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. 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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>
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			<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>
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			<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>
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			<td>Inverted microscope</td>
			<td>Olympus Corporation</td>
			<td>CKX 53</td>
			<td>Tokyo, Japan</td>
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			<td>Low temperature hardening epoxy resin adhesive</td>
			<td>ThreeBond Co., Ltd.</td>
			<td>TB2086M</td>
			<td>Tokyo, Japan</td>
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			<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>
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			<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>
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			<td>Three-terminal DC / DC regulator 3.3 V</td>
			<td>Marutsu Elec Co., Ltd.</td>
			<td>BR301</td>
			<td>Tokyo, Japan</td>
		</tr>
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			<td>Universal Flexible Arm</td>
			<td>Banggood Technology Co., Ltd.</td>
			<td>YP-003-2</td>
			<td>Hong Kong, China</td>
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			<td>USB cable USB-A - MicroUSB</td>
			<td>Akizuki Denshi Tsusho Co., Ltd.</td>
			<td>USB CABLE A-MICROB</td>
			<td>Tokyo, Japan</td>
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			<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.
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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.

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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

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) ...

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 ...

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

Radio Frequency Magnetron Sputtering of GdBa2Cu3O7ÃƒÂ¢Ã‹â€ 'ÃƒÅ½ ´/ 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 ...

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 ...

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 ...

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. ...