Name: fabrication of microscope stage for vertical observation with temperature control function
Uploaded: 2019-07-31T14:00:00
Description: Watch this Scientific Journal Video about Fabrication of Microscope Stage for Vertical Observation with Temperature Control Function at JoVE.com

The authors have no acknowledgements.

1. Design
<ol>
	<li>Fabrication of aluminum plates
	<ol>
		<li>Cut a 101 mm hole in the center of an aluminum plate of dimensions 150 mm x 200 mm x 2 mm to be used as the forefront plate with a laser processing machine. Machine claws at eight points to affix two rubber bands across the length, or two across the width of this plate (see Supplemental Figure 1A and Supplemental Figure 2A).</li>
		<li>Cut a 130 mm hole in the center of another 150 mm x 200 mm x 5 mm aluminum plate to be us...

<ol>
	<li>Drum, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Microscope+Observations+of+Diatos.">Electron Microscope Observations of Diatos.</a> Osterreichische Botanische Zeitschrift. 116, 321 (1969).</li><li>McBride, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+Random+Distributions+of+Datom+Values+on+Microscope+Slides.">Preparing Random Distributions of Datom Values on Microscope Slides.</a> Limnology and Oceangraphy. 33, 1627-1629 (1988).</li><li>Liu, X. Y., Lu, Z., Sun, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orientation+Control+of+Biological+Cells+Under+Inverted+Microscopy.">Orientation Control of Biological Cells Under Inverted Microscopy.</a> IEEE-ASME Transactions on Mechatronics. 16, 918-924 (2011).</li><li>Kahle, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+a+Compact,+Easy-to-Use+Inverted+Fluorescence+Microscope.">Applications of a Compact, Easy-to-Use Inverted Fluorescence Microscope.</a> American Laboratory. 43, 11-14 (2011).</li><li>Prunet, N., Jack, T. P., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+confocal+imaging+of+Arabidopsis+flower+buds.">Live confocal imaging of Arabidopsis flower buds.</a> Developmental Biology. , 114-120 (2016).</li><li>Nimchuk, Z. L., Perdue, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Shoot+Meristems+on+an+Inverted+Confocal+Microscope+Using+an+Objective+Lens+Inverter+Attachment.">Live Imaging of Shoot Meristems on an Inverted Confocal Microscope Using an Objective Lens Inverter Attachment.</a> Frontiers in Plant Science. 8, 10 (2017).</li><li>Hedde, P. N., Malacrida, L., Ahrar, S., Siryaporn, A., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=sideSPIM+-+selective+plane+illumination+based+on+a+conventional+inverted+microscope.">sideSPIM - selective plane illumination based on a conventional inverted microscope.</a> Biomedical Optics Express. 8, 3918-3937 (2017).</li><li>Crowe, W. E., Wills, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+Method+for+Monitoring+Changes+in+Cell+Height+using+Fluorescent+Microbeads+and+an+Ussing-type+Chamber+for+the+Inverted+Microscope.">A simple Method for Monitoring Changes in Cell Height using Fluorescent Microbeads and an Ussing-type Chamber for the Inverted Microscope.</a> Pflugers Archiv-Europian journal of Physiology. , 349-357 (1991).</li><li>Bavister, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Minichamber+Device+for+Maintaining+a+Constant+Carbon-Dioxide+in+Air+Atmosphere+during+Prolonged+Culture+of+Cells+on+the+Stage+of+an+Inverted+Microscope.">A Minichamber Device for Maintaining a Constant Carbon-Dioxide in Air Atmosphere during Prolonged Culture of Cells on the Stage of an Inverted Microscope.</a> In Vitro Cellular &amp; Developmental Biology. 24, 759-763 (1988).</li><li>Makler, 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+version+of+the+10-MU-M+Chamber+and+its+use+for+Semen+Analysis+with+Inverted+Microscope.">A New version of the 10-MU-M Chamber and its use for Semen Analysis with Inverted Microscope.</a> Archives of Andrology. 13, 195-197 (1984).</li><li>Xu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+microassembly+methods+for+micro/nanofluidic+chips+with+an+inverted+microscope.">Flexible microassembly methods for micro/nanofluidic chips with an inverted microscope.</a> Microelectronic Engineering. 97, 1-7 (2012).</li><li>Datyner, N. B., Gintant, G. A., Cohen, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+Temperature+Controlled+Tissue+Bath+for+Studies+of+Isolated+Cells+using+an+Inverted+Microscope.">Versatile Temperature Controlled Tissue Bath for Studies of Isolated Cells using an Inverted Microscope.</a> Pflugers Archive- Europian Journal of Physiology. 403, 318-323 (1985).</li><li>Claudet, C., Bednar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magneto-optical+tweezers+built+around+an+inverted+microscope.">Magneto-optical tweezers built around an inverted microscope.</a> Applied Optics. 44, 3454-3457 (2005).</li><li>Yamaoka, N., Suetomo, Y., Yoshihisa, T., Sonobe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motion+analysis+and+ultrastructural+study+of+a+colonial+diatom,+Bacillaria+paxillifer.">Motion analysis and ultrastructural study of a colonial diatom, Bacillaria paxillifer.</a> Microscopy. 65, 211-221 (2016).</li><li>Apoya-Horton, M. D., Yin, L., Underwood, G. J. C., Gretz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+modalities+and+responses+to+environmental+changes+of+the+mudflat+diatom+Cylindrotheca+closterium+(Bacillariophyceae).">Movement modalities and responses to environmental changes of the mudflat diatom Cylindrotheca closterium (Bacillariophyceae).</a> Journal of Phycology. 42, 379-390 (2006).</li><li>Bannon, C. C., Campbell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinking+towards+destiny:+High+throughput+measurement+of+phytoplankton+sinking+rates+through+time-resolved+fluorescence+plate+spectroscopy.">Sinking towards destiny: High throughput measurement of phytoplankton sinking rates through time-resolved fluorescence plate spectroscopy.</a> PLoS One. 12, 16 (2017).</li><li>Clarkson, N., Davies, M. S., Dixey, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diatom+motility+and+low+frequency+electromagnetic+fields+-+A+new+technique+in+the+search+for+independent+replication+of+results.">Diatom motility and low frequency electromagnetic fields - A new technique in the search for independent replication of results.</a> Bioelectromagnetics. 20, 94-100 (1999).</li><li>Iwasa, K., Shimizu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+of+Diatom,+Phaeodactylum-Tricornutum.">Motility of Diatom, Phaeodactylum-Tricornutum.</a> Experimental Cell Research. 74, (1972).</li><li>Edgar, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucilage+Secretions+of+Moving+Diatoms.">Mucilage Secretions of Moving Diatoms.</a> Protoplasma. 118, 44-48 (1983).</li><li>Edgar, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diatom+Locomotion.">Diatom Locomotion.</a> Computer-Assisted Analysis of Cine Film British Phycological Journal. 14, 83-101 (1979).</li><li>Iversen, M. H., Ploug, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effects+on+carbon-specific+respiration+rate+and+sinking+velocity+of+diatom+aggregates+-+potential+implications+for+deep+ocean+export+processes.">Temperature effects on carbon-specific respiration rate and sinking velocity of diatom aggregates - potential implications for deep ocean export processes.</a> Biogeosciences. 10, 4073-4085 (2013).</li><li>Riebesell, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Sinking+and+Sedimentation-Rate+Measurements+in+a+Diatom+Winter+Spring+Bloom.">Comparison of Sinking and Sedimentation-Rate Measurements in a Diatom Winter Spring Bloom.</a> Marine Ecology Progress Series. 54, 109-119 (1989).</li><li>Drum, R. W., Hopkins, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diatom+Locomotion+-+An+Explanation.">Diatom Locomotion - An Explanation.</a> Protoplasma. 62, (1966).</li></ol>

Trajectory analysis of moving diatom cells is a useful approach to evaluating diatom motility. However, while a normal inverted microscope observes samples horizontally, it is not suitable for observations of the influence of gravity or floating movement in the vertical direction. Developed and described here is a vertical microscope stage with temperature control and attached to an inverted microscope, which has been rotated by 90&#176;. This microscope stage with temperature control allows observation of temperature-de...

Optical microscopy is a technique employed to increase observable details via magnification of a sample with lenses and visible light. In optical microscopy, light is directed onto a sample, then transmitted, reflected, or fluorescent light is captured by magnifying lenses for observation. Various types of microscope are available that differ in design to accommodate different uses and observation methods. The different designs include an upright microscope, which is structured to illuminate a sample from below for observation from above, and an inverted microscope, which illuminates the sample from above for observation from below. Upright microscopes are the most common and widely used design. Inverted microscopes are often used to observe samples that cannot allow a lens close in distance from above, such as cultured cells adherent to the bottom of a container. Many research groups have reported observations in a wide range of fields using inverted microscopes1,2,3,4,5,6,7. Many additional devices have also been developed that take advantage of the features of inverted microscopes8,9,10,11,12,13.
Currently, in all conventional microscope designs, the microscope stage is horizontal and is therefore unsuitable for the observation of samples producing movement in the vertical plane, (due to gravity, buoyancy, motion, etc.). To make these observations possible, the microscope stage and light path must be rotated to vertical. The vertical stage is required to vertically mount glass slides or sample containers such as a Petri dishes to the stage. To address this, a sideways inverted microscope tilted by 90&#176; has already been devised. However, attaching samples with tape or other adhesives does not yield the necessary long-term immobility. Described here is a device that can achieve the necessary stability. This device permits observation over time of sample movement in the vertical plane. Mounting of a silicon rubber heater has also made it possible to observe the influence of temperature variation on sample behavior. Temperature data is transferred to an internet server by Wi-Fi, and temperature settings and log monitoring can be controlled remotely from a PC or smartphone. To our knowledge, the stage attached to a sideways tilted microscope tilted by 90&#176; has not yet been reported in previous studies.
The microscope stage is composed of three aluminum plates. The middle aluminum plate is mounted to the lower aluminum plate that attaches to the stage. The silicone rubber containing the temperature sensor is attached between the middle and upper aluminum plates. Rubber bands are used to affix the sample. Claws are attached in the left and right four points of the upper aluminum plate to secure the rubber bands. The control circuit of the temperature regulator receives a signal from the temperature sensor embedded in silicone rubber and modulates electric power by the pulse width modulation (PWM) method. The temperature can be gradually increased to 50 &#176;C in 1 &#176;C increments. This device is useful for applications in which vertical sample motions may be temperature-dependent.
This report provides examples of temperature effects on the floating phenomenon of diatoms. As examples of diatom observation studies, measurements of sedimentation velocity of cell clusters, motion analyses, ultrafine structure studies, etc. have been reported14,15,16,17,18,19,20,21,22,23. The specific gravity of diatoms floating in water with photosynthetic organisms is slightly higher than that of water, so they tend to sink; however, they will rise if even slight convection is occurring. To study this phenomenon, a glass slide is affixed vertically to a microscope stage, and the effects of increasing temperature on diatom vertical motion are observed.

<table>
	<tbody>
		<tr>
			<td>AC adapter 12V2A</td>
			<td>Akizuki Denshi Tsusho Co., Ltd.</td>
			<td>AD-D120P200</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>ADS1015 Substrate</td>
			<td>Akizuki Denshi Tsusho Co., Ltd.</td>
			<td>adafruit PRODUCT ID: 1083</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Alminium Plate (Back Side Plate)</td>
			<td>Inoval Co., Ltd.</td>
			<td>W 150mm&#215;L 200&#13212;&#215;T 1.5mm</td>
			<td>Gifu, Japan</td>
		</tr>
		<tr>
			<td>Alminium Plate (Forefront Plate)</td>
			<td>Inoval Co., Ltd.</td>
			<td>W 150mm&#215;L 200&#13212;&#215;T 2mm</td>
			<td>Gifu, Japan</td>
		</tr>
		<tr>
			<td>Alminium Plate (Middle Lower Plate)</td>
			<td>Inoval Co., Ltd.</td>
			<td>W 150mm&#215;L 200&#13212;&#215;T 4mm</td>
			<td>Gifu, Japan</td>
		</tr>
		<tr>
			<td>Alminium Plate (Middle Upper Plate)</td>
			<td>Inoval Co., Ltd.</td>
			<td>W 150mm&#215;L 200&#13212;&#215;T 5mm</td>
			<td>Gifu, Japan</td>
		</tr>
		<tr>
			<td>Aluminum Pedestal (Lower Plate)</td>
			<td>Inoval Co., Ltd.</td>
			<td>D 100mm&#215;T 3mm (30&#934;)</td>
			<td>Gifu, Japan</td>
		</tr>
		<tr>
			<td>Aluminum Pedestal (Upper Plate)</td>
			<td>Inoval Co., Ltd.</td>
			<td>D 100mm&#215;T 3mm (30&#934;)</td>
			<td>Gifu, Japan</td>
		</tr>
		<tr>
			<td>Bold Modified Basal Freshwater Nutrient Solution</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>B5282-500ML</td>
			<td>St. Louis, USA</td>
		</tr>
		<tr>
			<td>Controller Case</td>
			<td>Marutsu Elec Co., Ltd.</td>
			<td>pff-13-3-9</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>CPU</td>
			<td>Akizuki Denshi Tsusho Co., Ltd.</td>
			<td>ESP-WROOM-02D</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus Corporation</td>
			<td>CKX 53</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Low temperature hardening epoxy resin adhesive</td>
			<td>ThreeBond Co., Ltd.</td>
			<td>TB2086M</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Multi-turn semi-fixed volume Vertical type 500 &#937;</td>
			<td>Akizuki Denshi Tsusho Co., Ltd.</td>
			<td>3296W-1-501LF</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>OLED module</td>
			<td>Akihabara Inc.</td>
			<td>M096P4W</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Pressed Cork (For supporting electrode )</td>
			<td>Tera Co., Ltd.</td>
			<td>W 42mm&#215;L 30&#13212;</td>
			<td>Ishikawa, Japan</td>
		</tr>
		<tr>
			<td>Pressed Cork (Lower Disk)</td>
			<td>Tera Co., Ltd.</td>
			<td>D 100mm&#215;T 0.5mm (20&#934;)</td>
			<td>Ishikawa, Japan</td>
		</tr>
		<tr>
			<td>Pressed Cork (Upper Disk)</td>
			<td>Tera Co., Ltd.</td>
			<td>D 100mm&#215;T 2.5mm (20&#934;)</td>
			<td>Ishikawa, Japan</td>
		</tr>
		<tr>
			<td>Rotary encoder with switch with 2 color LED</td>
			<td>Akizuki Denshi Tsusho Co., Ltd.</td>
			<td>P-05772</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Silicone rubber heater</td>
			<td>Three High Co., Ltd.</td>
			<td>D 100mm&#215;T 2.5mm (20&#934;)</td>
			<td>Kanagawa, Japan</td>
		</tr>
		<tr>
			<td>Substrate</td>
			<td>Seeed Technology Co., Ltd.</td>
			<td>mh5.0</td>
			<td>Shenzhen, China</td>
		</tr>
		<tr>
			<td>Temperature sensor</td>
			<td>Akizuki Denshi Tsusho Co., Ltd.</td>
			<td>NXFT15XH103FA2B050</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Three-terminal DC / DC regulator 3.3 V</td>
			<td>Marutsu Elec Co., Ltd.</td>
			<td>BR301</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Universal Flexible Arm</td>
			<td>Banggood Technology Co., Ltd.</td>
			<td>YP-003-2</td>
			<td>Hong Kong, China</td>
		</tr>
		<tr>
			<td>USB cable USB-A - MicroUSB</td>
			<td>Akizuki Denshi Tsusho Co., Ltd.</td>
			<td>USB CABLE A-MICROB</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Video Canera</td>
			<td>Sony Corporation</td>
			<td>HDR-CX590</td>
			<td>Tokyo, Japan</td>
		</tr>
	</tbody>
</table>

fabrication of microscope stage for vertical observation with temperature control function

Samples are usually placed onto a horizontal microscope stage for microscopic observation. However, to observe the influence of gravity on a sample or study afloat behavior, it is necessary to make the microscope stage vertical. To accomplish this, a sideways inverted microscope tilted by 90&#176; has been devised. To observe samples with this microscope, sample containers such as Petri dishes or glass slides must be secured to the stage vertically. A device that can secure sample containers in place on a vertical microscope stage has been developed and is described here. Attachment of this device to the stage allows observation of sample dynamics in the vertical plane. The ability to regulate temperature using a silicone rubber heater also permits observation of temperature-dependent sample behaviors. Furthermore, the temperature data is transferred to an internet server. Temperature settings and log monitoring can be controlled remotely from a PC or smartphone.

Figure 2 shows the temperature distribution of the rubber heater. The surface temperature of the rubber heater was uniform at each temperature. Figure 3 shows the responsiveness of the measured temperature to set temperature changes. The orange line shows the set temperature and blue line shows the change of the sample temperature. The overshoot of the measured value to the setting change is small and the tracking is quick.
<...

Watch this Scientific Journal Video about Fabrication of Microscope Stage for Vertical Observation with Temperature Control Function at JoVE.com

Fabrication of Microscope Stage for Vertical Observation with Temperature Control Function

Presented here is a protocol using a temperature-controlled microscope stage that allows a sample container to be mounted on a vertical microscope.

Samples are usually placed onto a horizontal microscope stage for microscopic observation. However, to observe the influence of gravity on a sample or ...

This device can be used to fix a sample to a vertical microscope stage to allow observation of the influence of gravity and working on the sample of interest. Attachment of this device to the stage allows the observation of sample dynamics in the vertical frame and permits the observation of temperature-dependent sample behaviors. To fabricate the aluminum plates, use a laser processing machine to cut a 101-millimeter hole in the center of a 150 by 200 by two-millimeter aluminum plate to be used as the forefront plate.Make claws at eight points around the outside of the plate to allow two rubber bands to affixed across the length or the width of the plate. Next cut a 130-millimeter hole in the center of a 150 by 200 by five-millimeter aluminum plate to be used as the middle-upper plate, and make eight notches to allow the placement of two rubber bands across the length or width of the plate. Then cut a 130-millimeter hole in the center of a 150 by 200 by four-millimeter aluminum plate to be used as the middle-lower plate, and cut a 30-millimeter hole in the center of a 150 by 200 by 1.5-millimeter aluminum plate to be used as the base plate.To fabricate the pedestals, cut a 30-millimeter hole in the center of a 100-millimeter-diameter, three-millimeter-thick aluminum plate, and make a 42-millimeter-wide by 30-millimeter-deep notch on one side of the plate. Then cut a 30-millimeter hole in the center of a 100-millimeter-diameter, four-millimeter-thick aluminum plate, and drill three three-millimeter holes 25 millimeters from the center of the plate and spaced 120 degrees from each other. For fabrication of the pressed cork disks, use a water jet cutting machine to cut a 20-millimeter hole in the center of a 100-millimeter-diameter, two-millimeter-thick pressed cork disk, and make one 42-millimeter-wide by 30-millimeter-deep and one four-millimeter-wide by five-millimeter-deep notch on each side of the disk.Next cut a 20-millimeter hole in the center of a 100-millimeter-diameter, one-millimeter-thick pressed cork disk, and make one 42-millimeter-wide by 30-millimeter-deep and one four-millimeter-wide by 40-millimeter-deep notch on each side of the disk. Then cut a 42-millimeter-wide by 30-millimeter-deep pressed cork plate from a 100-millimeter-diameter disk. To fabricate a silicone rubber heater, cut a 20-millimeter hole in the center of a 100-millimeter-diameter, 2.5-millimeter-thick silicone rubber disk with a built-in nichrome wire.Then stack the fabricated parts as demonstrated, fixing the appropriate pieces with screws or adhesive as required. Use a dedicated cable to incorporate the rubber heater and the heater of the controller case to connect the microscope stage to the system, and use the controller to equip a Wi-Fi signal and control the current of the rubber heater. After building the system, connect the thermistor wire to the sensor terminal on the controller case, and receive the temperature signal measured by the thermistor.Use the knob on the controller to change the set temperature. Then transfer the measured temperature, set temperature, and time information at measurement from the controller to the server via the internet. To analyze the sample, place the sample on the microscope stage perpendicular to the ground surface, and use the four lengthwise claws to secure the sample with two rubber bands.Use the controller to set the temperature to 40 degrees Celsius, and check the temperature on the display. Then press the knob to start temperature control. The blue LED will light up, indicating the initiation of the heat supply.In these figures, representative temperature distributions of the rubber heater are shown. The surface temperature of the rubber heater was uniform at each temperature. Here, an example of the responsiveness of the measured temperature to set the temperature changes is shown.The orange line indicates the set temperature, and the blue line shows the change of the sample temperature. If the equipment is assembled correctly, the overshoot of the measured value to the setting change is typically small, and the tracking is quick. In this experiment, the temperature-dependent vertical motion of diatoms was successfully recorded, allowing the locus of the vertical motion of the diatoms to be detected.The effects of thermal convection on the vertical floating phenomenon of the diatom cells could then be visualized by direct observation. When the sensor is disconnected from the sample, or if the microcontroller does not operate properly, check whether the current to the heater has been cut off from the microcontroller. Using this method, the effect of temperature changes on the vertical movement of an organism in water can be observed.As the structure of the part of the stage that attaches to the microscope is complicated, future studies will address simplification of that structure. To cool samples to lower than room temperature requires a complicated cooling device which is also under consideration for future work.

fabrication-microscope-stage-for-vertical-observation-with

Research

JoVE Journal

Engineering

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 ...

Fabrication of Gate-tunable Graphene Devices for Scanning Tunneling Microscopy Studies with Coulomb Impurities

Owing to its relativistic low-energy charge carriers, the interaction between graphene and various impurities leads to a wealth of new physics and degrees of freedom to control electronic devices. In particular, the behavior of graphene&#8217;s charge carriers in response to potentials from charged Coulomb impurities is predicted to differ significantly from that of most materials. Scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) can provide detailed information on both the spatial and energy dependence of graphene&#39;s electronic structure in the presence of a charged impurity.&#160;The design of a hybrid impurity-graphene device, fabricated using controlled deposition of impurities onto a back-gated graphene surface, has enabled several novel methods for controllably tuning graphene&#8217;s electronic properties.1-8 Electrostatic gating enables control of the charge carrier density in graphene and the ability to reversibly tune the charge2 and/or molecular5 states of an impurity. This paper outlines the process of fabricating a gate-tunable graphene device decorated with individual Coulomb impurities for combined STM/STS studies.2-5 These studies provide valuable insights into the underlying physics, as well as signposts for designing hybrid graphene devices.

This paper details the fabrication process of a gate-tunable graphene device, decorated with Coulomb impurities for scanning tunneling microscopy studies. Mapping the spatially dependent electronic structure of graphene in the presence of charged impurities unveils the unique behavior of its relativistic charge carriers in response to a local Coulomb potential.

Owing to its relativistic low-energy charge carriers, the interaction between graphene and various impurities leads to a wealth of new physics and degrees ...

Fabrication of Low Temperature Carbon Nanotube Vertical Interconnects Compatible with Semiconductor Technology

We demonstrate a method for the low temperature growth (350 &#176;C) of vertically-aligned carbon nanotubes (CNT) bundles on electrically conductive thin-films. Due to the low growth temperature, the process allows integration with modern low-&#954; dielectrics and some flexible substrates. The process is compatible with standard semiconductor fabrication, and a method for the fabrication of electrical 4-point probe test structures for vertical interconnect test structures is presented. Using scanning electron microscopy the morphology of the CNT bundles is investigated, which demonstrates vertical alignment of the CNT and can be used to tune the CNT growth time. With Raman spectroscopy the crystallinity of the CNT is investigated. It was found that the CNT have many defects, due to the low growth temperature. The electrical current-voltage measurements of the test vertical interconnects displays a linear response, indicating good ohmic contact was achieved between the CNT bundle and the top and bottom metal electrodes. The obtained resistivities of the CNT bundle are among the average values in the literature, while a record-low CNT growth temperature was used.

A method for the growth of low temperature vertically-aligned carbon nanotubes, and the subsequent fabrication of vertical interconnect electrical test structures using semiconductor fabrication is presented.

We demonstrate a method for the low temperature growth (350 &#176;C) of vertically-aligned carbon nanotubes (CNT) bundles on electrically conductive ...

A Fabrication Method for Highly Stretchable Conductors with Silver Nanowires

Stretchable electronics are identified as a key technology for electronic applications in the next generation. One of the challenges in fabrication of stretchable electronic devices is the preparation of stretchable conductors with great mechanical stability. In this study, we developed a simple fabrication method to chemically solder the contact points between silver nanowire (AgNW) networks. AgNW nanomesh was first deposited on a glass slide via spray coating method. A reactive ink composed of silver nanoparticle (AgNPs) precursors was applied over the spray coated AgNW thin films. After heating for 40 min, AgNPs were preferentially generated over the nanowire junctions to solder the AgNW nanomesh, and reinforced the conducting network. The chemically modified AgNW thin film was then transferred to polyurethane (PU) substrates by casting method. The soldered AgNW thin films on PU exhibited no obvious change in electrical conductivity under stretching or rolling process with elongation strains up to 120%.

A simple synthesis method is used to chemically solder silver nanowire thin film to fabricate highly stretchable and conductive metal conductors.

Stretchable electronics are identified as a key technology for electronic applications in the next generation. One of the challenges in fabrication of ...

Free-form Light Actuators &#8212; Fabrication and Control of Actuation in Microscopic Scale

Liquid crystalline elastomers (LCEs) are smart materials capable of reversible shape-change in response to external stimuli, and have attracted researchers&#39; attention in many fields. Most of the studies focused on macroscopic LCE structures (films, fibers) and their miniaturization is still in its infancy. Recently developed lithography techniques, e.g., mask exposure and replica molding, only allow for creating 2D structures on LCE thin films. Direct laser writing (DLW) opens access to truly 3D fabrication in the microscopic scale. However, controlling the actuation topology and dynamics at the same length scale remains a challenge.
In this paper we report on a method to control the liquid crystal (LC) molecular alignment in the LCE microstructures of arbitrary three-dimensional shape. This was made possible by a combination of direct laser writing for both the LCE structures as well as for micrograting patterns inducing local LC alignment. Several types of grating patterns were used to introduce different LC alignments, which can be subsequently patterned into the LCE structures. This protocol allows one to obtain LCE microstructures with engineered alignments able to perform multiple opto-mechanical actuation, thus being capable of multiple functionalities. Applications can be foreseen in the fields of tunable photonics, micro-robotics, lab-on-chip technology and others.

Free-form Light Actuators &amp;#8212; Fabrication and Control of Actuation in Microscopic Scale

Here, we fabricate 3D polymeric micro/nano structures in which both the shape and the molecular alignment can be engineered with nanometer scale accuracy by the use of direct laser writing. Light induced deformation of several types of liquid crystalline elastomer microstructures can be controlled in the microscopic scale.

Liquid crystalline elastomers (LCEs) are smart materials capable of reversible shape-change in response to external stimuli, and have attracted ...

High Temperature Fabrication of Nanostructured Yttria-Stabilized-Zirconia (YSZ) Scaffolds by In Situ Carbon Templating Xerogels

We demonstrate a method for the high temperature fabrication of porous, nanostructured yttria-stabilized-zirconia (YSZ, 8 mol% yttria - 92 mol% zirconia) scaffolds with tunable specific surface areas up to 80 m2&#183;g-1. An aqueous solution of a zirconium salt, yttrium salt, and glucose is mixed with propylene oxide (PO) to form a gel. The gel is dried under ambient conditions to form a xerogel. The xerogel is pressed into pellets and then sintered in an argon atmosphere. During sintering, a YSZ ceramic phase forms and the organic components decompose, leaving behind amorphous carbon. The carbon formed in situ serves as a hard template, preserving a high surface area YSZ nanomorphology at sintering temperature. The carbon is subsequently removed by oxidation in air at low temperature, resulting in a porous, nanostructured YSZ scaffold. The concentration of the carbon template and the final scaffold surface area can be systematically tuned by varying the glucose concentration in the gel synthesis. The carbon template concentration was quantified using thermogravimetric analysis (TGA), the surface area and pore size distribution was determined by physical adsorption measurements, and the morphology was characterized using scanning electron microscopy (SEM). Phase purity and crystallite size was determined using X-ray diffraction (XRD). This fabrication approach provides a novel, flexible platform for realizing unprecedented scaffold surface areas and nanomorphologies for ceramic-based electrochemical energy conversion applications, e.g. solid oxide fuel cell (SOFC) electrodes.

High Temperature Fabrication of Nanostructured Yttria-Stabilized-Zirconia YSZ Scaffolds by In Situ Carbon Templating Xerogels

A protocol for fabricating porous, nanostructured yttria-stabilized-zirconia (YSZ) scaffolds at temperatures between 1,000 &#176;C and 1,400 &#176;C is presented.

We demonstrate a method for the high temperature fabrication of porous, nanostructured yttria-stabilized-zirconia (YSZ, 8 mol% yttria - 92 mol% zirconia) ...

Preparation of Large-area Vertical 2D Crystal Hetero-structures Through the Sulfurization of Transition Metal Films for Device Fabrication

We have demonstrated that through the sulfurization of transition metal films such as molybdenum (Mo) and tungsten (W), large-area and uniform transition metal dichalcogenides (TMDs) MoS2 and WS2 can be prepared on sapphire substrates. By controlling the metal film thicknesses, good layer number controllability, down to a single layer of TMDs, can be obtained using this growth technique. Based on the results obtained from the Mo film sulfurized under the sulfur deficient condition, there are two mechanisms of (a) planar MoS2 growth and (b) Mo oxide segregation observed during the sulfurization procedure. When the background sulfur is sufficient, planar TMD growth is the dominant growth mechanism, which will result in a uniform MoS2 film after the sulfurization procedure. If the background sulfur is deficient, Mo oxide segregation will be the dominant growth mechanism at the initial stage of the sulfurization procedure. In this case, the sample with Mo oxide clusters covered with few-layer MoS2 will be obtained. After sequential Mo deposition/sulfurization and W deposition/sulfurization procedures, vertical WS2/MoS2 hetero-structures are established using this growth technique. Raman peaks corresponding to WS2 and MoS2, respectively, and the identical layer number of the hetero-structure with the summation of individual 2D materials have confirmed the successful establishment of the vertical 2D crystal hetero-structure. After transferring the WS2/MoS2 film onto a SiO2/Si substrate with pre-patterned source/drain electrodes, a bottom-gate transistor is fabricated. Compared with the transistor with only MoS2 channels, the higher drain currents of the device with the WS2/MoS2 hetero-structure have exhibited that with the introduction of 2D crystal hetero-structures, superior device performance can be obtained. The results have revealed the potential of this growth technique for the practical application of 2D crystals.

Through the sulfurization of pre-deposited transition metals, large-area and vertical 2D crystal hetero-structures can be fabricated. The film transferring and device fabrication procedures are also demonstrated in this report.

We have demonstrated that through the sulfurization of transition metal films such as molybdenum (Mo) and tungsten (W), large-area and uniform transition ...

Radio Frequency Magnetron Sputtering of GdBa2Cu3O7&#8722;&#948;/ La0.67Sr0.33MnO3 Quasi-bilayer Films on SrTiO3 (STO) Single-crystal Substrates

Here, we demonstrate a method of coating ferromagnetic La0.67Sr0.33MnO3 (LSMO) nanoparticles on (001) SrTiO3 (STO) single-crystal substrates by radio frequency (RF) magnetron sputtering. LSMO nanoparticles were deposited with diameters from 10 to 20 nm and heights between 20 and 50 nm. At the same time, (Gd) Ba2Cu3O7&#8722;&#948; ((Gd) BCO) films were fabricated on both undecorated and LSMO nanoparticle decorated STO substrates using RF magnetron sputtering. This report also describes the properties of GdBa2Cu3O7&#8722;&#948;/ La0.67Sr0.33MnO3 quasi-bilayer films structures (e.g., crystalline phase, morphology, chemical composition); magnetization, magneto-transport, and superconducting transport properties were also evaluated.

Radio Frequency Magnetron Sputtering of GdBa2Cu3O7ÃƒÂ¢Ã‹â€ &#039;ÃƒÅ½ ´/ La0.67Sr0.33MnO3 Quasi-bilayer Films on SrTiO3 STO Single-crystal Substrates

Here, we present a protocol to grow LSMO nanoparticles and (Gd) BCO films on (001) SrTiO3 (STO) single-crystal substrates by radio frequency (RF)-sputtering.

Here, we demonstrate a method of coating ferromagnetic La0.67Sr0.33MnO3 (LSMO) nanoparticles on (001) SrTiO3 (STO) single-crystal substrates by radio ...

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 ...

Fabrication of Soft Pneumatic Network Actuators with Oblique Chambers

Soft pneumatic network actuators have become one of the most promising actuation devices in soft robotics which benefits from their large bending deformation and low input. However, their monotonous bending motion form in two-dimensional (2-D) space keeps them away from wide applications. This paper presents a detailed fabrication method of soft pneumatic network actuators with oblique chambers, to explore their motions in three-dimensional (3-D) space. The design of oblique chambers enables actuators with tunable coupled bending and twisting capabilities, which gives them the possibility to move dexterously in flexible manipulators, to become biologically inspired robots and medical devices. The fabrication process is based on the molding method, including the silicone elastomer preparation, chamber and base parts fabrication, actuator assembly, tubing connections, checks for leaks, and actuator repair. The fabrication method guarantees the rapid manufacturing of a series of actuators with only a few modifications in the molds. The test results show the high quality of the actuators and their prominent bending and twisting capabilities. Experiments of the gripper demonstrate the advantages of the development in adapting to objects with different diameters and providing sufficient friction.

Here we present a fabrication method of soft pneumatic network actuators with oblique chambers. The actuators are capable of generating coupled bending and twisting motions, which broadens their application in soft robotics.