An Inexpensive, 3D-Printable, Arduino- and Blu-Ray-Based Confocal Laser and Fluorescent Scanning Microscope

Abstract

There is a growing field that is devoted to developing inexpensive microscopes and measurement devices by leveraging low-cost commercial parts that can be controlled using smartphones or embedded devices, such as Arduino and Raspbery Pi. Examples include the use of Blu-ray optical heads like the PHR-803T to perform cytometry, spinning disc microscopy, and lensless holographic microscopy. The modular or disposable nature of these devices means that they can also be used in contaminating and degrading environments, including radioactive environments, where replacement of device elements can be expensive. This paper presents the development and operation of a confocal microscope that uses the PHR-803T optical device in a Blu-ray reader for both imaging and detection of temperature variations with between 1.5 and 15 µm resolution. The benefits of using a PHR-803T confocal system include its relatively inexpensive design and the accessibility of the components that are used in its construction. The design of this scanning confocal thermal microscope (SCoT) was optimized based on cost, modularity, portability, spatial resolution, and ease of manufacturability using common tools (e.g., drill press, 3D printer). This paper demonstrated the ability to resolve microscale features such as synthetic spider silk and measure thermal waves in stainless steel using a system requiring <USD 1000 in material costs.

1. Introduction

In the study of nuclear materials, post-irradiation examination (PIE) is a critical part of understanding the impact that radiation damage has had on the material performance [1]. The process is broken up into different stages (Figure 1) based on the size and radioactivity of the sample [2]. The materials of interest are pulled from the reactor core and allowed to cool in a pool, after which they are moved into a hot cell, where handling of materials can only be accomplished with “requester–responder” manipulators [3]. The sample preparation process begins with dismantling the assembly and ends with samples being cut into the appropriately sized specimens for further analysis [2] within a hot cell. The current paper presents the development of an inexpensive microscope (the scanning confocal thermal (SCoT) microscope) that has the potential to be used in these post-irradiation examination (PIE) processes.

Prior to more expensive testing, which often involves electron or X-ray microscopy, metallography or ceramography is performed within the hot cell using optical microscopes to understand the macroscopic nature of the material [4]. This allows the 2D morphology of the material to be measured at the $\mathsf{\mu }$m to mm scale, with a focus on the detection of grains, porosity, bubbles, and cracking [5]. Recent developments in thermal characterization microscopy [6] have also allowed measurements of the spatial distribution of the material’s thermal properties to be taken [7]. Confocal microscopy, where either reflected or emitted light is collected from a single optical plane of the sample, has been used to recreate the fracture behavior of ferritic/martensitic steel by measuring the surface topology of the fracture surface [8]. It has also been used to understand the erosion of plasma-facing components in fusion tokamaks [9] by again measuring the topography of the irradiated sample. A microscope that can observe both thermal and topological changes [10] within the material would be a valuable tool.

Confocal microscopy [11] involves the use of an illumination or excitation light source that is focused through a pinhole (or iris) onto a sample that is capable of fluorescing and the collection of the emitted light through a pinhole (Figure 2). The locations of the pinholes are such that only light emitted from the sample at a particular plane is collected into the detector, as opposed to standard microscopy, where light from multiple planes is collected by the detector. This allows for higher-contrast images, while movement of the sample along the focal axis allows “slices” of the sample at different depths to be imaged to understand the topography of the material [12].

As the devices in the hot cell are in a radioactive environment, expensive radiation-resistant materials have to be used to prevent browning of the optical components (i.e., going opaque) and to shield any electrical equipment to ensure the device continues to operate [13]. For imaging, the standard approach is to buy a single, expensive, custom-made radiation-hardened camera from companies such as Optique Peter. A second approach is to buy a commercial off-the-shelf (COTS) webcam, fully expecting it to have a short lifespan. The latter “disposable” model has been implemented successfully at the hot cell in Pacific Northwest National Laboratory [14], but this approach has not yet been applied to other imaging or measurement systems. This paper presents the proof-of-concept of a dual-purpose microscope based on COTS devices and low-cost, modular electronics that is meant to perform optical (confocal in particular) and thermal measurements.

In order to develop inexpensive do-it-yourself (DIY) confocal microscopes, researchers have leveraged a variety of low-cost, commercially available equipment, such as by modifying existing epifluorescence or total internal reflection fluorescence microscopes [15]. As an example, Gong [16] developed a simple LED-powered, camera-imaged, partially 3D-printed, and non-automated confocal microscope for USD 5188, while commercial devices cost between USD 10 k and USD 100 k. Further developments of cheap microscopes involved a scanning point fluorescence microscope that imaged biological samples rapidly by using the disc drive and PHR-803T optical head of a Blu-ray player [17]. An optical head is the part of an optical disc reader that contains the laser diodes, optical components (mirrors, filers, lenses), and detectors to measure light on the optical disc. Another researcher used the Blu-ray PHR-803T optical head to determine impurities in crystals at cryogenic temperatures [18].

In further support of this low-cost, COTS, and expendable approach to instrumentation, there is a surprisingly large, and growing, body of work in the literature on the use of the Arduino platform in the development of measurement systems. The applications of this are varied, including allowing for remote health monitoring [19], remote measurements on UAVs [20], small satellites [21], high-altitude prototypes [22], and educational tools to train students for internet of things [23] or laboratory systems [24]. The Arduino platform has also been used to control fluorescent microscopes using the PHR-803T, a low-cost, compact optical system [25,26].

The PHR-803T is the optical component that is used to read disc data in Blu-ray and HD-DVD players and is often used in many DIY projects by hobbyists and academic researchers [27]. This has ensured that there is a large amount of existing software and hardware to make the optical head function. This is because the optical head is relatively cheap (USD 15) and contains 405 nm, 660 nm, and 785 nm lasers in a small, pre-engineered package [27]. This hacking has been useful in biosensing with bioassays on the Blu-ray disc [27], high-contrast label-free imaging of cells [28], optical tweezers in microfluidic devices controlled by an Arduino [29], atomic force microscope imaging [30], digital lenseless holographic microscopy [31], and as a Blu-ray scanning microscope [17]. Another DIY microscope (not laser scanning) was able to be constructed from an open-source series of modules based on Rasperry Pi and Arduino systems [32]. Our previous research [26] used the PHR-803T in a stationary fluorescent scanning thermal microscope to provide optical illumination, focusing, and optical transmission to a separate photodiode. However, it could not scan the surface of the sample to determine its surface characteristics.

This paper presents the design and operation of a modular, low-cost, confocal laser scanning microscope using the PHR-803T and controlled by an Arduino that would fit within this “disposable” model for hot cell usage to characterize nuclear materials. The ability of the SCoT microscope to measure in reflection mode, confocal fluorescent mode, and temperature-sensitive mode will be demonstrated. The final spatial resolution of the SCoT microscope is 12.5 $\mathsf{\mu }$m in the horizontal and 1.5 $\mathsf{\mu }$m in the vertical directions.

2. Device Overview

This section provides an overview of the SCoT microscope (Figure 3), including its basic features, such as the optical system (including the PHR-803T Toshiba PHR-803T, Tokyo, Japan), the positioning system, and the electronics. The design has been guided by constraints on cost, modularity, portability, spatial resolution, and ease of manufacturability using common tools (e.g., drill press, 3D printer).

2.1. Hardware—Structural Frame

Most of the physical components making up the housing of the device were 3D-printed using fused deposition modeling to supply the diversity of structural frame shapes involved in the design. Having a printable design also allows for easy construction of the device for diverse academic or development purposes, not just those with manufacturing capabilities. A 3D-printed housing contains the optics, motors, and linear bearings for 3D positioning, while the device skeleton consists of two thick (0.5″) plates of aluminum that are joined in an L-shape to provide rigidity to the system, as Figure 3.

2.2. Hardware—Optics and Lasers

The optics of the device include several more expensive components (e.g., <USD 600) that make up the majority of the cost. These components have been mounted into a structural system as shown in Figure 4 and will be discussed in detail below.

2.2.1. PHR-803T

The main optical component of the system is the PHR-803T, which will henceforth be referred to as the PHR. The PHR was designed to read HD-DVDs and other discs. Included in the optics of the device are three lasers: a red, a blue, and an infrared (IR). It also has its own focusing system that is capable of making minute adjustments using a small lens within a voice-coil that can be manipulated by a fast PWM signal to the PHR driver board (Figure 5a). The microscope has been designed to have manual control of this voice-coil-based focusing via manual buttons to reach an optimal intensity, because the auto focusing function is less reliable for non-mirror-like finishes. This system involves two buttons and a switch. The two buttons control the vertical actuation, and the switch changes between a “fine” and a “course” movement speed. The microscope is considered to be in focus when the output of the photodiode at the top of the optical stack reaches a maximum from the emitted blue laser.

The optics of the PHR (Figure 5c) allow red/IR (660/785 nm) and blue (405 nm) light to be transmitted within the optical head and to the sample while transmitting midrange visible wavelengths through its dichroic mirror towards the photodiode. This light is used to create the images of the microscope. The light from the blue laser is reflected through a turning mirror onto the sample. The resultant fluorescent light (in the middle of the visible range) transverses back through the focusing lens before being collected in the photodiode, which is mounted directly above the PHR and the optical stack. This can be seen in Figure 4. To locally heat the sample, the 785 nm laser in the PHR can be removed, and a more powerful external IR laser can be inserted in the 3D-printed PHR housing. Sinusoidally modulating the intensity of the IR laser with a function generator and detecting sinusoidal changes in the fluorescent signal can enable the detection of small changes in temperature, as will be shown in Section 4.3.

The benefits of using a PHR-803T confocal system include its relatively inexpensive design and the accessibility of the components that are used in its construction. The majority of its structure being 3D-printed allows for easy adjustments to the design, and the printing of these can be carried out in any environment with suitable 3D printing equipment, such as a college campus. With easily replaceable components, the quality of the results can be modulated to fit the needs of those working with the designs of the device.

2.2.2. Optical Stack

An optical stack is used to filter light and act as the confocal pinhole; Figure 4b shows the elements in the stack. To remove the blue light that is used to excite the fluorescent dye, a band pass filter (Thorlabs FESH0750, 400 nm to 740 nm passing band, Thorlabs, Newton, NJ, USA) was implemented. To reduce the possible out-of-plane light, a focusing lens and a small (∼1 mm diameter), 3D-printed pinhole were added to the optical stack. All 3D-printed parts were printed using acrylonitrile butadiene styrene (ABS) on a fused deposition modeling (FDM) printer.

For the purposes of the confocal fluorescent microscope, only a single filter is needed (450 nm highpass filter, as shown in Figure 4b). Two filters are used for the ambient red light in the thermal application (IR and 750 nm lowpass filters). Any filters in use are placed in a filter mount that is connected to the photodiode surface. A pinhole is used to block light coming into the area of the photodiode at odd angles. Even with less-reflective surfaces, trace amounts of this ambient light can be observed going through the filters if the microscope is not covered, creating a need to calibrate the obtained results with the assumption that the ambient light remains static.

The focusing of the laser, performed specifically by the fine-focus voice-coil actuator as opposed to the vertical motion stepper motor, is the final stage of the focusing process and ensures that the area of laser effect on the sample matches the predetermined calculated values during readings. If a 3D image is needed, the fine-focus is able to be lowered to alternative depths of the sample (see Section 4.2.2).

2.2.3. Photodiode

The microscope uses a Thorlabs amplified photodiode (PDA36A2, Thorlabs, Newton, NJ, USA) to collect light from the microscope, which is set to a maximum output amplification (70 dB). The position of the photodiode is able to be changed to account for any error in the location of the focused fluorescent signal. This is achieved manually by use of a screw on the back of the system. The location is fixed in place at the point where the highest signal is measured. Further details of the photodiode circuit are provided in Section 2.4.3.

2.2.4. External IR “Heating” Laser

The existing IR laser in the PHR does not have sufficient power to heat the surface of sample materials (e.g., stainless steel) enough to detect heat diffusion in the samples via changes in the fluorescent intensity of deposited dyes. An external IR laser (Aziz, Wuhan, China, 780 nm 150 mW Laser Module 12 × 30 mm) was added into the system to provide heating with the requisite power. It was placed externally to the PHR optical head after the existing 785 nm laser was removed from the optical head (Figure 6b). The IR laser was housed in a cylinder that was pinned to the 3D-printed holder, and a servo motor arm was connected to the cylinder to allow the laser positions to change relative to the blue laser spot. A continuous rotation servo motor was controlled by an Arduino Mega (Arduino, Scarmagno, Italy) to adjust the position of the IR laser relative to the fluorescent emission. IR light was used because it did not induce fluorescence and was also outside the emission spectrum of the Rhodamine B and quantum dot fluorescent materials used in this work.

Integrating the external IR laser required three key changes to the optical system that was used for basic confocal microscopy. The first inclusion was a collimator lens on the 785 nm laser, which was used to ensure that the laser beam is parallel and focused onto the sample. This better allows the PHR’s built-in lens to focus the beam and more precisely target the area of interest on the sample.

The second addition is a set of filters that are mounted just below the photodiode, which is used to filter out all other wavelengths of light except for the desired emission from the fluorescent dye. The fluorescent dye has a peak emission wavelength of roughly 600 nm when excited with radiation from the 405 nm laser probe from the PHR-803T. The specific wavelength band of the filter was selected to more effectively filter out the 405 nm wavelength while still allowing the 600 nm wavelength to reach the photodiode. This ensures that only the emission of interest is collected by the detector.

Finally, a 27.5 mm focal length lens was added to focus the emitted light from the sample onto the photodiode detector. This lens is specifically designed to maximize the amount of light that reaches the detector, improving the sensitivity of the system and allowing for the detection of even smaller temperature differences (which manifested as changes in fluorescent intensity) in the sample. To further improve the optical accuracy, an additional pinhole (Thorlabs ID15, Thorlabs, Newton, NJ, USA with a minimum hole size of 1.2 mm) was used to ensure that the spatial distribution of the laser remained as a clean Gaussian beam. Because the thermal analysis occurs in the frequency domain, only relative instead of absolute changes in temperature are needed to see heat diffuse through the sample [33].

2.3. Hardware—Positioning Motors

Figure 7 shows the sample tray with its associated motor systems. The design of the position system uses motors that are mounted to the sample tray to move the “stage” in the x and y directions while the laser position remains fixed, which is easier to accomplish with a 3D-printed support structure. This approach is based on the work of Arunkarthick [34], who used a confocal microscope to create 3D images of pollen.

Three linear actuating stepper motors control the x and y location of the sample (on the sample tray), as well as the z position of the optics (for the vertical direction). NEMA 8 linear actuators with a 100 mm long lead screw and a 0.3 mm screw lead were used (Pololu Corporation, Las Vegas, NV, USA). The motors themselves have a 1.8° step angle and function at 0.5 A/Phase. These provide precision movement without having to invest in an expensive high-precision adjustable sample tray. To avoid unnecessary bending or shaking of the platform, the threaded rods of the linear actuators are centered on the tray table and run through the center of the tray table (see Figure 7). This custom stage allows the sample to be repositioned at various locations to take measurements. Section 4.1 will discuss some issues with FDM printing of the platforms and how the SCoT microscope accounts for this.

The motor control process incorporates the time that it takes to read the analog input pin with data from the photodiode into the time delay that is required for the motor to take a single step. This means that the motor is able to continuously move while the measurements are being taken, greatly decreasing the time it takes to map an image of the fluorescing sample.

2.4. Electronics Overview

The Arduino Mega board was used as the main processing unit because it is cheap, easy to use, and easily replaceable. Outside of the Arduino, several other important circuits are used to control the device. Overall, the electronics (Figure 8) can be broken up into the following sections: the PHR-803T driver, the stepper motors, the thermal hardware, and the amplification circuit.

As shown in Figure 9, at the center of all the electronics functionality is the Arduino. It controls the flow of all information throughout the system. It communicates with nearly all other components of the device, including the blue laser control board, stepper motor driver board, laser focus system, photodiode, and MATLAB programs on the PC. After receiving the proper information from the Arduino via a mix of PWM and digital signals, the blue laser control board commands the intensity of the blue laser on the sample to remain stable. This is the source of the fluorescent signal and has limited change in function after testing has initiated.

The stepper motor driver boards (Polulu 8834, Pololu Corporation, Las Vegas, NV, USA) govern the positioning of the stepper motors. This is facilitated by the use of the full stepper motor driver PCB. This PCB simplifies the distribution of the signal from the Arduino’s digital pins. It also includes voltage regulation to avoid any component burnout.

Data from the photodiode in the optical stack are passed as an analog signal to the Arduino, which converts it to a digital signal using its own internal analog-to-digital (ADC) converter (10 bit, 4.88 mV resolution). A USB cable is used for the Arduino-to-PC communication. Protocalls for this communication are governed via the MATLAB and Arduino code programming. The power for all the devices is regulated by a central power board derived from an XBox 360 disc player (Microsoft, Redmond, WA, USA) and split into 5-volt or 12-volt sources depending on the voltage demand. The only other power connection is for the photodiode, which takes a 120 VAC external wall connection.

2.4.1. Motor Control

The sample tray’s horizontal motion and system’s vertical motion are controlled using a system of three stepper motors. The motors are controlled using external motor driver boards that are mounted on a custom-built PCB. The motor driver board step configuration pins are set up on its lowest 32nd of a step setting. The PCB is designed as a central hub for all signal control. It is provided with general commands from the Arduino such as speed, step, and directional information. This can all be controlled by preprogramming; however, buttons to manually control each motor were also included for debugging purposes.

2.4.2. Laser Control

A few functional capabilities of the PHR, such as the laser intensity and fine-focus actuation, are only possible through a designated laser driver control board. The design for this board was developed by DiYouware, last accessed on 14 December 2023 (http://www.diyouware.com/), with some minor modifications to allow for fabrication simplicity. A 12 V output of the Xbox360 disc player power supply was connected to a 5 V voltage regulator, with the addition of a large heat sink to help dissipate the extra power that otherwise would have caused fluctuations in the laser intensity.

2.4.3. Amplification Circuit

The circuit, using a conventional setup with an LM324 amplifier (TI, Dallas, TX, USA), takes the analog signal produced by the photodiode and increases it to the measuring range of the Arduino. As the signal intensity is dependent on the setup of the system, the gains of the amplifier are tunable to either the confocal or thermal imaging configuration of the SCoT microscope. This signal is given by the photodiode and should account for a significant portion of the fluorescence that is emitted by the Rhodamine B (Millipore-Sigma, Darmstadt, Germany) or quantum dot (QD, Millipore-Sigma, Darmstadt, Germany)-coated sample, or reflected light from the PCB image. The amplifier circuit zeroes the signal to a set minimum and then amplifies it to readable levels. The amplifier gain was set using a 10–100 k$\Omega$ potentiometer. This sets the gains at about 100× based on a resistor value of 1000 $\Omega$. The offset potentiometer is able to match an output voltage to ensure a reasonable range.

The Arduino’s maximum measurable voltage is 5 V. Calibration was needed to set the photodiode output’s minimum and maximum voltage range to match those of the Arduino input. This value was determined iteratively, because the output maximum and minimums are influenced by the ambient light in the room during testing, the position of the sample during testing, any minute changes in the photodiode position, and the amount of fluorescent dye on the sample.

2.4.4. Thermal Hardware

The thermal hardware consists of a servo motor, an external heating laser (Figure 6), a waveform generator to produce a sinusoidal analog signal, and a lock-in amplifier. The servo motor is directly controlled by the Arduino Mega and was included to change the relative position between the external heating laser and the fluorescent emission on the sample caused by the PHR’s blue laser.

The external IR laser is used as a modulated heating source. The intensity of the IR laser is controlled with an input sine wave signal from the AD9833 function generator (Analog Devices, Wilmington, MA, USA). By modulating the heating source, the SCoT microscope is able to detect the diffusion of heat through the sample by locking it in to changes in the fluorescent intensity of the sample at the modulation frequency. Because the fluorescent intensity is a function of temperature, this allowed the relative changes in temperature to be measured. Additionally, locking in to the temperature-sensitive signal allows for small perturbations in temperature to be measured that would normally be below the noise floor of the measurement system. These variations in temperature are termed thermal waves and fall under a class of measurements called photothermal methods [35], which are often used to measure the thermal properties of materials or detect microscale defects in materials.

An SRS 830 lock-in amplifier (LIA, Stanford Research Systems, Sunnyvale, CA, USA) was used to perform the lock-in detection. The amplified output of the photodiode was recorded by the LIA as the amplitude and phase of the fluorescent light that was collected by the SCoT microscope. The diffusion of heat from the IR laser could then be observed as a decrease in the amplitude and phase as the distance between the IR and blue laser spots were changed. The lock-in amplifier requires that the temperature fluctuations reach a steady state, which takes between 2 and 40 s, based on the modulation frequency of the laser. This time is the factor that defines the scanning speed of the SCoT microscope.

3. Operation of the Device

This section will describe the basic operation of the SCoT microscope and debugging processes, as well as component replacement considerations.

3.1. Operation

The operation of the SCoT microscope consists of two fundamental procedures (Figure 10): system positioning and data acquisition. The device is first powered on by connecting to the power supply board. The Arduino is then reset to start the control program and data collection until the PC is enabled. A sample is placed on the mounting stage. The system positioning process begins with the tray table stepping through a set number of locations, performing data readings at each location as it moves. The fluorescent emission readings are taken from the amplified photodiode. The system guides the laser focus location across an initial row, steps to the next row, changes direction, and continues to collect emission data in the opposite direction. This process is repeated until the full designated collection area has been traversed. The amplified photodiode readings are written either directly to an external SD card for post-processing or transferred over a USB-to-serial connection for analysis on the PC. The benefit of the SD-card-based data collection is that it has less compatibility issues with the programs and electronics used.

To use the SCoT microscope in thermal detecting mode, the lock-in amplifier is connected to a BNC adapter that is located on the motor driver board to provide the modulated sine wave that is sent to the external heating IR laser. The output of the photodiode is also connected to the lock-in amplifier. Amplitude and phase data are transmitted to the PC, and the servo motor is used to move the IR laser to another location.

3.2. Debugging and Replacement Parts

During the development of the SCoT microscope, the device was dismantled and reconstructed multiple times during troubleshooting of the device’s operation. This provided insights into several debugging processes when setting up the device, as well as highlighting which components need more frequent replacement.

3.2.1. Debugging

The most common issues during assembly were with the electronic components compared to the physical hardware. The general debugging process for the electronics was broken down into the following categories in the recommended order to follow when troubleshooting: (1) checking wire connections; (2) checking soldered connections; (3) checking the quality of the output data; and (4) checking the functionality of each board (power, motor control, laser focusing, Arduino Mega, etc.) and replacing if necessary. Standard test procedures were established so that the instrument had a consistent performance after component replacement.

3.2.2. Component Replacements

This section will discuss the most common components that needed to be replaced during testing and those that would be expected to need more frequent replacement when exposed to harsh conditions.

The most common component that needed to be replaced was the PHR-803t because of the blue laser diode and the IR laser. This happened because the laser would overheat when it was operated at full power for multiple days. Limiting the output of the laser and/or providing cooling helped extend its lifetime.

The second most commonly replaced components during testing were the Arduino Mega and the laser driver board. The Arduino Mega failure was often caused by a defect in the board because of the low production quality of the device. The laser driver board issues were focused mainly on soldering problems and fluctuations in laser intensity when powering both the IR laser and the PHR-803t using a 5 V source. The soldering issues were resolved by increasing the size of the PCB and using screen printing with soldering paste and surface mount electronics. The fluctuations in the laser intensity were fixed by powering the board with a 12 V source into a 5 V regulator and dissipating extra heat with a heat sink.

When exposed to harsh environments, the optical lenses will brown and need to be replaced, as will the Arduino Mega if it is not properly shielded against radiation. The 3D-printed housing, motor spindle housings, and PHR-803t holder will likely be the next most replaced components based on embrittlement or swelling. However, the stresses on the parts is low, so the swelling will likely cause the most issues with the motor spindle housings.

4. Results

This section will discuss the use of the microscope in reflection mode (without a filter) and fluorescent mode (with a filter). Both modes use the pinhole shown in the optical stack in Figure 4 to limit in-plane light. Additionally, the detection of a weak thermally sensitive signal is also presented when adding a modulated heating source by an IR laser.

4.1. Reflective Imaging

The microscope was used to image text on a PCB. During initial scanning, the device followed the “rastering” path given by Figure 11a. This had the side effect of a noticeable and consistent drift, as observed in Figure 11b, where the number “6” that was being imaged is skewed by a consistent amount. The corrected image uses a diagonal shift, as illustrated in Figure 11e. This shift is tuned to the current setup and remains constant throughout testing. Additionally, the corrected image, shown in Figure 11f, can be recreated and is comparable to the image produced by an Opti-Tekscope Digital USB Microscope Camera (Opti-Tekscope LLC, Chandler, AZ, USA).

This slight drift occurs due to marginal inconsistencies in the motorized baseplates, resulting in a change in the motion of the sample tray. Because the scanning resolution is on the order of $\mathsf{\mu }$m, these inconsistencies result in a slight drift in the measurements. The change in the motion of the sample tray was caused by the distortion of the used 3D-printed components and imperfections in the bearing rods governing the motion of the tray. Although the use of 3D-printed parts is a root cause of this issue, one of the goals of this work was to produce a low-cost and modular device. To avoid expensive machining solutions, this issue was solved by modifying the imaging path that is taken by the microscope (Figure 11c). This greatly improved the drift issue caused by the distortion of the 3D-printed components. Figure 11d shows the improved imaging with this path, but a consistent sawtooth pattern was still observed in the image. To overcome this, a consistent shift in both the x- and y-axes was performed after a non-imaging scan, resulting in the imaging path shown in Figure 11e. The use of both of these imaging compensations resulted in the imaging of the numeral “1” in Figure 11f at a spatial resolution of 12.5 $\mathsf{\mu }$m in the horizontal and 1.5 $\mathsf{\mu }$m in the vertical directions. The resolution can be further improved with more expensive motor systems, and greater accuracy in the resolution could be obtained using a resolution target like 1951 USAF.

4.2. Fluorescent/Confocal Imaging

This section focuses on the use of the microscope to create a simple planar image of Rhodamine B on an opaque, brass sample to test the contrast of the fluorescent image against the background. This will be followed by a series of depth images through a transparent synthetic spider silk [36] sample that is treated with quantum dots as the fluorophore. The spider silk images are compared to the results of images taken by a commercial Leica scanning confocal microscope of those same silks.

4.2.1. Imaging a Reflective Surface Coated with a Fluorescent Dye

A brass sample was coated in Rhodamine B (>95% HPLC from Millipore-Sigma, Darmstadt, Germany), as shown in Figure 12. The droplets of water containing the dyes were each applied roughly 1 month apart to test the sensitivity of the SCoT microscope to fluorescent intensity changes. The photobleaching effect of the dye is clearly seen in the colored microscopic image in Figure 12. Although this effect is not easily observed visually because the red color does not look that different, the generated image shows a significant difference in fluorescence, where the fresher, less photobleached dye provides a stronger fluorescent signal. This demonstrates the ability to differentiate between fluorescent intensities based on changes in the fluorophore.

The movement of the sample tray was calculated to be approximately 1.5 $\mathsf{\mu }$m. This was determined as an average step size when the system was jogged through a 100-step cycle. The final distance averaged to 151 $\mathsf{\mu }$m, with an error of approximately 5 $\mathsf{\mu }$m. The size and accuracy limitations were, respectively, limited due to the resolution of the stepper motors and the 3D-printed construction of the device. This resolution can be easily improved by decreasing the step size of these motors at the expense of speed. Improvements to the accuracy of the step size can possibly be achieved by the integration of better 3D printing methods (such as resin-based stereolithography, SLA) and higher-accuracy components into the device.

4.2.2. Multi-Planed Fluorescent Scanning

To test the confocal capabilities of the SCoT microscope, the focusing lens in the optical head was moved to allow different imaging planes to be the focus of the blue laser. The sample used for this testing was a silk thread that was mounted to a cover slip, which was previously treated with quantum dots. The synthetic silk was created from spider silk protein produced in transgenic goats that underwent the extrusion and pulling process described in Refs. [36,37]. This dye-coating process is similar to that described in our previous work [38]. The silk fiber was then imaged with the SCoT microscope, and the resulting image is shown in Figure 13a.

The axial focus of the images are rather poor quality, as each step is roughly 18 $\mathsf{\mu }$m apart. This focus resolution can be decreased to 9 $\mathsf{\mu }$m, but the quality of the images can only be increased by the inclusion of an improved iris that is located at the collection point to further restrict out-of-plane light. The iris will help to filter out the light from all other focus locations than the one of interest. An iris of a sufficient size would require high-resolution printing, which is achievable by a micro-3D printer [39] or the purchase of precision pinholes such as ThorLabs stainless steel foils.

Figure 13b shows the same silk fiber when it is imaged using a commercial Leica scanning confocal microscope with a much higher resolution. The images provided are roughly 4 $\mathsf{\mu }$m apart in focus depth and show significantly better resolution in the individual images. However, the change in width of the silk is observable with both microscopes as the imaging plane moves through the fiber.

4.3. Detection of Temperature-Sensitive Fluorescent Signal

This section shows the capability of the SCoT microscope to detect relative temperature changes in the sample as a function of the spacing between a heating laser and the blue laser. The photoluminescence that is emitted by a fluorescent probe (illuminated by the blue laser) is dependent on the temperature of the probe, where decreases in light intensity are associated with increases in temperature.

To allow the SCoT microscope to image temperature-dependent processes, several additions to the optical stack and electronic hardware were required. Full details were provided in the device overview (Section 2). These modifications are designed to more efficiently focus the desired light towards the photodiode and eliminate any reflected light from the IR heating laser with additional filters, as shown in Figure 14. Additionally, they provide a higher intensity of IR laser light to be transmitted to the sample surface to increase the local temperature. The data acquisition system records a batch of amplitude and phase data for each position of the servo motor moving the IR heating laser.

To prepare a sample for measuring relative temperature changes, Rhodamine B in an ethanol solution was spin-coated onto a stainless steel 304 disc (Figure 15a). The thickness of the coating was measured using a Zeta 20 profilometer (KLA, Ann Arbor, MI, USA) as 20.7 $\mathsf{\mu }$m thick. Stainless steel was selected as the sample material, because the current design of the SCoT microscope cannot be used with ferromagnetic materials. This is to avoid any interaction with the magnetic actuator that is located on the PHR that is used for focusing the lasers.

The sinusoidal modulation of the IR heating laser’s intensity results in a thermal wave, which is a periodic variation in the local temperature of the material [38]. This temperature variation occurs at the same frequency as that of the modulated heating but is delayed in time, as the heat needs to diffuse from the heating spot. The use of lock-in amplification allows the temperature-sensitive fluorescent signal at the modulation frequency to be isolated and amplified, but this also results in the signal being mapped into the frequency domain. As fluorescent data are collected at increasing distances from the heating spot, the thermal wave amplitude decreases, and the time delay between the heating wave and the temperature response increases. This results in the phase delay shown in Figure 15b.

Figure 15b shows a linear trend in the phase delay when the IR and blue lasers are spaced more than 80 $\mathsf{\mu }$m apart. Thermal wave theory predicts that at distances greater than about 5× the diameter of the laser spot size, a thermal wave has a linear slope in the phase delay between the modulated heating source and the temperature of the material away from the heating source [6]. This phase delay is large for thermally insulating materials and small for thermally conductive ones. The outlier that deviates from linearity when the laser spot spacing is about 75 $\mathsf{\mu }$m could have been caused by the presence of a microscopic defect in the sample, such as the scratch shown in Figure 15a. Hua demonstrated that grain boundaries can create a phase jump of about 1° in the thermal wave, and larger defects are expected to create large phase delay jumps [40]. The results in Figure 15b demonstrate that the SCoT microscope is able to detect these thermal waves.

5. Conclusions

This paper presents the design and operation of a scanning confocal thermal (SCoT) microscope. Using inexpensive components, the device was able to create microscopic images using fluorescent emissions by means of the PHR-803T optical head, optical filters and lenses, positioning motors, and a 3D-printed structural frame. This was demonstrated by using the microscope in a reflective mode (imaging writing on a PCB board without fluorescent probes) and in a confocal mode (observing the photobleaching of Rhodamine B and imaging different planes of synthetic spider silk). Additional features in the design of the system include post-processing techniques and construction calibration features that are key to improving the quality of the system but are not necessary for its functionality. This research also demonstrated how the device can be used as a thermal microscope by using additional filters and a heating laser to detect thermally induced fluctuations in fluorescent dyes.

One improvement of the device could be the use of a smaller pinhole print, located at the collection sight of the laser. Based on the function of the laser, an accurately located and sized pinhole can improve the vertical resolution in 3D imaging applications. Additionally, modeling specialized components could also greatly reduce the cost of the system while maintaining the integrity and precision of the results.