Controlled Photothermal Therapy 2 (or just Controlled PTT 2) is the second version of software and hardware setup to study thermal effects of nanoparticles under laser irradiation in vitro (on living cells) or in vivo (in small animals). The primary aim is to control the temperature if cell media in vitro, but the setup can be applied for animal studies. Depending on time of laser irradiation, nanoparticle type, their photothermal conversion efficiency, different thermal dose can be delivered to the irradiated object. Thus, different biological responses can be triggered depending on the maximum achived temperature and gained thermal dose. More about thermal dose can be found in our publication.

Controlled PTT 2 consists of two main parts: hardware part and software part. The hardware part consists of temperature sensor(s) and a laser. Depending on the application, different sensor parts and laser parts can be used. You can also develop your own sensor or laser parts, or they can be developed specifically for you upon a request. Please contact Konstantin Tamarov to discuss your requirements.

Controlled PTT 2 was developed by Konstantin Tamarov, PhD, Emilia Happonen, MSc, Mikke Varis, and Yiannis Constantinou at Department of Applied Physics at University of Eastern Finland. Please see Credits section for the correct referencing when using the software.

The Controlled PTT 2 consists of 3 main parts: (1) the PC program written in C# programming language; (2) the board part which reads the temperature sensor data and sends it to the PC program; (3) the laser part which controls the laser hardware if it is attached. The following requirements are in place:

• .NET Framework 4.7.2 to run the software on a PC with Windows OS. .NET Framework 4.7.2 is included by default in Windows 7 or higher.
• Arduino Studio 1.8.13 or higher. Arduino Studio is needed to install necessary drivers and upload the program to the board. Refer to the sensors section or to the full Sensors documentation for more information about temperature sensors and boards.
• Necessary drivers for laser hardware. Drivers for the available laser parts are included in the distribution. Refer to the lasers section or to the full Lasers documentation.

In order to use Controlled PTT 2 software, at least one sensor hardware part must be assembled and programmed. The available sensor parts, detailed instruction to assemble and program them are located in Sensors directory. Arduino Studio must be installed in any case to upload sketch to the sensor board and provide necessary drivers.

The Controlled PTT 2 software requires either its executables to be downloaded from the latest release or it can be built from the source.\

If software does not start, check troubleshooting.

To use the Controlled PTT 2 executables directly do the following:

1. Download Release-v2.x.x.zip archive from the latest release.
2. Unpack the contents of Release-v2.x.x.zip to any suitable location (e.g. Program Files, Documents, Desktop, etc.).
3. Navigate to Sensors directory and select the appropriate sensor. Sketches and necessary libraries for boards are located in Board* directory for each sensor.
4. Upload the selected sensor's sketch to the board using Arduino Studio. The detailed instruction for boards are in the Sensors.
5. Run the ControlledPTT.App.exe. More information about its capabilities and interface is in App.
6. Select the correct sensor in the CotrolledPTT.App and press "Start" button. In the opened Sensor interface press "Connect to Board" button and verify that the sensor starts to sent the temperature to the ControlledPTT.App.
7. The basic setup is ready to record temperature and thermal dose. No laser is connected in the basic setup.

To build the Contolled PTT 2 from source do the following:

1. Install Visual Studio 2019 with C# and Windows Forms as development environment.
2. Download source code from the latest release or clone the repository using the git command git clone https://github.com/naitsok/controlled-ptt.
3. Navigate to controlled-ptt/Sensors directory and select the appropriate sensor. Sketches and necessary libraries for boards are located in Board* directory for each sensor.
4. Upload the selected sensor's sketch to the board using Arduino Studio. The detailed instruction for boards are in the Sensors.
5. Run Visual Studio and open the controlled-ptt/ControlledPTT.sln file. There should be 10 C# project including ControlledPTT.App.
6. Make sure that Startup Project is ControlledPTT.App in the dropdown list in the panel below menu.
7. Select either Debug or Release.
8. In the Visual Studio interface press "Start" to start building solution. If there are no errors, ControlledPTT.App will launch after a while.
9. The built executables together with the sketches can be then found in the "Build" directory inside the cloned controlled-ptt directory.

The curently developed sensor parts are based on the Melexis infrared temperature sensors:

1. MLX90621ESF-BAD-000. 16x4 array sensor with FOV 40x10 degrees (40 - 50 €).
2. MLX90614ESF-BCF-000. Single pixel sensor with FOV 10 degrees (25 - 35 €).
3. MLX90614ESF-BCI-000. Single pixel sensor with FOV 5 degrees (30 - 40 €).

The latter two single point sensors can be used on their own with a basic Arduino board (Uno, RedBoard, etc.). See MLX Sensor part for more details.

Furthermore, two single point sensors can be simultaneously attached to one board for better precision. See Two MLX Sensors part for more details.

Array sensor requires a board with more memory than Arduino. In our case a Teensy 3.6 board was used. See Array MLX Sensor part for more details.

In order to use a selected sesnor part, the sensor must be correcly mounted to board and the appropriate sketch must be uploaded to the board. The detailed description of board wiring and sketch uploading can be found on the links above.

If there is no part suitable for your hardware, new sensor part can be developed upon a request or by yourself.

• Software does not start. Software does not start if Windows (for example through organization policies) does not allow to laungh software from unverified publisher. In this case all the executales (ControlledPTT.App, executables for each sensor in Sensors folder and executables for each laser in Lasers folder) must be unblocked via the following procedure. First, right click the executable file and select "Properties". Next, on "Security" tab select Unblock and Apply -> ok. Repeat on all executables. Now the software should start.
• Make sure that the board connected via USB is recognized by PC. It can be done using Windows Device Manager and checking if there are unrecognized USB devices.
• After uploading the sketch to the board using Arduino Studio, Serial Monitor there can be used to check if board is sending data to PC.
• The compiled Windows Forms executables for each sensor part can be run separately without running ControlledPTT.App. By clicking "Connect to Board" button, the received data from board can be verified. Note that Serial Monitor of Arduino Studio must be closed and the appropriate COM port must be selected.

The currently developed laser parts are:

1. CNI LED Laser with the MDL-III-LED controller. Controlled PTT 2 sends the power data to the controller via COM port connection. The Prolific USB-to-Serial Comm Port is Lib folder.
2. Connect a laser diode to Agilent N5768A or similar power supply, which is in turn connected to PC and Controlled PTT 2. The power supply sets the current to the laser diode and thus regulates its power.

If you have a specific laser hardware which can be connected to PC, new laser part can be developed upon a request or by yourself.

Controlled PTT 2 aims at making photothermal experiments with nanoparticle suspensions easy, controlled, reproducible and versatile. Easiness is gained through using a unified interface of the ControlledPTT.App, which is independent from sensor or laser part used. Control is achieved by possibility to perform different types of experiments and applying different experiment stop conditions. Reproducibility is obtained through keeping calibrations and having different configuration profiles for different users. Versatility is aquired through independence of the ControlledPTT.App from the laser and sensor part. Virtually any sensor and any laser can be connected to the ControlledPTT.App considering its executable inherits the necessary base classes. Thus, the experiments can be performed in any imaginable experimental setup, including in situ measurements, in vitro experiments with live cells, or in vivo experiments with animals, provided that the suitable laser and sensor parts are available.

Overall, to perform its functions properly Controlled PTT 2 requires the necessary hardware and software.

Hardware of Controlled PTT 2 consists of two main parts:

1. Sensor part. This part is responsible for getting data from sensors and sending it to PC via any suitable connection. At this point, the developed sensor parts are dedicated to in vitro experiments in 96-well plates. These sensor parts consist of one or two infrared temperature sensors connected to an Arduino or similar board that each second sends the measured temperature data to a PC. On the PC, the sensor executable written in C# reads the data, parses it if necessary and sends to ControlledPTT.App for calibration and further processing.

2. Laser part. This part is resonsible for controlling laser hardware, e.g. initializing connection to laser, setting necessary parameters and controlling the laser power. At this point, Agilent power supply and CNI Laser laser parts are programmed.

Controlled PTT 2 Software consists of three parts:

1. ControlledPTT.App(.exe) is the main application executable. It performes connection to sensor and laser parts, manages sensor and laser, creates and keeps calibration for sensor data, and performes the experiment. More details can be found in the related folder.

There are three main types of experiments:

• Experiment with only sensor part connected. ControlledPTT.App receives the data from sensor, uses calibration if applicable, writes the data to file. This experiment type can be used if laser is operated manually or there is no laser at all.
• Experiment with a full laser power. The power of laser is set by the user in laser window and ControlledPTT.App only switches laser on when experiment is started and off when experiment is over.
• Experiment with controlled laser power to achieve a specific temperature of the irradiated target. The power of laser is controlled using a built in Proportional-Integrtal-Differential (PID) controller in the limits set by the user in the laser window. The PID coefficients must be calibrated for the specifically used laser in order to be efficient.

There are there different stop conditions for an experiment:

• Stop when time expires.
• Stop when specified thermal dose of the object is gained. More about thermal dose can be found in our publication.
• Stop when either timer expires or specified thermal dose is gained.

The important option is "Discretization Time Interval" in ms; it can be modified in the Experimetal Setting menu. The "Discretization Time Interval" governs the time interval between the events during the experiment. In particular, it controls how often the temperature data from Sensor part is sent to the ControlledPTT.App, how often the data is processed and saved, and how often Laser power is set. For example, if the "Discretization Time Interval" equals to 1000 ms, then each second data is obtained from a Sensor part, the data is modified according to the loaded calibration, the laser power is calculated and sent to the Laser part if the appropriate experiment type is selected.

2. ControlledPTT.Sensors.(MLXSensor|TwoMLXSensors|ArrayMLXSensor)(.exe) are executables for sensor parts. Inside of the directories for each sensor executable there is a folder with sketches for Arduino Studio and detailed instruction of board wiring and sketch upload guide. In the top of ControlledPTT.App interface sensor part can be selected and loaded. Below is the image of all sensor parts available now.

3. ControlledPTT.Lasers.(Agilent)(.exe) is the only available laser executor for now. It connects to Agilent N5768A or similar power supply to set current for a laser diode, which is in turn connected to the power supply.

The development of Controlled PTT 2 is directed by Konstantin Tamarov at Department of Applied Physics, Univeristy of Eastern Finland. The roadmap of Controlled PTT 2 is to add more options for sensor and laser parts depending on the required applications. Therefore, Konstantin Tamarov can be conacted with a request to discuss and develop the necessary part. Further development as well includes bug fixes, more documentation, and examples of hardware assemly with 3D printed parts.

The proper way to contribute the Controlled PTT 2 development is to use GIT and github functionality for software development. This repository can be forked to a contributor's account and pull requests are warmly welcome.

The correct implementation of a new sensor part requires the development of hardware and software parts. The hardware part is completely under one's specification. The software part must be developed using Visual Studio 2019 and .NET Framework 4.7.2. The sensor part executable must be inherited from the BaseSensor class, which is in turn inherited from the System.Windows.Forms.Form class.

The correct implementation of a new laser part requires the development of hardware and software parts. The hardware part is completely under one's specification. The software part must be developed using Visual Studio 2019 and .NET Framework 4.7.2. The laser part executable must be inherited from the BaseLaser class, which is in turn inherited from the System.Windows.Forms.Form class.

Controlled PTT 2 has been developed by Konstantin Tamarov and Mikke Varis.

Licence: GPLv3.

When using please give the following references:

1. Cite the published paper, where the first version of Controlled PTT was used:

• E. Happonen, K. Tamarov, M.-V. Martikainen, K. Ketola, M. Roponen, V.-P. Lehto, W. Xu, Thermal dose as a universal tool to evaluate nanoparticle-induced photothermal therapy, 2020, 587, 119657.

2. Cite the Controlled PTT 2 and this repository:

To cite the latest version:

K. Tamarov, E.Happonen, M. Varis, Y.Constantinou, W. Xu, V.-P. Lehto. 2021. Controlled Photothermal Therapy 2. DOI: 10.5281/zenodo.4629678.

To cite the specific version, select the DOI of specific version on Zenodo page:

K. Tamarov, E.Happonen, M. Varis, Y.Constantinou, W. Xu, V.-P. Lehto. 2021. Controlled Photothermal Therapy 2. DOI: (Use DOI of specific version).

• Fixed crash on start bug when calibration file in the selected configuration is not found.
• Fixed bug when converting received temperature from one and two MLX sensors.

• ArrayMLXSensor part now also shows the ambient (shell) temperature of the sensor.
• Updated README to troubleshoot the issue of Windows blocking software.

• Added documentation for CNI Laser and Agilent Power Supply laser parts.
• Discretiztion Time Interval now gets disabled during experiment run.
• Slighlty reduced sizes of the ArrayMLXSensor, Agilent Power Supply laser, CNIMDLIII laser and ControlledPTT.App to fit 1440x900 resolution screens.
• Fixed runtime errors during building when switching from Debug to Release mode and vice versa.

• CNI Laser with PSU-III-LED controller is added.
• Discretization Time Interval is added to the Experiment Settings menu.
• Minor updates and bug fixes, such as correct configuration saving for laser and sensor parts.
• Updated README.md files.

• App. App controls sensor and laser parts. Allows to perform experiments with and without connected laser. Laser power can be controlled to reach specific temperature. Experiment can stop either by time expiration or gained thermal dose.
• Sensor. There are three different sensor parts included. The first two are based on either one or two MLX90614ESF-BCF-000 sensor connected to Arduino compatible board. The third type of sensor part is based on the 16x4 pixel array MLX90621ESF-BAD-000 sensor. For the array sensor a more advanced board with larg memory such as Teensy is needed.
• Laser. Laser part for now is based on the connection to Agilent N5768A or similar. A simple diode laser such as HS-808-1000 infrared handheld laser. The laser must be calibrated beforehand to verify its optical power dependence on the applied current from the power supply.