Name: compact lens-less digital holographic microscope for mems inspection and characterization
Uploaded: 2016-07-05T15:00:00
Description: Watch this Scientific Journal Video about Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization at JoVE.com

The authors have no acknowledgements.

1. Preliminary Preparation of the Measurement
Note: The sample used for the experiment is a MEMS electrode. The gold electrodes are fabricated on a silicon wafer using lift off process. The sample is an 18 mm x 18 mm wafer with periodic structures (electrodes) with 1 mm period
<ol>
	<li>Sign into the logbook before using the system.</li>
	<li>Turn on the computer, LASER and translation stage power.</li>
	<li>Place the MEMS electrode/micro-diaphragm sample.
	<ol>
		<li>Place the MEMS sample in the middle of the s...

<ol>
	<li>Maluf, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An introduction to Microelectromechanical Systems. , (2002).</li><li>Novak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEMS+metrology+techniques.">MEMS metrology techniques.</a> Proc. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+digital+microholo+interferometric+system+for+micromechanical+testing.">Development and validation of digital microholo interferometric system for micromechanical testing.</a> Proc. SPIE. 4778, 11-20 (2002).</li><li>Qu, W., Bhattacharya, K., Choo, C. O., Yu, Y., Asundi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+digital+holographic+microscopy+based+on+a+beam-splitter+cube+interferometer.">Transmission digital holographic microscopy based on a beam-splitter cube interferometer.</a> Appl. Opt. 48 (15), 2778-2783 (2009).</li><li>Potcoava, M. C., Kim, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fingerprint+biometry+applications+of+digital+holography+and+low-coherence+interferography.">Fingerprint biometry applications of digital holography and low-coherence interferography.</a> Appl. Opt. 48 (34), 9-15 (2009).</li><li>Kolman, P., Chmel&#236;k, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherence-controlled+holographic+microscope.">Coherence-controlled holographic microscope.</a> Opt. Express. 18 (21), 21990-22003 (2010).</li><li>Lee, M., Yaglidere, O., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field-portable+reflection+and+transmission+microscopy+based+on+lensless+holography.">Field-portable reflection and transmission microscopy based on lensless holography.</a> Biomed. Opt. Express. 2 (9), 2721-2730 (2011).</li><li>Mico, V., Zalevsky, Z., Garc&#236;a, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common-path+phase-shifting+digital+holographic+microscopy:+a+way+to+quantitative+phase+imaging+and+superresolution.">Common-path phase-shifting digital holographic microscopy: a way to quantitative phase imaging and superresolution.</a> Opt. Commun. 281 (17), 4273-4281 (2008).</li><li>Singh, V. R., Miao, J., Wang, Z., Hedge, G. M., Asundi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+characterization+of+MEMS+diaphragm+using+time+averaged+in-line+digital+holography.">Dynamic characterization of MEMS diaphragm using time averaged in-line digital holography.</a> Opt. Commun. 280 (2), 285-290 (2007).</li><li>Singh, V. R., Anderi, A., Gorecki, C., Nieradko, L., Asundi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+MEMS+cantilevers+lensless+digital+holographic+microscope.">Characterization of MEMS cantilevers lensless digital holographic microscope.</a> Proc. SPIE. 6995, 69950F-1 (2008).</li><li>Schmidt, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+Simulation+of+Optical+Wave+Propagation+with+Examples+in+MATLAB.">Numerical Simulation of Optical Wave Propagation with Examples in MATLAB.</a> SPIE PRESS BOOK. , (2010).</li><li>Zhao, M., Huang, L., Zhang, Q. C., Su, X. Y., Asundi, A., Qian, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality-guided+phase+unwrapping+technique:+comparison+of+quality+maps+and+guiding+strategies.">Quality-guided phase unwrapping technique: comparison of quality maps and guiding strategies.</a> Appl. Opt. 50 (33), 6214-6224 (2011).</li><li>Wang, Z., Qu, W., Yang, F., Wen, Y., Asundi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+autofocus+method+based+on+angular+spectrum+method+in+digital+holography.">A new autofocus method based on angular spectrum method in digital holography.</a> Proc. SPIE. 9449, 2-7 (2015).</li></ol>

In this review, we provide a protocol to accurately recover the quantitative morphology of different MEMS devices by using a compact system relying on digital holography. MEMS characterization in both static and dynamic mode is demonstrated. Quantitative 3D data of a micro channel MEMS is obtained. In order to validate the accuracy of the system, results have been compared between the CDHM and the AFM. Good agreement is found meaning that digital holography can be a reliable technique for 3D imaging. Results indicate tha...

Metrology of micro and nano objects is of great importance for both industry and researchers. Indeed, miniaturization of objects represents a new challenge for optical metrology. Micro electro mechanical systems (MEMS) are generally defined has miniaturized electromechanical systems and usually comprises components such as micro sensors, micro actuators, microelectronics and microstructures. It has found many applications in diverse field such as biotechnology, medicine, communication and sensing1. Recently, the increasing complexity as well as the progressive miniaturization of test object features call for the development of suitable characterization techniques for MEMS. High throughput manufacturing of these complex microsystems requires the implementation of advanced inline measurement techniques, to quantify characteristic parameters and related defects caused by the process conditions2. For instance, the deviation of geometrical parameters in a MEMS device affects the system properties and has to be characterized. In addition, industry requires high resolution measurement performance, such as full three dimension (3D) metrology, large &#64257;eld of view, high imaging resolution, and real time analysis. Thus, it is essential to ensure a reliable quality control and inspection process. Moreover, it requires the measuring system to be easily implementable on a production line and thus relatively compact to be installed on existing infrastructures.
Holography, which was first introduced by Gabor3, is a technique that allows the recovery of the full quantitative information of an object by recording the interference between a reference and an object wave into a photosensitive medium. During this process known as recording, the amplitude, phase and polarization of a field are stored in the medium. Then the object wave field can be recovered by sending the reference beam onto the medium, a process known as optical reading of the hologram. Since a conventional detector only records the intensity of the wave, holography has been a subject of great interest in the past fifty years since it gives access to additional information on the electric field. However, several aspects of conventional holography make it unpractical for industry applications. Indeed, photosensitive materials are expensive and the recording process generally requires a high degree of stability. Advances in high resolution camera sensors such as charged coupled devices (CCD) have opened a new approach for digital metrology. One of those techniques is known as digital holography4. In Digital Holography (DH), the hologram is recorded on a camera (recording medium) and numerical processes are used to reconstruct the phase and intensity information. As with conventional holography, the result can be obtained after two main procedures: the recording and reconstruction as shown in Figure 1. However, if the recording is similar to conventional holography, the reconstruction is only numerical5. The numerical reconstruction process is shown in Figure 2. Two procedures are involved in the reconstruction process. Firstly, the object wave field is retrieved from the hologram. The hologram is multiplied with a numerical reference wave to get the object wavefront at the hologram plane. Secondly, the complex object wavefront is numerically propagated to the image plane. In our system, this step is performed using the convolution method6. The reconstructed field obtained is a complex function and thus phase and intensity can be extracted providing quantitative height information on the object of interest. The capability of whole &#64257;eld information storage in holography method and the use of computer technology for fast data processing offer more flexibility in experimental configuration and significantly increase the speed of the experimental process, opening up new possibilities to develop DH as a dynamic metrological tool for MEMS and micro-systems7,8.
Use of digital holography in phase contrast imaging is now well established and was first presented more than ten years ago9. Indeed, investigation of microscopic devices by combining digital holography and microscopy has been performed in many studies10, 11, 12, 13. Several systems based on high coherence14 and low coherence15 sources as well as different types of geometry13, 16, 17 (in line, off axis, common path&#8230;) have been presented. In addition, in line digital holography has been used previously in characterization of MEMS device18, 19. However, those systems are generally difficult to implement and bulky, making them unsuitable for industrial applications. In this study, we propose a compact, simple and lens free system based on off axis digital holography capable for real time MEMS inspection and characterization. The Compact Digital Holographic microscope (CDHM) is a lens less digital holographic system developed and patented to obtain the 3D morphology of micro-size specular objects. In our system, a 10 mW, highly stable, temperature controlled diode laser operating at 638 nm is coupled into a mono-mode fiber. As shown in Figure 3, the diverging beam emanating from the fiber is split into a reference and an object beam by a beam splitter. The reference beam path comprises a tilted mirror to realize the off axis geometry. The object beam is scattered and reflected by the sample. The two beams interfere on the CCD giving the hologram. The interference pattern imprinted onto the image is called a spatial carrier and permits the recovery of the quantitative phase information with only one image. The numerical reconstruction is performed using a common Fourier transform and convolution algorithm as stated previously. The lens-less configuration has several advantages making it attractive. As no lenses are used, the input beam is a diverging wave providing a natural geometrical magnification and thus improving the system resolution. Moreover, it is free of aberrations encountered in typical optical systems. As can be seen in Figure 3B, the system can be made compact (55x75x125 mm3), lightweight (400 g), and thus can be easily integrated into industrial production lines.

<table border="0" cellpadding="0" cellspacing="0" width="1228">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2 MP Camera</td>
			<td style="width:251px;">Imaging Source</td>
			<td style="width:251px;">DMX 41BU02</td>
			<td style="width:512px;">used to record the hologram. 4.65 microns pixel size</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Motorized X,Y,Z Translation Stage</td>
			<td style="width:251px;">Zaber Technology&#160;</td>
			<td style="width:251px;">TLS28-M</td>
			<td>Holder for the system&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beam splitter</td>
			<td style="width:251px;">Edmund optics</td>
			<td style="width:251px;">49-003</td>
			<td>Cube Beam splitter. Separate and recombine the object and reference beam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laser&#160;</td>
			<td style="width:251px;">Micro Laser Systems, Inc.</td>
			<td>SRT-F635S-20/OSYS</td>
			<td>Diode laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mirror</td>
			<td style="width:251px;">Edmund Optics</td>
			<td style="width:251px;">#43-412-566</td>
			<td>1&#34; Dia. Protected Gold, &#955;/20 Flat Zerodur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">monomode Fiber</td>
			<td style="width:251px;">Thorlabs</td>
			<td style="width:251px;">S405-XP</td>
			<td style="width:512px;">Single Mode Optical Fiber, 400 - 680 nm, &#216;125 &#181;m Cladding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sample holder</td>
			<td style="width:251px;">Edmund Optics</td>
			<td style="width:251px;">#39-930</td>
			<td>Ideal Positioning Platform,&#177;35mm Travel in Both X and Y</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hotplate</td>
			<td style="width:251px;">Thermolyne Mirak hotplate</td>
			<td style="width:251px;">Barnstead International HP72935-60</td>
			<td>temperature range&#160;&#160; 40-370 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Holoscope Software</td>
			<td style="width:251px;">d&#39;Optron Pte Ltd</td>
			<td style="width:251px;">NA</td>
			<td>software developed by the NTU researchers&#160;</td>
		</tr>
	</tbody>
</table>

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.

The protocol described above was designed to inspect and characterize MEMS and Micro devices using CDHM system. In our system, a mono-mode fiber is coupled to a diode laser operating at a 633 nm wavelength. Due to the diverging beam configuration, it is important to match the object beam and reference beam path in order to obtain a hologram that can be reconstructed. This is achieved through careful vertical positioning of the sample with respect to the system. In the calculated wrapped p...

Watch this Scientific Journal Video about Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization at JoVE.com

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

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 ...

The overall goal of this procedure is to test micro-electro-mechanical structures to be used in static and dynamic applications using optical full-field measurements with computational and experimental techniques. This method can help answer key questions in the semiconductor industry and more specifically for micro-electro-mechanical system inspection such as the characterization of the mechanical properties of MEMS structures at different stages of manufacturing. The main advantage of this technique is that it is full-field, non-contact, real-time, high-resolution, and it provides a fully quantitative 3D map of the reflective object.The system have been made with no lens and, hence, is really compact. So this method can provide insight into MEMS characterization. It can also be used for wafer inspection or optics reference testing.If set up in a transparent geometry, it is also possible to inspect micro-optics, diffractive optics, or even used for bioimaging. This procedure uses a compact digital holographic microscope, or CDHM, to characterize a micro-electro-mechanical system or MEMS device. For this demonstration, an 11 square millimeter silicon wafer with square gold electrodes positioned every 0.25 millimeters will be characterized.Using tweezers, place the MEMS sample on the sample holder. Adjust the sample holder so that the laser beam targets the electrodes. The largest possible field of view is limited by the camera sensor, and in this case, is 2.3 by 1.8 millimeters.Vertically position the system about 1.5 centimeters from the sample, and proceed with setting up the 3D View software. This package was developed in C+Begin with clicking on the green box icon to select the video imaging device. Choose the DMx 41BU02 imaging source camera in the drop-down menu.Next, under the Device Settings, Select the Y800 Video Format option, and set the capture rate to 15 Frames Per Second. Press OK, and start the camera using the yellow play button. A live video image of the sample should appear.The displayed image should be a defocus image of the sample. Adjust the exposure time and contrast of the image if needed. Now, center the sample as best as possible and access the Settings options.There, set the System Type to Reflection or Transmission. Check that the wavelength of the laser is set to 633 nm, The Pixel size of the camera to 4, 650 nm, and the magnification is times two. Next, select the Convolution Reconstruction algorithm, and set the Reconstruction Distance to 100 mm and the Reconstruction Step to one.The reconstruction distance can be adjusted later to adjust the intensity image to a sharp focus, while the Step parameter indicates how many times the convolution operation is performed to simulate the beam propagation. This parameter can be changed to fine-tune the reconstruction distance in the intensity image. Lastly, under the Post Processing options, set the Unwrapping algorithm to the Quality mapped option.Make sure that the Intensity filter and the Phase filter options are set to None. Then, press OK.Begin with accessing the 3D View software and opening the Fourier spectrum window. If one zero order and two plus one minus one order spectrums do not appear, check that the sample is placed below the red laser beam.The laser reflection can easily be seen from the sample. Now, stop the live measurement mode and select one of the diffracted orders using the filter tool. Select an area large enough to encompass all the frequencies needed for the phase retrieval.Select the SET option to apply the filter. Then, restart the live measurement mode. With the selection made, and imaging live, open the Phase window and make sure that the unwrapped mode is not enabled.Next, adjust the vertical stage to reduce the number of fringes in the phase image so only one or two fringes remain. Let the system adjust after each stage movement. To find the best reconstruction distance, use the auto-focus option.Several refocus steps may be needed to get to the optimal reconstruction distance. Ultimately, the image should become sharp and clear. Fine focus adjustments can be made using the focus slider bar if the auto-focus is not providing the best result.For very fine focus adjustments, the Reconstruction Step can be changed in the settings. With the image prepared, enable the unwrapped mode to see the unwrapped phase image. In the 3D View software, open the 3D image window to see the final image of sample, and use the available image adjustments to observe the result.On the unwrapped phase image, select the ruler icon and draw a line on the area of interest to get a cross-sectional plot in the line plot window. Now, in the line plot window, use the two green line markers to get an approximate height of the object. To arrange the windows for simultaneous viewing, select the Tile Windows option.Surface roughness can also be calculated on the flat part of the sample using MATLAB software. Save the final phase image as a bitmap for further viewing in other software. To prepare the sample for dynamic analysis, position it on the sample stage for analysis.In this case, the sample is a micro diaphragm. Connect the sample electrodes to the crocodile clips of the generator. Following the described procedures, record a hologram of the micro diaphragm at the ambient temperature for reference.Click on the Delta icon to remove the initial phase, and thus only observe deformation. Then, turn on the DC generator and gradually increase the voltage from zero to 12 volts, taking phase map images at every 1 volt increment. Later, using a simple MATLAB code, plot the different phase map deformations into one graph to better observe the total deformation, and characterize the MEMS deformation to electrical loading.A CDHM system was used to characterize a MEMS device as described, using a monomode fiber coupled to a diode laser operating at a 633 nanometer wavelength. The object beam and reference beam path were matched to obtain a hologram image of the MEMS device. The yellow line represents the cross-section location on the sample, and the two green marker lines were used to estimate the sample height.To validate the results of the digital holographic system, an atomic force microscope was used to measure the same structure. A height difference of 2.1 nanometers was found between the atomic force microscope measurement and the CDHM measurement. A MEMS electrode made using a lift-off process is subject to sample morphology variance that needs quantification.Using the described protocol, a def map was made for this purpose. A plot in the other dimension shows that surface roughness of the electrode is also observable using the system. In a dynamic system, with the temperature rising from 50 to 300 degrees Celsius, morphological changes were measured in a micro diaphragm fabricated by bonding a thin-plate onto an SOI wafer sample.This thermal deformation was then summarized in a line plot showing a cross-sectional view of the different deformation states. After watching this video you should have a clear understanding of how to set up the system and carry out morphological studies related to reflective samples such as 3D profile, deformation maps, and surface roughness. Once trained, anyone can use the software and the system so that it can be easily implemented on production chains and operated by technicians.While attempting this procedure, it's important to remember to place the sample at the right distance from the system. This is an important step. And it has a real impact on the final results.After it's development, this technique b-v for researchers in the field of optical and electrical engineering to categorize the behavior of MEMS samples for static and dynamic conditions. Following this procedure, other experiments like observation of resonant mode while your playing high-frequency alternative current or quantifying cantilever deflection can be performed in order to provide a calibration of a packed column MEMS device.

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Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

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 ...

Real-Time DC-dynamic Biasing Method for Switching Time Improvement in Severely Underdamped Fringing-field Electrostatic MEMS Actuators

Mechanically underdamped electrostatic fringing-field MEMS actuators are well known for their fast switching operation in response to a unit step input bias voltage. However, the tradeoff for the improved switching performance is a relatively long settling time to reach each gap height in response to various applied voltages. Transient applied bias waveforms are employed to facilitate reduced switching times for electrostatic fringing-field MEMS actuators with high mechanical quality factors. Removing the underlying substrate of the fringing-field actuator creates the low mechanical damping environment necessary to effectively test the concept. The removal of the underlying substrate also a has substantial improvement on the reliability performance of the device in regards to failure due to stiction. Although DC-dynamic biasing is useful in improving settling time, the required slew rates for typical MEMS devices may place aggressive requirements on the charge pumps for fully-integrated on-chip designs. Additionally, there may be challenges integrating the substrate removal step into the back-end-of-line commercial CMOS processing steps. Experimental validation of fabricated actuators demonstrates an improvement of 50x in switching time when compared to conventional step biasing results. Compared to theoretical calculations, the experimental results are in good agreement.

The robust device design of fringing-field electrostatic MEMS actuators results in inherently low squeeze-film damping conditions and long settling times when performing switching operations using conventional step biasing. Real-time switching time improvement with DC-dynamic waveforms reduces the settling time of fringing-field MEMS actuators when transitioning between up-to-down and down-to-up states.

Mechanically underdamped electrostatic fringing-field MEMS actuators are well known for their fast switching operation in response to a unit step input ...

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.

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 ...

Comprehensive Characterization of Extended Defects in Semiconductor Materials by a Scanning Electron Microscope

Extended defects such as dislocations and grain boundaries have a strong influence on the performance of microelectronic devices and on other applications of semiconductor materials. However, it is still under debate how the defect structure determines the band structure, and therefore, the recombination behavior of electron-hole pairs responsible for the optical and electrical properties of the extended defects. The present paper is a survey of procedures for the spatially resolved investigation of structural and of physical properties of extended defects in semiconductor materials with a scanning electron microscope (SEM). Representative examples are given for crystalline silicon. The luminescence behavior of extended defects can be investigated by cathodoluminescence (CL) measurements. They are particularly valuable because spectrally and spatially resolved information can be obtained simultaneously. For silicon, with an indirect electronic band structure, CL measurements should be carried out at low temperatures down to 5 K due to the low fraction of radiative recombination processes in comparison to non-radiative transitions at room temperature. For the study of the electrical properties of extended defects, the electron beam induced current (EBIC) technique can be applied. The EBIC image reflects the local distribution of defects due to the increased charge-carrier recombination in their vicinity. The procedure for EBIC investigations is described for measurements at room temperature and at low temperatures. Internal strain fields arising from extended defects can be determined quantitatively by cross-correlation electron backscatter diffraction (ccEBSD). This method is challenging because of the necessary preparation of the sample surface and because of the quality of the diffraction patterns which are recorded during the mapping of the sample. The spatial resolution of the three experimental techniques is compared.

The optical, electrical, and structural properties of dislocations and of grain boundaries in semiconductor materials can be determined by experiments performed in a scanning electron microscope. Electron microscopy has been used to investigate cathodoluminescence, electron beam induced current, and diffraction of backscattered electrons.

Extended defects such as dislocations and grain boundaries have a strong influence on the performance of microelectronic devices and on other applications ...

Fabrication of 3D Carbon Microelectromechanical Systems (C-MEMS)

A wide range of carbon sources are available in nature, with a variety of micro-/nanostructure configurations. Here, a novel technique to fabricate long and hollow glassy carbon microfibers derived from human hairs is introduced. The long and hollow carbon structures were made by the pyrolysis of human hair at 900 &#176;C in a N2 atmosphere. The morphology and chemical composition of natural and pyrolyzed human hairs were investigated using scanning electron microscopy (SEM) and electron-dispersive X-ray spectroscopy (EDX), respectively, to estimate the physical and chemical changes due to pyrolysis. Raman spectroscopy was used to confirm the glassy nature of the carbon microstructures. Pyrolyzed hair carbon was introduced to modify screen-printed carbon electrodes ; the modified electrodes were then applied to the electrochemical sensing of dopamine and ascorbic acid. Sensing performance of the modified sensors was improved as compared to the unmodified sensors. To obtain the desired carbon structure design, carbon micro-/nanoelectromechanical system (C-MEMS/C-NEMS) technology was developed. The most common C-MEMS/C-NEMS fabrication process consists of two steps: (i) the patterning of a carbon-rich base material, such as a photosensitive polymer, using photolithography; and (ii) carbonization through the pyrolysis of the patterned polymer in an oxygen-free environment. The C-MEMS/NEMS process has been widely used to develop microelectronic devices for various applications, including in micro-batteries, supercapacitors, glucose sensors, gas sensors, fuel cells, and triboelectric nanogenerators. Here, recent developments of a high-aspect ratio solid and hollow carbon microstructures with SU8 photoresists are discussed. The structural shrinkage during pyrolysis was investigated using confocal microscopy and SEM. Raman spectroscopy was used to confirm the crystallinity of the structure, and the atomic percentage of the elements present in the material before and after pyrolysis was measured using EDX.

Fabrication of 3D Carbon Microelectromechanical Systems C-MEMS

Long and hollow glassy carbon microfibers were fabricated based on the pyrolysis of a natural product, human hair. The two fabrication steps of carbon microelectromechanical and carbon nanoelectromechanical systems, or C-MEMS and C-NEMS, are: (i) photolithography of a carbon-rich polymer precursor and (ii) pyrolysis of the patterned polymer precursor.

A wide range of carbon sources are available in nature, with a variety of micro-/nanostructure configurations. Here, a novel technique to fabricate long ...

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 ...

Design and Characterization Methodology for Efficient Wide Range Tunable MEMS Filters

Here, we demonstrate the advantages of the laser Doppler vibrometer (LDV) over conventional techniques (the network analyzer), as well as the techniques to create an application-based microelectromechanical systems (MEMS) filter and how to use it efficiently (i.e., tuning the tuning-capability and avoiding both failure and stiction). LDV enables crucial measurements that are impossible with the network analyzer, such as higher mode detection (highly sensitive biosensor application) and resonance measurement for very small devices (fast prototyping). Accordingly, LDV was used to characterize the frequency tuning range and resonance frequency at different modes of the MEMS filters built for this study. This wide range frequency tuning mechanism is based simply on Joule heating from the embedded heaters and relatively high thermal stress with respect to the temperature of a fixed-fixed beam. However, we demonstrate that another limitation of this method is the resulting high thermal stress, which can burn the devices. Further improvement was achieved and shown for the first time in this study, such that the tuning capability was increased by 32% through an increase in the applied DC bias voltage (25 V to 35 V) between the two adjacent beams. This important finding eliminates the need for the extra Joule heating at the wider frequency tuning range. Another possible failure is through stiction and requirement of structure optimization: We propose a simple and easy technique of low frequency square wave signal application that can successfully separate the beams and eliminates the need for the more sophisticated and complicated methods given in the literature. The above findings necessitate a design methodology, and so we also provide an application-based design.

A protocol for a fixed-fixed beam design using a laser Doppler vibrometer (LDV), including the measurement of frequency tuning, modification of tuning capability, and avoidance of device failure and stiction, is presented. The superiority of the LDV method over the network analyzer is demonstrated due to its higher mode capability.

Here, we demonstrate the advantages of the laser Doppler vibrometer (LDV) over conventional techniques (the network analyzer), as well as the techniques ...

Intermediate Strain Rate Material Characterization with Digital Image Correlation

The mechanical response of a material under dynamic load is typically different than its behavior under static conditions; therefore, the common quasistatic equipment and procedures used for material characterization are not applicable for materials under dynamic loads. The dynamic response of a material depends on its deformation rate and is broadly categorized into high (i.e., greater than 200/s), intermediate (i.e., 10&#8722;200/s) and low strain rate regimes (i.e., below 10/s). Each of these regimes calls for specific facilities and testing protocols to ensure the reliability of the acquired data. Due to the limited access to high-speed servo-hydraulic facilities and validated testing protocols, there is a noticeable gap in the results at the intermediate strain rate. The current manuscript presents a validated protocol for the characterization of different materials at these intermediate strain rates. Strain gauge instrumentation and digital image correlation protocols are also included as complimentary modules to extract the utmost level of detailed data from every single test. Examples of raw data, obtained from a variety of materials and test setups (e.g., tensile and shear) is presented and the analysis procedure used to process the output data is described. Finally, the challenges of dynamic characterization using the current protocol, along with the limitations of the facility and methods of overcoming potential problems are discussed.

Here we present a methodology for the dynamic characterization of tensile specimens at intermediate strain rates using a high-speed servo-hydraulic load frame. Procedures for strain gauge instrumentation and analysis, as well as for digital image correlation strain measurements on the specimens, are also defined.

The mechanical response of a material under dynamic load is typically different than its behavior under static conditions; therefore, the common ...

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...