Fabrication of Microscope Stage for Vertical Observation with Temperature Control Function

Summary

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

Abstract

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° 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.

Introduction

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 microscopes^{1,2,3,4,5,6,7}. Many additional devices have also been developed that take advantage of the features of inverted microscopes^{8,9,10,11,12,13}.

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° 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° 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 °C in 1 °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 reported^{14,15,16,17,18,19,20,21,22,23}. 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.

Protocol

1. Design

1. Fabrication of aluminum plates
   1. 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).
   2. Cut a 130 mm hole in the center of another 150 mm x 200 mm x 5 mm aluminum plate to be used as the middle upper plate with a laser processing machine. Machine eight notches for attaching rubber bands at two points across the length, or two across the width of this plate (see Supplemental Figure 1B and Supplemental Figure 2B).
   3. Cut a 130 mm hole in the center of a 150 mm x 200 mm x 4 mm aluminum plate to be used as the middle lower plate with a laser processing machine (see Supplemental Figure 1C and Supplemental Figure 2C).
   4. Cut a 30 mm hole in the center of a 150 mm x 200 mm x 1.5 mm aluminum plate to be used as the base plate (see Supplemental Figure 1D and Supplemental Figure 2D).
2. Fabrication of two aluminum pedestal
   1. Cut a 30 mm hole in the center of the aluminum plate (100 mm diameter, 3 mm thickness) and make a notch from one side with the dimensions 42 mm wide x 30 mm deep (see Supplemental Figure 3A).
   2. Cut a 30 mm hole in the center of the plate in an aluminum plate (100 mm diameter, 4 mm thickness) and drill three 3 mm holes located 25 mm from the center, spaced 120° from each other (see Supplemental Figure 3B).
3. Fabrication of three pressed cork disc
   1. Cut a 20 mm hole in the center of the pressed cork disc (100 mm diameter, 2 mm thickness) with a water jet cutting machine. Make one cut 42 mm across x 30 mm deep, then one cut 4 mm wide x 5 mm deep (see Supplemental Figure 4A).
   2. Cut a 20 mm hole in the center of the pressed cork disc of dimensions 100 mm diameter, 1 mm thickness with a water jet cutting machine. Make a cut 42 mm across x 30 mm deep, a cut 4 mm wide x 40 mm deep (see Supplemental Figure 4B).
   3. Cut a pressed cork plate from a 100 mm diameter disc with a 42 mm width and 30 mm depth. Two sheets of 1 mm thickness and one sheet of 2 mm thickness are required (see Supplemental Figure 4C).
4. Fabrication of silicone rubber heater
   1. Fabricate a heater using a 100 mm diameter disc of 2.5 mm thick silicon rubber with built-in Nichrome wire and cut a 20 mm hole in the center of the disc (see Supplemental Figure 5).
5. Assemble parts described in steps 1.1–1.4 by stacking them as shown in Supplemental Figure 6.
6. To construct a microscope stage, refer to Supplemental Figure 6, cross-section of the microscope stage. Fix and , then and with screws. Fix and with screws. Fix and , and , and , and , and and with adhesive.

2. Hardware design outlines

1. Prepare a “power supply and programming circuit” as shown in Supplemental Figure 7. Supply 12 V DC to the heater controller from the J4 terminal connected to the AC adapter. Decrease the voltage from 12 V DC to 3.3 V DC for the circuit power supply using a regulator because the CPU supply voltage is 3.3 V DC.
   NOTE: USB 1 is a terminal for 5 V DC and serial signal of development PC. Although 5 V DC is not essential, it is used as the power source to program the CPU. This is also converted to 3.3 V DC by the regulator. J1 is a control signal terminal at the time of programming. This circuit is housed in the controller case shown in Supplemental Figure 8.
2. Prepare a “heater control circuit” as shown in Supplementary Figure 7. Switch to 12 V DC with Q5 (P channel MOS FET) and supply it to the heater. Q5 is a switching element that controls 12 V DC with PWM to adjust the amount of power supplied to the heater.
   NOTE: The circuit includes an LED to visually confirm that voltage is being supplied to the heater. This drive signal (HEATER_C) is a PWM signal from the CPU. When an overheat signal is detected by the protection circuit, the BREAKER signal switches to LOW, and the operation of the MOS-FET stops. This circuit is housed in the controller case shown in Supplemental Figure 8.
3. Prepare a “connector circuit for heater unit” as shown in Supplementary Figure 7. Install a USB connector for connection with the heater section.
   NOTE: This circuit is housed in the controller case shown in Supplemental Figure 8.
4. Prepare a “connector circuit for temperature sensor” as shown in Supplemental Figure 7. Mount the connector (Euroblock receptacle 2P) to connect the temperature sensor.
   NOTE: This circuit is housed in the controller case shown in Supplemental Figure 8.
5. For a “A/D converter” as shown in Supplemental Figure 7, use ADS 1015 as an AD conversion device.
   NOTE: AD conversion device converts the values of the temperature sensor and overheat detection sensor from voltage to digital values. This is a 12-bit multiplexer AD conversion device and is connected to the CPU with the I2C interface. This circuit is housed in the controller case shown in Supplemental Figure 8.
6. Make a “protect circuit” as shown in Supplemental Figure 7 by connecting the overheat detection sensor (OHS) signal to the inverting input of the OP amp. Compare this signal with the voltage of the trimmer resistor connected to the noninverting input.
   1. Ensure that when the voltage becomes lower than the voltage of the trimmer resistor, the output of the OP amplifier goes HIGH, the connected NPN transistor Q2 turns ON and the BREAKER signal goes LOW.
   2. Ensure that at the same time, Q4 turns ON and the connected overheat indicator LED D6 lights up.
      NOTE: This circuit is housed in the controller case shown in Supplemental Figure 8.
7. For a “display section” as shown in Supplemental Figure 7, use 192 x 64 dots for OLED. Connect with the CPU via the I2C interface.
   1. Reset the OLED by separating the GND of the OLED by the CPU signal IO0 using an NPN transistor Q1 connected to the GND of the OLED.
      NOTE: This OLED displays various types of information. This circuit is housed in the controller case shown in Supplemental Figure 8.
8. For a “LED & rotary encoder with push switch” in Supplemental Figure 7, mount a rotary encoder by solder that functions as a push switch and incorporates two LEDs.
   1. Connect one LED to VCC for use as a power LED. The other is connected to the CPU for use as an indicator during heater operation.
   2. Use a push switch contact for heater START/STOP that is connected to CPU. Connect the A and B outputs of the rotary encoder to the IO input set in the CPU interrupt.
      NOTE: This circuit is housed in the controller case shown in Supplemental Figure 8.
9. For the CPU in Supplemental Figure 7, use the CPU of WROOM - 02D.
   1. Output from IO12, IO13 to the “display unit” because the interface of the display is I2C standard. Connect IO0 to "display unit" and reset the OLED.
   2. Connect IO15 to "heater control unit" and control the power supplied to the heater by PWM output.
   3. Connect IO2 to "LED & rotary encoder with push switch" and light the START LED. Connect IO4 and IO14 to "LED & rotary encoder with push switch" and receive the signals (REA and REB) from the rotary encoder to determine the set temperature. Connect IO5 to "LED & rotary encoder with push switch" and start/stop the heater.

3. Software design outline

1. Use Arduino CORE for WROOM - 02D for the CPU as the controller for this system.
   NOTE: As input devices, the start/stop switch, rotary encoder, temperature sensor (thermistor) are used. As output devices, an LED, character display (OLED), and heater are used. The communication device uses Wi-Fi.
2. Outline of the operation
   1. Detect the operation of the rotary encoder as shown in the LED & rotary encoder with the push switch in Supplemental Figure 7, store it as the set temperature, and display it on the OLED. Set the input terminal of the CPU to which the phase terminals REA and REB are connected as an interrupt input terminal and process the rotation (forward and reverse) of the rotary encoder by interrupt. Set it to +1 for forward rotation and -1 for reverse rotation. Write the set temperature to the global variable and use it for the heater temperature control. At the same time, update the set temperature display of the OLED.
   2. Identify start and stop by CPU IO 5 by start/stop switch (SW-S) as shown in the CPU of Supplemental Figure 7. The state of the start/stop switch is a timer interrupt process every 50 ms.
      NOTE: Since the switch is a momentary switch, it reverses the state of start/stop when it is pushed and released. This state is stored in the global variable.
   3. Use a thermistor for the temperature sensor. Read the measured values from the sample sensor (refer to “connector circuit for heater connection” in Supplemental Figure 7) into the CPU after A/D converter (refer to “A/D converter” in Supplemental Figure 7). Supply the current to the heater by turning on the IO15 port in the “CPU” of Supplemental Figure 7.
      NOTE: There are two types of temperature sensors. One is used to measure the temperature of the sample and control the heater on the set temperature, and the other is attached to a heater and used for heat prevention. Connect the thermistor to 3.3 V via a resistor and record the change in resistance as a change in voltage. Remove a noise by the moving average method.
   4. Use a thermistor for temperature prevention temperature sensor. Overheat detection is performed using a thermistor (R2) (“connector circuit for heater connection” in Supplemental Figure 7), and when the set value is exceeded, the heater current is shut off (“protect circuit” in Supplemental Figure 7).
      NOTE: This sensor is incorporated into a circuit and not through the CPU. This sensor is independent from the CPU and compared with the resistance value set by the resistor trimmer by a differential amplifier in an analog manner. When it detects that the temperature has exceeded the set value, it intervenes in the FET switch, which controls the current to the heater and forcibly stops the current supply. The purpose is to prevent the temperature of the heater from exceeding a certain level even in a situation where the CPU does not work properly.
   5. Turn on the LED in the “LED & rotary encoder with push switch” of the Supplemental Figure 7 by the CPU (in the “CPU” of Supplemental Figure 7), when the equipment is in operation.
   6. Display the set temperature and measured value to OLED in the "display section" of the Supplemental Figure 7 by the CPU (in the "CPU "of Supplemental Figure 7).
   7. Drive the FET switch in the "heater control circuit" of Supplemental Figure 7 with PWM from CPU to control the heater.
   8. Control the heater by PID, based on measured temperatures acquired by the temperature sensor. Use Arduino's pid_v1.h library for PID processing.
      NOTE: The CPU calibrates the time, communicates with the server, transmits data, and receives instructions from the server. When the sensor temperature exceeds the set temperature, the current to the heater is set to 0, and overshoot is suppressed.
   9. Use the built-in Wi-Fi connection function of the CPU and connect to the Internet. Transmit set the temperature, heater temperature, etc. to the designated server by Wi-Fi.

4. System configuration

1. Build the system according to Supplemental Figure 9.
2. Equip a Wi-Fi with the controller.
3. Use a thermistor as a sensor for temperature measurement. Connect the thermistor wire to the "SENSOR" terminal on the controller case. Receive the temperature signal measured by the thermistor.
4. Connect a microscope stage incorporating the rubber heater and the "HEATER" of the controller case with a dedicated cable. Control the current to the rubber heater.
5. Change the set temperature with the knob on the controller.
   NOTE: Temperature log monitoring, temperature setting can be operated remotely from a PC or smartphone.
6. Transfer the measured temperature, set temperature, and time information at measurement from the controller to the server via the internet. The data measurement cycle time is 5 s and cycle time for data transfer to the server is 1 min.
7. Access the server from the controller side at regular intervals and transfer the measurement data stored in the CPU of the controller to the server for analysis and graphing.
8. Refer to the supplementary material for how to operate the server.

5. Design of the sideways inverted microscope

1. Fix two aluminum plates of 15 mm in thickness vertically with screws to create a basic mount.
2. Attach a jig (one place) to the horizontal part of the base mount.
3. Place the microscope stage part vertically, attach the jigs (two places) to the vertical part of the base stand and fix the bottom of the microscope to the base stand.
4. Fix the microscope stage with screws.

6. Method of operation

NOTE: Here, the sample used is a mixture of Bold Modified Basal Freshwater Nutrient Solution liquid culture medium, sodium metasilicate, vitamins, and sterile water. 800 μL of this sample is diluted in 10 mL of fresh water medium.

1. Observation method
   1. Inject 1,000 μL of the prepared sample into a self-made glass chamber.
      NOTE: The self-made glass chamber arranges two slide glasses in parallel and fixes them with an adhesive. A normal Petri dish has a large thickness and cells escape in the depth direction in the chamber, making it difficult to observe with a microscope. To prevent this, the chamber with a small depth direction is made, which makes it possible to prevent the cells from escaping in the depth direction in the chamber. A low temperature curable epoxy resin adhesive is used to bond around the glass to prevent the sample dropping from the chamber.
   2. Attach a separately prepared video camera to the microscope. Connect a video camera using the microscope's dedicated lens adapter and shoot the sample.
   3. Use a microscope with a 10x eyepiece and 200x objective.
   4. Attach the vertical microscope stage to a microscope in four locations with 4 mm screws.
      NOTE: Refer to Supplemental Figure 1A and Supplemental Figure 2A for design drawings of aluminum plates. In this experiment, an inverted microscope was used. This was tilted by 90°, and the fabricated microscope stage was affixed with screws. Refer to Figure 1.
   5. Secure the sample with two rubber bands using the four claws made lengthwise. Place a sample on a microscope stage perpendicular to the ground surface.
   6. Set the temperature to 40 °C with the controller shown in Supplemental Figure 8. Turn the controller knob to set the temperature. Check the set temperature on the display. Press the knob to start temperature control, and the blue LED will light up. Press the knob again to turn off the LED and stop temperature control.
      NOTE: The measured temperature is displayed in real-time, and the heater is controlled to reach the set temperature. When the temperature control starts, the blue LED lights up and remains so while the heater is in operation. When the heater overheats, the red LED lights, and the heater automatically stops.
   7. Refer to "The Server Operation Manual" in the supplementary information for server operation.
      NOTE: A server for data storage is required. The server's database uses My-SQL.

7. Measurement of surface temperature distribution of rubber heater

1. Measure the distribution of the rubber heater surface temperature by thermography to check the temperature uniformity.
2. Attach the microscope stage which incorporated a rubber heater with a stand.
3. Change the setting temperature of the rubber heater surface to 35 °C, 45 °C, 55 °C, and 65 °C, and measure by the thermography from the front (refer to Supplemental Figure 10).

8. Temperature response test

1. Start temperature control by setting the sample set temperature to 30 °C. Wait until the measurement value reaches 30 °C and stabilizes. Increase the preset temperature stepwise by 5 °C from 30 °C to 50 °C and wait until the measured value stabilizes following the respective preset temperature.
2. Decrease the preset temperature stepwise by 5 °C from 50 °C to 30 °C and detect the tracking ability of the measured value.

Results

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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Discussion

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°. This microscope stage with temperature control allows observation of temperature-de...

Materials

List of materials used in this article

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