology, the question of how to arrange and assemble such building ...

Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and nanotechnology. Although near-field scanning microscopy and superlenses have improved the resolution in the near-field region, far-field imaging in real-time remains a significant challenge. Recently, the hyperlens, which magnifies and converts evanescent waves into propagating waves, has emerged as a novel approach to far-field imaging. Here, we report the fabrication of a spherical hyperlens composed of alternating silver (Ag) and titanium oxide (TiO2) thin layers. Unlike a conventional cylindrical hyperlens, the spherical hyperlens allows for two-dimensional magnification. Thus, incorporation into conventional microscopy is straightforward. A new optical system integrated with the hyperlens is proposed, allowing for a sub-wavelength image to be obtained in the far-field region in real time. In this study, the fabrication and imaging setup methods are explained in detail. This work also describes the accessibility and possibility of the hyperlens, as well as practical applications of real-time imaging in living cells, which can lead to a revolution in biology and nanotechnology.

The use of a hyperlens has been regarded as a novel super-resolution imaging technique due to its advantages in real-time imaging and its simple implementation with conventional optics. Here, we present a protocol describing the fabrication and imaging applications of a spherical hyperlens.

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and ...

All-electronic Nanosecond-resolved Scanning Tunneling Microscopy: Facilitating the Investigation of Single Dopant Charge Dynamics

The miniaturization of semiconductor devices to scales where small numbers of dopants can control device properties requires the development of new techniques capable of characterizing their dynamics. Investigating single dopants requires sub-nanometer spatial resolution, which motivates the use of scanning tunneling microscopy (STM). However, conventional STM is limited to millisecond temporal resolution. Several methods have been developed to overcome this shortcoming, including all-electronic time-resolved STM, which is used in this study to examine dopant dynamics in silicon with nanosecond resolution. The methods presented here are widely accessible and allow for local measurement of a wide variety of dynamics at the atomic scale. A novel time-resolved scanning tunneling spectroscopy technique is presented and used to efficiently search for dynamics.

We demonstrate an all-electronic method to observe nanosecond-resolved charge dynamics of dopant atoms in silicon with a scanning tunneling microscope.

The miniaturization of semiconductor devices to scales where small numbers of dopants can control device properties requires the development of new ...

Single-Digit Nanometer Electron-Beam Lithography with an Aberration-Corrected Scanning Transmission Electron Microscope

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by employing an aberration-corrected scanning transmission electron microscope as the exposure tool. Here, we present results of single-digit nanometer patterning of two widely used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. The method achieves sub-5 nanometer features in poly (methyl methacrylate) and sub-10 nanometer resolution in hydrogen silsesquioxane. High-fidelity transfer of these patterns into target materials of choice can be performed using metal lift-off, plasma etch, and resist infiltration with organometallics.

We use an aberration-corrected scanning transmission electron microscope to define single-digit nanometer patterns in two widely-used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. Resist patterns can be replicated in target materials of choice with single-digit nanometer fidelity using liftoff, plasma etching, and resist infiltration by organometallics.

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by ...