Arduino Servo Motor Projects

Abstract

In optical experiments, shutters are devices that open or close a path of light. They are often used to limit the duration of light exposure onto a target or onto a detector in order to reduce possible light-induced damage. Many commercial shutters are available for different applications – some provide very fast opening and closing times, some can handle large optical powers, and others allow for fail-safe operation.

Many of these devices are costly and offer limited control options. Here we provide an open-source design for a low-cost, general purpose shutter system based on ubiquitous servo motors that are connected to an Arduino-based controller. Several shutters can be controlled by one controller, further reducing system cost. The state of the shutters can be controlled via a display built into the controller, by serial commands via USB, or by electrical control lines. The use of a microcontroller makes the shutter controller adaptable – only control options that are used need to be included, and the design accommodates a selection of display and servo options. We provide designs for all required components, including 3D print files for the servo holders and cases, software for the Arduino, libraries for serial communication (C and python), and example graphical user interfaces for testing.

Keywords

Optical shutter, Laser safety, Microscopy, Arduino, Open hardware

Source File Repository

GitHub repo link: https://github.com/MCFLab/Shutter

1. Hardware In Context

In many studies that involve a light source, light exposure is intermittent with “on”- and “off”-periods. Switching could be required for safety reasons (turning the light off while observing the sample), to limit the amount of deposited energy, to switch between different light paths, or to observe a time-dependent response. Repeatedly turning the light source on or off is often too slow (as with halogen lamps) or detrimental to the source (as with some mercury lamps). Mechanical shutters provide a convenient route to control the exposure window by inserting a mechanical block into the light path.

Many designs are in use for the type of mechanical blade, the way the blade is moved into and out of the light path, and the means of controlling the open/closed state. Common blade designs are irises (diaphragms) that open/close radially, or rigid blades that sweep across the path linearly. The blades can block the light by absorbing it (e.g., absorption on dark blades) or by redirecting it (e.g., scattering or reflection on bare metallic blades). The most common options for moving the blades are to attach them to solenoids or to rotary motors. Because of the mechanical motion, there is a tradeoff between the size of the obstructed light path and the time required to open/close it (and the frequency of opening/closing cycles). In some applications, the opening and closing times can be shortened by reducing the size of the light path by focusing and placing the shutter at the focal position. The use of a continuously rotating shutter wheel can offer vastly increased cycle frequencies, but this approach is restricted to cases where opening and closing occurs at regular intervals. Our design is not aimed at high- speed applications, but at the most common scenarios where the shutter merely provides a means to turn light on and off at arbitrary times.

Here we provide a brief overview of the available commercial shutters, which cover different applications areas. Devices with very fast opening/closing times and low latencies are available from Vincent Associates or Thorlabs , but a single shutter with the associated driver electronics generally costs in excess of a thousand US dollars. Lower-cost devices are available that sacrifice some performance, mostly opening/closing speeds, or control options. Solenoid-based shutters are available from Brandstrom Instruments , EOPC , DACO Instruments , and KENDRION with a wide range of blade options; Picard Instruments offers a stepper-motor based shutter. Some of these devices are available with controllers and/or a programming interface. Radiant Dyes offers a device based on a servo motor and a controller that opens/closes the shutter via manual switches, digital inputs, or a serial communications port. However, all these lower-cost devices still cost several hundred dollars to operate a single shutter and configuration and control options are very limited.

Fig. 1. Schematic overview of the shutter system.

3

Many do-it-yourself (DIY) shutter designs have been published in scientific literature. They vary greatly in performance, cost, and ease of assembly; few provide easy-to-replicate build instructions. Early designs relied on magnetic coils, relays, or solenoids , DC motors [12, 13], or loudspeakers . Faster blade motion with lower timing jitter can be achieved with voice coils derived from hard disk drives [15, 16] or with a piezo cantilever design . A simple servo-based shutter has been demonstrated as part of a (only partially completed) spectrometer design, and in a quantum optics lab , but no details on the implementation could be found. Of these designs, the 3D printed, DC motor-based design contains part files and electronic design files that aid in replication; for the voice coil design instructional material and videos [22, 23] are available.

Here we describe a simple, easy-to-build, low-cost, and open-source shutter design based on an RC servo with an Arduino-based controller that offers a wide range of configuration options. Several shutters can be controlled with one controller – we tested the design with four shutters, but extensions to 12 or more shutters are straightforward. Blades sizes are adaptable up the several centimeters. Small- beam opening/closing times of sub-10 ms and on/off cycle rates in the 10 Hz range are easily achievable.

The shutter controller can be configured to receive input from a display (LCD with push buttons or a touch screen display), from hardware digital control inputs, through USB serial communication, or the shutters can be moved manually. We provide the control software (Arduino code, a C or python-based library for serial communication, and a python GUI) and the mechanical designs for two servo sizes and controller enclosures. We use our system to control the light paths in our laser-scanning microscope, for example to protect the sample from exposure when the acquisition is stopped, switch the light paths for different experimental conditions, and as a laser safety measure when the microscope is not acquiring data, but we are hopeful that our design will prove useful for many other applications.

2.1. Overall Implementation And Design

The shutter system consists of the mechanical part and the association control electronics. The mechanical shutter is based on commercial RC servos mounted in a 3D-printed holder. A black anodized aluminum blade is mounted on the servo horn (the rotatable portion) to block the light path for a given servo position. The associated control electronics is based on an Arduino Uno with a pulse width modulation (PWM) shield for controlling several servos and a display shield for displaying and switching the state of the shutters. Control of the position of the shutters can occur through user control on the display, serial communication, or TTL-compatible digital control inputs. The provided enclosures and the default hard/software configuration is designed for up to 4 channels, but the design can accommodate tens of shutters with trivial modifications.

2.2. Shutter Mechanics

The blocking action of the shutter is achieved by rotating an opaque blade into the light path. Here, the blade is made from black anodized aluminum, which absorbs most of the light, exhibits some scattering, but transmits no light. The metallic blade dissipates moderate amounts of absorbed light as heat to the surrounding air. For high-intensity beams, care must be taken not to overheat the servo (horn and gear) and gradual bleaching of the dye used in the anodization process can be expected. For such cases, a mirror may be mounted on the blade to redirect, rather than absorb the light.

4

Fig. 2. Schematic of the shutter blade. The red circle indicates the light beam. The speed at which the shutter opens/closes the light path depends on the rotational speed of the servo, 𝑟 (measured in degrees/s), and the required angle of the blade to traverse the light, 𝜃. The angle 𝜃, in turn, depends on the diameter of the light path, 𝑑, and its distance, 𝐿, from the servo axis. If 𝑑≪𝐿, the

Eqn. 1

where we assumed a uniform beam and neglected acceleration effects of the servo. Hence, a faster servo, a longer distance between light path and servo axis, and a small light path diameter (maybe even by focusing through a lens) decreases the opening/closing times. For a laser beam of a few mm in diameter, a convenient 𝐿 of a few cm, and typical servo speeds (60° / 100 ms), opening and closing times are on the order of several ms. The time delay between an open/close request and the actual light increase/decrease is determined by the width of the blade, 𝑡, the limited acceleration of the servo, and the delay of the control electronics. Even for a minimal blade width (𝑡= 𝑑) the frequency of the servo control signal limits the response to a delay of several tens of ms and timing jitter of several ms (example performance data are given below). In the accompanying material we provide part files for the mechanical holders for two servo sizes (we tested the larger size with the ubiquitous MG 996R and HiTEC HS-322; the smaller one with the faster Savöx SH-0262MG).

2.3. Controller

through USB or via an external power supply. To drive the servos, we utilize a dedicated, external pulse width modulation (PWM) shield with a separate power supply for two reasons. First, the current that the Arduino 5V pin can supply is quite limited (800 mA if using the power input, even less if connected to an unpowered USB hub), whereas a single, medium-sized servo can already temporarily draw several hundred mA. Second, even though the servos can be PWM-controlled directly by the Arduino’s digital outputs, controlling several servos requires careful sharing of the Arduino’s resources (especially timers).

Hence, using a dedicated, external PWM board provides enough current for many servos and significantly simplifies the programming. An optional display board serves to display the status of the shutters and to provide a way for user input. The user can also control the shutters via serial commands (such as open/close positions, labels, and control port mapping) is stored in the Arduino’s electrically erasable programmable read-only memory (EEPROM). The Arduino, shields, and input BNC connectors are housed within a 3D printed enclosure. Below we describe each of these components in more detail.

2.3.1. Servo Shield

The servo shield is a 16-channel PWM shield with an Inter-Integrated Circuit (I2C) interface (Adafruit Product ID: 1411). For servo position updates, commands are sent from the Arduino to the shield over the I2C bus. In between updates, the shield holds the servo positions and does not require Arduino involvement. Several shields can be stacked if more than 16 channels are required (though some changes in case design and software would be required). If a display shield is used on top of the servo shield, the required headers limit the space for servo connectors (see build instructions). The Arduino is

5

not able to supply enough power for the use of several servos simultaneously, so the servo shield utilizes a separate 5V power supply. To provide enough peak current for the simultaneous movement of several servos, place for a storage capacitor is provided on the shield circuit board – its capacity should be matched to the expected number of servos utilized.

2.3.2. Display Shield

If the use of a display shield is desired, we provide two options: an LCD screen with buttons or a touch screen. The liquid-crystal display (LCD) shield (Adafruit Product ID: 772) contains a 16x2 character LCD and push buttons. On the display, the first line displays the shutter name, the second line the shutter status (open, closed, or inactive). The up/down buttons cycle through the selected shutter, the left/right buttons change the state (left for close, right for open, an optional timeout sets the shutter to an inactive state).

The touchscreen shield (Adafruit Product ID: 1947) contains a 2.8", 240x320 pixel thin-film- transistor (TFT) LCD with capacitive touch sensing. The display portion uses the Arduino’s Serial Peripheral Interface (SPI) bus, the touch sensing the I2C bus. Each line displays the shutter label in the center, and touch areas (“buttons”) for opening and closing the shutter on either side. For ease of touching the correct buttons, the number of lines is currently restricted to four. However, if more shutters are desired, smaller fonts could be chosen (at the expense of smaller buttons) or line scrolling by swiping motions could be implemented.

2.3.3. Digital Input

To control the shutters with an electrical signal, we implemented an interrupt-based monitoring routine of some of the Arduino digital input ports. The ports are configurable as 0 – 5V (TTL) inputs with (optional) input pull-up resistors that also allow the use of simple mechanical single-pole switches. The current case and software allow for four control lines, but this can be easily extended. Each shutter can be mapped to any (or no) control line and each control line can control several shutters. Note that while the inputs can be used as an interlock signal for laser safety applications, the RC servos keep their position in case of a power failure and hence do not provide a fail-safe closed state.

2.3.4. Serial Communication

Serial communication via the Arduino’s USB port allows for programming of the shutter parameters, controlling the shutters, and checking the shutter status. Communication is done by sending three- character ASCII commands (with following command parameters, if applicable). A list of commands is provided in Table 1. The command format and example responses for the serial commands are listed in Table 2.

Gtdx

Gets the stored transit delay of shutter number x in ms

Gprx

Gets all the stored parameters for shutter number x

Sspx

Sets the current servo position of shutter number x

Sprx

Sets all the parameters stored for shutter number x

Save The Current Shutter Parameters To Eeprom

Table 1. List of serial commands.

“St=1”

Note: 0 -> closed, 1 -> open, 2 -> manual position set, -1 -> inactive

Gpr1

“PR1,11,2,110,130,800, ShutterLabel2”.

Format:

PRx,PWMChannel,digInputChannel,posOpen,posClosed,transitDelay_ms,

Opens Shutter Number 0

Returns “OK” if successful, an error otherwise.

Shutterlabel2

Sets the parameters for servo number 1. Returns “OK” if successful, an error otherwise.

Format: See Gprx

Note: if x=-1 then a new shutter is added after the existing shutters

Sav

Saves only if at least one shutter is defined. Returns “OK” if successful, an error otherwise. Table 2. Format and example responses for the serial commands.

•

For the communication to the Arduino, line termination is a line feed (‘\n’, LF, 0x0A) by default, but can be changed to a carriage return (CR, ‘\r’, 0x0D). The response from the Arduino is the standard CR/LF (“\r\n”).

•

For commands that address a specific shutter (e.g. GSTx) the shutter number x has a zero-based index (0->first shutter, 1->second shutter). The same applies to the input control lines.

•

Another SPR example: “SPR-1,3,-1,255,315,400,Name1” adds a new shutter labelled “Name1” after the existing shutters. The new shutter uses the servo shield channel 3, is not controlled by input lines, and has an open/close position of 255/315, and a transit delay of 400 ms.

2.3.5. Parameter Storage

Each shutter is associated with a range of parameters:

•

“PWMChannel” is the assigned hardware PWM channel number of the servo shield. Range: 0 to 15.

•

“digInputChannel” is the input control line that controls the shutter state. Range: 0 to 3 and -1 (not controlled).

•

“posOpen” and “posClosed” are the servo positions (PWM value) corresponding to the open/closed position.

•

“transitDelay_ms” is the delay in ms that the shutter requires to fully open/close. This value is not used by the Arduino controller, but simply stored and can be returned upon request to implement wait times in a control sequence. This should be measured experimentally for each shutter.

•

“Label” is the label displayed on the display. By default, this is limited to 7 characters (to fit on the touch screen display) but this can be extended in the configuration file. These shutter parameters are stored in the EEPROM of the Arduino to retain their values after an Arduino reset.

2.3.6. Arduino Control Modules And Sequence

The shutter control in the Arduino is split into modules, which can be utilized independently: the serial communication module, the display module, the control input module, and the idle check module. In the Arduino main loop, these modules (if utilized) are repeatedly queried for change requests.

The optional serial communication module handles serial communication between the Arduino and a computer through a USB connection. For the optional display module, the LCD or TFT module can be utilized. Either will display the status of the shutter and let the user change it. A debounce mechanism is included for either device to avoid accidental multiple button presses. After a period of inactivity, either screen can dim and can be brightened again by any touch (for the TFT) or button press (for the LCD).

In the control input module, the control input lines are mapped to the Arduino’s pin change interrupt mechanism. Even though interrupts can suspend all other Arduino activity when called, we decided to simply queue the state changes to be handled in the main loop. Given the relatively slow mechanical response time of an RC servo, the much more involved handling within the interrupt routine would not provide a noticeably improved response time. As in the display module, a debounce mechanism is included to avoid rapid erroneous state change requests (for example with a mechanical switch).

The idle check module (if enabled) checks when the controller last received a state change request and disengages the servo motors if an idle time has been exceeded. This can allow the user to move the shutter positions manually, which is only possible when the servos are disengaged. We found this capability to be convenient especially during optics alignment, where the shutter controller always seemed to be just out of easy reach.

2.3.7. Library

We provide a library for serial communication with the Arduino in both C and Python. The provided functions in the libraries handle the low-level serial communication and provide easy-to-use access functions. Both libraries utilize the Virtual Instrument Software Architecture (VISA) standard and provide wrapper functions (e.g. to set the shutter parameters or to open/close the shutters).

In addition to these libraries, we provide an example graphical user interface (GUI) in python, based on the tkinter library. Even though NI LabWindows/CVI is a commercial program (not freely available), it is a C IDE that is used in many labs (including ours) and as a convenience we provide the source to build a GUI using this platform.

Mendeley Data

The Servo Mounts directory contains CAD files (both STEP and STL files) to 3D print mounts for attaching the RC servos to an optical post. Included files: ServoPostMount, ServoPostMount_Small The Enclosures directory contains CAD files (both STEP and STL files) to 3D print an enclosure for the shutter controller. Included files: Enclosure_Bottom, Enclosure_Top_TFT, Enclosure_Top_LCD,

8

The Arduino Code directory contains the C source code for the Arduino shutter controller. Included files: ShutterDriverUniversal.ino, Common.h, SerialComm.cpp, SerialComm.h, LCD.cpp, LCD.h, TFT.cpp, TFT.h, Parameters.cpp, Parameters.h, DigInput.cpp, DigInput.h The C Library directory contains source files for the C library that handles communication with the shutter controller. Included files: ArdShutter.c, ArdShutter.h The C GUI directory contains source and resource files to build a test GUI to configure and test the shutter controller. Depends on the C library above and needs NI LabWindows/CVI to be installed.

Included files: ArdShutterTest.c, ArdShutterTest.h, ArdShutterTest.uir The Python Library directory contains source files for the python library that handles communication with the shutter controller. The logging level can be adjusted to include informational messages for debugging. Included files: ard_shutter.py The Python GUI directory contains source files to build a test GUI to configure and test the shutter controller. Depends on the python library above. Included files: ard_shutter_test.py,

Metal

For example, a shutter controller with four shutters using large RC servos, an LCD panel, and serial control would require an Arduino, a servo shield, an LCD shield, a servo power supply, four servos, and various electronics. The cost for this shutter system would amount to about $120.

General Safety Notice:

The assembly of the shutters and shutter controller involves 3D printing, mechanical assembly, electrical wiring, and soldering. To prevent damage to the electrical components, test power supply voltages before wiring cables to the Arduino and/or servo shield. All usual safety precautions should be taken when working with electronics or while soldering.

5.1 Shutter

Fig. 3. Mounting of the servo and the shutter. Small and large servo mounts (a), back and front view of the mounted blade (b), and shutters mounted vertically (axis up and axis down) and horizontally (c). Two sizes of shutter mounts (small and large) are provided for two common sizes of shutters (Fig. 3a).

Small tabs are provided to fix the servo cables, if desired. For the blade, we drill a central hole large enough to clear the ledge on the mounting horn, and two small holes to attach it to the horn with self- tapping screws (Fig. 3b). For mounting, the servo slides in the U-shaped opening in the mount and is secured by four self-tapping screws (usually provided with the servo). Depending on the servo, rubber grommets are provided to minimize vibration transmitted from the servo to the mount. The servo can be mounted with its axle near the post mounting screw holes or opposite, depending on the light path requirements. (Fig. 3c). Finally, the assembled servo mount can be fixed with socket head screws (8-32 or M4 for the small mount, ¼-20 or M6 for the large mount) to a post.

5.2. Controller Hardware

Fig. 4. Electronic modules: Arduino (a), LCD shield (b), TFT shield (c), and servo shield (front (d) and back(e)). Areas on the servo shields marked with color are: headers (A1), servo power connector (A2), right-angle RC servo connectors (A3), servo power supply capacitor (A4), right-angle control input connector (A5), and reset bypass capacitor and jumper (A6). Simplified electrical schematic (f), indicating electrical hookup in areas A5 and A6.

5.2.1. Arduino

In order to mount the servo shield, a set of female headers need to be soldered into the two rows of pins (Fig. 4a).

5.2.2. Servo Shield

Technical details for this shield are provided by Adafruit . To accommodate the display shield, two rows of female through headers and the upside-down SPI female through header need to be soldered into the servo shield (Fig. 4d, A1). A 2-pin screw terminal is soldered onto the shield for the power supply cable (A2), while the other end of the cable is attached to a power supply connector. The use of a cable connector reduces the risk of accidentally plugging the servo power supply into the Arduino power connector and vice versa (both power supplies have the same connector but a different voltage). Leaving all solder pads for the I2C address open results in the default address of 0x40. Because the display shield sits on top of the servo shield, use of angled servo connectors is necessary (A3), unless the cables are soldered directly into the board. We face the first connector outward for ease of access. The remainder of the connectors (if installed) need to face inwards because of the installed headers. Note that installing the header in the opposite direction reverses the order of the servo connector pins (GND on the top vs GND on the bottom). A capacitor to provide surge current for the servos can be installed on the board (A4); the

Authors:

Peder EZ Larson 1, 2,* , Jenna ML Bernard1, James A Bankson 3, Nikolaj Bøgh 4, Robert A Bok1, Albert P. Chen 5, Charles H Cunningham 6,7, Jeremy Gordon1, Jan-Bernd Hövener 8, Christoffer Laustsen 4, Dirk Mayer 9,10, Mary A McLean11 12, Franz Schilling13, James Slater1, Jean-Luc Vanderheyden5, 14, Cornelius von Morze 15, Daniel B Vigneron1, 2, Duan Xu1, 2, and the HP 13C

94143, Usa.

Denmark. 5 GE Healthcare, Menlo Park, California, USA. 6 Physical Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada.

8 Section Biomedical Imaging, Molecular Imaging North Competence Center (MOIN CC), Medicine, Baltimore, MD, USA. Cambridge, United Kingdom.

14Jlvmi Consulting Llc, Dousman, Wi, Usa

#See Acknowledgements for a list of all HP 13C MRI Consensus Group Members This work was supported by the ISMRM Hyperpolarized Media MR Study Group, the ISMRM Hyperpolarization Methods & Equipment Study Group, and the Hyperpolarized MRI Technology Resource Center (NIH/NIBIB grant P41EB013598).

Abstract

MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of hyperpolarized agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate - by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation, (2) MRI system setup and calibrations, (3) data acquisition and image reconstruction, and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the “HP 13C MRI Consensus Group” as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods & Equipment study groups. It further aims to provide a comprehensive reference for future consensus building as the field continues to advance human studies with this metabolic imaging modality.

Keywords: Hyperpolarized MRI, metabolic imaging, carbon-13, pyruvate, dissolution dynamic

Introduction

MRI with hyperpolarized 13C agents, also known as hyperpolarized (HP) 13C MRI, has shown great potential as a novel imaging modality, particularly for its ability to probe metabolic processes in real time. The first human studies with HP [1-13C]pyruvate were performed in 2011 in prostate cancer patients (1).

Since then, there have been over 60 papers published with imaging results of human subjects from 13 different sites, with applications including prostate cancer, brain tumors, breast cancer, kidney cancer, pancreatic cancer, metastatic disease, liver disease, ischemic heart disease, diabetes and cardiomyopathies. The vast majority of these studies used [1-13C]pyruvate (1–63), where [2-13C]pyruvate (64) and 13C-urea (56) have been demonstrated too.

As clinical HP 13C MRI advances, there is a growing need to build consensus for best practices, which are critical for comparing data across sites, performing multi-site trials,deploying methods to new sites, partnering with vendors, and potentially for obtaining broader regulatory approvals.

In March 2022, we initiated an effort to build consensus within the HP 13C MRI community with this opportunity in mind, and it was greeted with strong enthusiasm. The “HP 13C MRI Consensus Group”, containing over 55 members from 27 sites, identified the area of greatest need and opportunity for consensus building to be HP [1-13C]pyruvate human

●

Pyruvate is the most mature and widely used HP agent and has the most significant translational evidence emphasizing the potential clinical impact.

●

Clinical trials, particularly multi-site trials, have the strongest need for consensus methods to ensure that data can be combined across sites. This work is a Position Paper for which the goal is to describe current successful practices and study methods for HP [1-13C]pyruvate human studies along with justification to support those practices. This is divided into four major topic areas: (1) HP 13C-pyruvate preparation, (2) MRI system setup and calibrations, (3) data acquisition and image reconstruction, and (4) data analysis and quantification (Fig. 1). The current successful practices and study methods include a literature review of published peer-reviewed journal papers showing human HP [1-13C]pyruvate study data, up to September 2022 (1–63), as well as new unpublished information from surveys of HP 13C study sites. Based on this information, we also highlight the evidence gaps, strengths, and limitations of current practices which are summarized at the end of each section.

Figure 1: Illustration of the HP 13C MRI human study process, including the 4 major areas covered in this paper: Hyperpolarized 13C-pyruvate preparation, MRI system setup and calibration, Acquisition and Reconstruction, and Data Analysis and Quantification.

Figure 2: Anatomical targets of HP [1-13C]pyruvate MRI human studies published up to September 2022.

Hyperpolarized 13C-Pyruvate Preparation

This section covers the processes for creating the HP agent, 13C pyruvate, and will include many aspects and considerations that are needed to safely and effectively prepare doses for metabolic imaging studies in human subjects. These include material, personnel, equipment and facility, fluid path preparation, quality control, and release.

It is helpful to understand that the specifications of a dose of 13C pyruvate suitable for in vivo MR HP metabolic imaging were shaped in part by early preclinical studies performed by GE HealthCare summarized in Ref. (65). In short, the safety of the two novel drug components, 13C pyruvate and the electron paramagnetic agent (EPA) AH111501, were demonstrated in those studies. The more precise formulation of the dose suitable for human use was then determined from clinical studies (66) that included two Phase 1 clinical trials in young and elderly healthy volunteers without hyperpolarization of the 13C nuclei and another Phase 1/2a dose escalation and imaging feasibility study with HP 13C pyruvate in 31 prostate cancer patients at the With the exception of the first HP 13C imaging clinical trial, which utilized a prototype device in a cleanroom (1), all HP 13C studies performed in humans to date have utilized the SPINlab polarizer (manufactured by GE HealthCare). Consequently all doses of the HP 13C pyruvate delivered by SPINlab have been produced using the “SPINlab Pharmacy Kit” that serves as the container-closure system for the various drug components (13C pyruvic acid and EPA mixture, dissolution medium, and neutralization and dilution medium) during sample polarization, dissolution and quality control (QC) processes. Thus many aspects of the HP sample preparation considerations discussed below are related to the SPINlab instrument and the consumables designed to be used with it (67).

General Considerations

While more than 860 patients or healthy subjects having been injected with HP 13C pyruvate as of January 2022 without reports of any serious adverse events (68), HP 13C pyruvate injection remains an investigational MR contrast agent and can only be administered by those with Investigational New Drug (IND) exemption from the Food and Drug Administration (FDA) in the USA, a Clinical Trial Application (CTA) in Canada, approval from National Research Ethics Committee Services in the UK, or approval from the relevant local regulatory body. Thus, methods and processes involved to produce a dose should have patient safety as the first priority. Since utilizing dissolution dynamic nuclear polarization (dissolution-DNP) for human use is still a relatively new development, there are no existing published regulatory guidelines specifically for this method.

There are two major production styles that determine how various sites approach the agent preparation. In the US, the most common approach is to rely on a sterilizing filter (“Terminal Sterilization”) to ensure sterility of the final product, akin to PET tracer production, where a starting molecule with a radioisotope is processed using various other ingredients to make the final, desired and injectable contrast agent within a necessarily short amount of time (69). For these sites, sterilization of the components and accessories upstream of this filter are not required, although many of them were manufactured and tested following Good Manufacturing Practice (GMP) or Good Laboratory Practice (GLP) requirements. The filling process is usually performed under an ISO 5 laminar flow hood, but a clean room or an isolator is not required.

This approach is typically accompanied by testing the integrity of the sterilizing filter prior to release of the dose for injection. Typically, post release endotoxin and sterility tests are performed using an aliquot reserved from each released dose.

In the UK and EU, the most common approach is to more-closely follow sterile pharmaceutical compounding guidelines (70), where all components and ingredients are required to be sterile or manufactured under GMP guidelines and are assembled and filled within a clean room environment or an isolator system (“Sterile Preparation”). Typically a batch of Pharmacy Kits for HP 13C pyruvate injection are prepared together. The sterility of the final dose is also ensured by batch validation testing, in addition to the sterility of the ingredients and the sterile compounding process. The endotoxin and sterility testing are performed for the process validation but are not performed for each injected dose.

Some institutions fill and assemble the Pharmacy Kit required for a specific study on the same day or the day prior to polarization, dissolution, and patient administration, but others have also demonstrated the feasibility of preparing a batch of kits, keeping them in a -20ºC freezer and using them over a period of a few months.

Beyond the obvious requirements that the process and the facility has to ultimately produce a dose that is safe to inject into a human, regulatory authorities will also focus on the question “Are you in control of your processes?”. To be in control of your process requires an in-depth and broad understanding of all processes involved in pre, post, and during the production process.

Personnel

It is typical and may be required to have licensed personnel involved in the production process depending on local regulations.Typically a pharmacist, radiopharmacist or other similarly qualified person (QP), in charge of the facility where the Pharmacy Kit filling and preparation is taking place, is responsible for the overall process and the release of the injectable dose.

Qualified cleanroom technicians are often involved in the Pharmacy Kit filling under the supervision of the pharmacist or QP. As is required for pharmaceutical compounding or PET tracer production, training requirements and training records for all personnel need to be maintained and available for audit by the FDA or equivalent.

Equipment And Facility

The facility and all equipment need to have standard operating procedures (SOPs) that describe how equipment is used, maintained, and calibrated to comply with relevant legislation. Currently, almost all the filling of the Pharmacy Kit takes place within a compounding laminar flow hood or isolator (typically ISO 5). At some sites, the filling is conducted within a cleanroom, while at others, it is conducted in a dedicated non-cleanroom space, reflecting differences in cleanroom approach and specifications between regulators worldwide (71). Some equipment or facilities, such as the compounding hood or cleanroom, may require external certified laboratories for testing.

Material Handling

Material handling guidelines (69,70) require SOPs detailing a system to track all of the materials involved in the HP production process for a particular patient dose, similar to current good manufacturing practice (cGMP) requirements for material handling for drug compounding. This includes acceptance standards, storage conditions, amount used in the patient dose for each ingredient and materials used in the assembly of the fluid path and Pharmacy Kit. Currently some users choose to open and inspect and sometimes modify the Pharmacy Kits upon arrival, but some users keep them in the sealed packaging until they are required for dose preparation.

Pharmacy Kit Filling And Assembling

As required by an IND or its equivalent, the preparation of the doses of HP 13C agent are detailed in the Chemistry, Manufacturing, and Control (CMC) section of an applicable regulatory submission; an example of this has been made available (72). It describes the processes of filling the Pharmacy Kit with the different components that make up the final drug product, and of assembling the final kit for either storage or immediate use in the polarizer. Special attention should be given to the laser welding process in order to satisfy installation qualification (IQ) and operational qualification (OQ). Typically, the final developed process is validated by process qualification (PQ) runs, during which 3 or more Pharmacy Kits are filled and used and the final HP 13C products are tested for endotoxin and sterility and to confirm that they meet the dose specifications for injections (usually including pyruvate concentration, residual EPA concentration, pH, liquid state polarization level and dose temperature). The data from 3 consecutive PQ runs are submitted as part of the IND submission (or its equivalent), and are often also reviewed by the Institutional Review Board (IRB) where the studies are conducted.

Quality Control And Dose Release

The quality control (QC) and dose release can be separated into two aspects: one is the QC and release of the filled Pharmacy Kit, and second is the QC and release of the HP 13C agent for injection, after polarization and dissolution. For institutions filling a batch of kits and storing them to use over a period of time, typically the batch can be released based on initial validation, environmental monitoring data from the day of kit production, and if filters are used during preparation of any of the components, filter integrity testing. But in some cases one or more kits are used for validation before the batch of kits are released for future use. For institutions that fill only the kits required for specific studies shortly before the experiment, the filled kits often do not go through separate release tests before they are used.

The quality control of the HP 13C pyruvate solution post dissolution is primarily performed to ensure that the agent meets the dose specifications (Table 1) before it is administered to the subject. These specifications target both safety (pH, residual EPA, temperature) and efficacy (pyruvate concentration, polarization, volume). Typically, the pyruvate concentration, residual EPA concentration, pH, dose temperature, dose volume, and liquid state polarization are measured by the QC accessory associated with the SPINlab polarizer. Some users perform a secondary measurement for one of the parameters, such as pH, using a different instrument or pH paper. For sites that do not go through a separate release testing process for batch filled kits, the integrity of the sterilization assurance filter, a part of the Pharmacy Kit, is typically tested as a part of the dose release. It is also common for these users to preserve an aliquot of the final HP 13C pyruvate solution for post-release endotoxin and sterility testing. This testing cannot be completed fast enough to test an individual dose prior to injection, but this is why other processes such as PQ runs and validation testing are done to minimize the chance a subject could be injected with a contaminated dose.

The Final Dose Release And Injection

should be done under the supervision of a licensed professional, based on local regulations.

Some Key Challenges

Many of the challenges associated with HP 13C pyruvate preparation can be attributed to the conditions required for the dissolution-DNP method of high magnetic field (~3-7 T) and very low temperature (~1 K) during polarization, with pressurized and superheated water necessary for the rapid dissolution event. These extreme conditions are quite challenging for the design of the container-closure and fluid path system. In particular, the cryogenic temperature in the polarizer requires special attention to any moisture or ambient (moist) air introduced into that portion of the fluid path, which can form an ice block at ~1 K. This ice can lead to flow restriction during the dissolution event and reduce the strength of the laser welded bond between the cryovial and its cap. This can ultimately produce failures in the dissolution step, including variations in final pyruvate concentration and pH that may fail to meet QC release criteria as well as fluid path ruptures that provide no available dose and result in polarizer down-time.

The polarization of the HP 13C pyruvate sample decays quickly over the span of a few minutes after dissolution, and thus the process of dissolution, QC for release, and injection should be completed as fast as possible to preserve the high polarization level achieved. Any delays in the preparation process, such as transportation time or equipment malfunction, can significantly reduce the final polarization and result in lower quality imaging data.

Current Practices

A summary of data collected from all sites performing clinical trials with HP 13C-pyruvate is shown in Fig. 3 and Table 1, including the specification of the final dose and how the quality control and release of the final dose are performed. There is a split in the Production Style, described in the General Considerations section above, with 8/13 sites using Sterile Preparation versus 5/13 using Terminal Sterilization. While many of the dose specifications show notable differences in acceptable ranges, all of these variations listed in tables have been successfully and safely been used to perform HP 13C pyruvate studies in humans. Their differences depend on the institutions’ preferences, resources and their particular regulatory situation. There is high similarity in pyruvate ranges, temperature ranges, EPA limits, and volume limits. There is modest variability in pH ranges and large variability in the endotoxin test limit. There is a 3-fold difference in acceptable polarization levels, which are measured to ensure a futile dose is not injected since the polarization is directly proportional to SNR. This reflects the decision by several sites to believe that useful data can be still be obtained with suboptimal polarizations.

Figure 3: Hyperpolarized agent preparation methods reported by sites currently performing HP

In House

Table 1: HP 13C-pyruvate preparation parameters, methods, and dose specifications used for quality control testing and release as well as validation. These were obtained from a survey of all sites performing clinical trials with HP [1-13C]pyruvate. The parameters used for product release are noted in bold text, otherwise these parameters are measured for batch validation or other QC measurements. The endotoxin and sterility testing are performed during process validation of the batch and/or post-injection, and largely depends on the agent production approach.

Summary

The overall safety record of HP 13C-pyruvate has been very strong, and the SPINlab hyperpolarizer has proven to provide high polarizations at human sized doses while meeting numerous QC and release criteria. A weakness remains the failure modes of the SPINlab Phamacy Kits (e.g. ice blocks, path ruptures), which are placed under extreme requirements particularly during dissolution. The preparation process still requires a high degree of expertise.

Therefore, there is a significant need to improve the reliability, robustness, and ease of operation for generating HP 13C-pyruvate doses for human studies. Furthermore, there is a divide between manufacturing and sterile compounding style preparation as well as other site-specific practices, resulting in variations in SOPs and justification required to relevant regulatory bodies. There have also been no comparisons between these approaches. It is also unclear what release criteria and QC parameters are truly required to ensure patient safety.

However, all of the reported methods are acceptable and approved by the appropriate regulatory authorities, and have led to the rapid expansion of successful human studies in recent years.

Mri System Setup And Calibrations

This section covers the MRI system setup, including the imaging system, RF coils, phantoms, and prescan calibration methods.

Imaging System

The main prerequisite for a given MRI scanner to be capable of supporting studies with HP 13C is its “broadband” capability to transmit and receive radiofrequency (RF) signal at the frequency of 13C, which is around 4 times lower than 1H. This does not come as a default on clinical MR devices. The transmit power of the broadband amplifier should also be sufficient to support the intended flip angle and RF pulse shape with the employed transmission RF coil(s) for 13C. Most studies to date use relatively low flip angles (< 90 degrees) for HP 13C in order to preserve polarization for time-resolved imaging. The capability to receive 13C signal on multiple channels is also desirable to increase SNR, as discussed further in the “RF coils” section.

The choice of magnetic field strength is primarily dependent on the metabolites’ frequency separation due to chemical shift dispersion and 1H imaging. High field strengths do not enhance hyperpolarized 13C signal as they do for 1H because the signal strength in a HP experiment relies on manipulating the population of quantum energy states outside of the MRI scanner.

However, the injected HP 13C-pyruvate and its metabolic products have greater frequency separation at higher fields, and it may thus be easier to separate and quantify these resonances at higher fields. This comes at the cost of a reduction in the achievable T2* and often reduced T1. As the initial polarization is independent of the imaging field strength it has been proposed that the increased T2* at 1.5T can potentially be exploited to increase SNR by adapting the acquisition bandwidth or reduce off-resonance imaging effects in cases when the decay of the transverse magnetization is dominated by T2* (73). In practice, 3T has been used in all published human 13C-pyruvate studies surveyed (Supporting Table S1), and comprises the majority of scanners currently in use for human studies (Table 3). A field strength of 3T is well-suited for 1H MRI anatomical reference and correlative imaging.

Stronger and more rapidly slewing magnetic field gradients support more rapid spatial encoding, particularly for metabolite-specific single-shot imaging using echo-planar imaging (EPI) or spiral imaging (See “Acquisition and Reconstruction”). Although the spatial resolution acquired for HP 13C imaging is typically much coarser than for 1H MRI, the factor of ~4 in gyromagnetic ratio leads to the same reduction factor in performance of the gradient system, so 13C experiments are potentially more limited by gradient hardware performance. To date, all human studies have used the commercially-available integrated gradient systems provided in clinical MRI scanners.

Optimization of scanner design has understandably focused on minimization of artifacts in 1H MRI, where devices such as room lights, the gradient amplifiers, and the motors driving the patient bed are checked to ensure that they do not produce RF interference at the 1H frequency, but artifacts may arise at other frequencies. Eddy current compensation is also not always appropriately adjusted for nuclei at other frequencies (74). In order to optimize for 13C, many sites have performed checks on phantoms for RF interference, gradient artifacts, and eddy currents (74), including the use of post-hoc gradient impulse response function characterisation and correction, and some vendors have fixed these issues as well.

Rf Coils

For HP 13C imaging studies in humans, RF coils for both 1H and 13C nuclei are needed, with 1H MRI providing an anatomical reference for registration and optional additional multiparametric MRI readouts. At the Larmor frequency of 13C nuclei, the relative contributions from coil noise compared to sample noise increase compared to 1H (73,75), although sample noise still is likely the dominant contributor for human-sized coils at 32.1MHz - the resonance frequency of 13C nuclei at 3T.

The key requirement for human 13C-pyruvate RF coils are that the coil geometry and sensitive volume must cover the volume of interest in the subject. Table 2 and Figure 4 shows coil configurations that have been used and optimized for applications in different anatomic regions.

Volume resonators are most commonly used for transmit, as they surround the subject to

Provide B1 Transmit Across The Fov (B1

+). While 1H relies on a large birdcage (“body”) coil built into the scanner, 13C transmit coils must be placed inside the bore. This takes up valuable space within the magnet, and also has led to the use of designs with relatively inhomogeneous

B1

+. Many human studies have used Helmholz pair resonators for transmit, including the “clamshell coil”, which has a notably inhomogeneous B1

+ Profile But Has Been Used Because Of

relatively easy integration into the scanner bore. B1

+ Variation Results In Variations In The Flip

angles that control the use of the hyperpolarized magnetization and creates errors in common HP metrics (9,76). The exception are head coils, where birdcage designs with highly

Homogeneous B1

+ can be placed around the head while easily fitting inside the bore. As with 1H MRI, higher SNR can typically be achieved by smaller receive coil elements, such as surface coils or phased arrays, and the majority of 13C receive coils used have layouts similar to 1H phased arrays.

RF coil quality control is important to ensure proper functioning of the coils to provide consistent imaging quality, especially with limited natural abundance 13C signal in vivo. It typically involves 1) a physical integrity check of the coil cables and connectors and 2) phantom SNR tests to check the coil’s performance and to monitor it over time (see Phantoms below). An useful reference for RF coil quality control is outlined in the MRI accreditation program of the American College of Radiology (77) and can be adapted for 13C coils.

Notably, configurations for brain and prostate studies used dual-tuned 1H/13C coil designs, which greatly simplify workflow and registration of 1H and 13C images, as no switching of coils is needed.

(1)

Table 2: RF coil configurations reported for human HP [1-13C]pyruvate studies.

Tx = Transmit

coil, RX = receive coil. The commonly used “clamshell” TX coil is a Helmholz pair design. For 1H RF configurations, all used the Body coil for TX unless otherwise noted, and “repositioned” indicates the 13C coil was removed for 1H imaging. One representative reference is listed for each configuration. The RF coil configurations reported in the reviewed papers are shown in Supporting Table S1.

Figure 4: Examples of RF coil configurations used for human HP [1-13C]pyruvate brain studies. (A,B) 13C Clamshell TX (Helmholz pair) and 2× 4-channel paddle RX arrays. (C) 13C Birdcage volume TX and 32-channel RX array (RX array slides into TX coil). (D) 13C Birdcage volume TX and 24-channel RX array, combined with a 1H 8-channel RX array. Image reproduced with permission from Ref (16).

Phantoms

Since hyperpolarized magnetization is non-renewable, phantoms containing 13C nuclei are important to: 1) test the multi-nuclear capabilities of the imaging system, including all parts of the signal excitation and receive chain; 2) perform calibration measurements before a scan with hyperpolarized nuclei; and 3) perform necessary pre-scan adjustments (see “Prescan Calibration” section). The phantoms currently in use are listed in Table 3. Their composition must provide sufficient 13C signal, with additional considerations of conductivity, stability, chemical shift(s) present, potential for dynamic imaging, and cost. The phantom geometries are typically either compact, in order to be used alongside the subject during a HP scan, or large enough to mimic the inner volume of a RF coil for system testing.

One popular compact design contains enriched 13C-urea at high concentration, typically 8 M, which provides a single resonance, placed inside a small container ~1 mL. The most common recipe mixes 13C-urea in a 90% water/10% glycerol solution, with glycerol used to increase the urea solubility and doping with a Gd-based contrast agent to shorten T1 which increases the potential SNR per unit time. For example, when Dotarem is added at a 3:1000 volume ratio the 13C-urea T1 is around 500 ms and T2 is around 100 ms. However, when testing pulse sequences influenced by T1 and T2, doping should be used carefully. This phantom is suitable for frequency calibration, transmit gain calibration, sequence testing, and as a fiducial marker when placed next to a patient. However, enriched 13C-urea has a relatively high cost compared to natural abundance compounds.

For larger volumes (>100 ml), the phantoms most often used contain undiluted ethylene glycol, glycerol, or dimethyl silicone. These compounds have sufficiently high carbon concentrations to provide sufficient 13C signal even with the 1.1% natural abundance of 13C. These larger phantoms matching the inner volume of an RF coil are useful for coil testing, including transmit

+) And Receive (B1

-) coil profile mapping, as well as to mimic acquisitions using in vivo FOV requirements. In this case, size and conductivity should match the expected subject size in order to mimic coil loading and get a realistic estimation of B1+. Large-volume natural abundance urea phantoms have also been used by some sites, but suffer from higher conductivity compared to biological tissues. Typically, it is easier to increase the conductivity and hence coil loading of the non-conductive phantom by adding NaCl to match physiological loading (16,78).

Dynamic phantoms that aim to mimic metabolite kinetics have also been developed (79–81), and have the potential to more closely mimic the HP experiment, but so far these are not widely used.

Prescan Calibration

Prior to performing an MRI acquisition, the so-called prescan procedure is used to set the shim parameters to maximize B0 homogeneity over the field of view (FOV) or a specific region of interest (ROI), the scanner center frequency (CF), the RF transmit gain, and the receiver gain.

While this calibration procedure is usually automated for 1H, the lack of sufficient natural abundance 13C signal prevents use of automated methods. (Although natural abundance 13C lipid signal has been detected, there are so far no reports on using this signal for prescan.) Table 3 shows current practices across sites.

Maximizing B0 homogeneity is independent of the nucleus and is therefore performed prior to 13C imaging using the 1H water signal and existing shimming tools, such as by a standard automated process (“Auto Shimming”) or using high order shimming routines. Similarly, the 13C CF can be calculated from the 1H CF using a predetermined scaling factor that depends on the target chemical shift (82). Another common approach used is to have a small, high-concentration 13C phantom, e.g. 8M 13C-urea, integrated in the RF coil or placed next to the scan subject (1). The reference frequency can also be based on real-time measurements after the HP injection but prior to imaging (83). Both the CF and B0 shimming are critical when using spectrally-selective RF pulses, as inmetabolite-specific imaging methods, where the desired excitation bandwidths are typically very narrow and frequency offsets can lead to a failure mode that is only apparent after injection.

The calibration of the RF transmit power is typically performed on a small, high-concentration 13C phantom placed near the region of interest during the scan or on a large 13C phantom of similar size and coil loading as the subject, prior to the subject scan. Reference power is often done by sweeping the power in a pulse-acquire sequence (53,62), or the Bloch-Siegert method (52,84). When using a small phantom, the location of the phantom, B1

+ Inhomogeneity As Well

as any shielding effects, e.g., when the phantom is integrated into a coil (1), may degrade the accuracy. Other methods include real-time Bloch-Siegert method measurements after the HP injection (83), and using the stronger natural abundance 23Na signal that is close enough to the 13C resonance frequency to be detected by 13C coils (82).

The receiver gain is predetermined, either systematically based on independent phantom measurements and assuming the dose and polarization of the HP compound is known prior to injection, or based on past HP imaging studies.

Power [Kw]

Phantom(s) - during study Phantom(s) - before study 13C Frequency

8

13C-bicarbonate doped with dimethyl silicone, various

Maximum Values

Table 3: Summary of the imaging systems, phantoms, and prescan procedures used at sites currently performing HP 13C-pyruvate human studies. These were obtained from a survey of all sites performing clinical trials with HP [1-13C]pyruvate. *Previously performed studies with a Siemens 3T Tim Trio. The imaging systems, phantoms, and prescan procedures reported in the reviewed papers are shown in Supporting Table S1.

Summary

Commercially available 3T MRI systems are by far the most commonly used for human HP 13C-pyruvate studies, although a systematic investigation of the impact of B0 has only recently been investigated (73). The multi-nuclear RF transmit and receive chain has proven sufficient for current acquisition strategies, although many sites have observed artifacts due to RF interference, gradient interference, and residual eddy currents when operating at the 13C frequency. A variety of 13C RF coils, tailored for numerous anatomical targets, have been successfully demonstrated, with the main limitation that most transmit coils take up a lot of additional space inside the bore and provide relatively inhomogeneous B1

+ Profiles. The

phantoms used have converged into generally 2 categories - small phantoms containing 13C-enriched compounds that can be used during the study and human-sized phantoms containing compounds with high carbon concentrations but without 13C enrichment that are used to test and calibrate the coils. There are no standardized compositions or geometry, and dynamic phantoms that recapitulate in vivo kinetics would be desirable but are still an emerging area. Prescan calibration procedures were not well defined in most publications, so we surveyed individual sites to determine current practices. Calibration procedures for the B0 field (13C CF and shimming) for most sites take advantage of 1H signal and methods, while methods

For Calibration Of B1

+ is more variable across sites, likely a reflection of remaining challenges in how to perform this calibration. Standardization of both phantoms and calibration procedures would synergistically improve the robustness and reproducibility of HP 13C studies.

Acquisition And Reconstruction

Data acquisition strategies in human HP [1-13C]pyruvate MRI studies must account for multiple chemical shifts, efficiently utilize the non-renewable HP magnetization, and acquire data quickly relative to metabolism and relaxation decay processes. These studies require spectral encoding to separate metabolites, necessitating pulse sequences that efficiently encode up to 5D data (3 spatial + 1 spectral + 1 temporal dimension). RF pulses must efficiently sample without immediately saturating the non-renewable HP magnetization, and sequences must acquire data quickly and be robust to both experimental and physiologic variation (e.g. B1

+ Inhomogeneity,

variation in perfusion) to ensure reproducibility and minimize scan-to-scan variability. This section covers current successful practices for data acquisition in human [1-13C]pyruvate studies, and accompanying 1H imaging, from different anatomic regions, including scan parameters and image reconstruction.

Acquisition And Reconstruction Methods

The acquisition methods used in human [1-13C]pyruvate studies can be classified into 3 categories: 1) MR spectroscopy or MR spectroscopic imaging (“MRS/I”), 2) chemical shift encoding methods, and 3) metabolite-specific imaging (Fig. 5).

Mrs/I Methods Specifically

resolve a spectrum that can be analyzed to extract expected as well as unexpected resonances, making this approach very robust. It was used in many initial studies (1).

Chemical Shift

encoding methods, most commonly the Iterative Decomposition of water and fat with Echo Asymmetry and Least-squares estimation (IDEAL) method, use imaging sequences acquired with multiple TEs and rely on a model-based separation of expected chemical shifts (85).

Metabolite-specific imaging methods use specialized RF pulses that are spatially and spectrally selective to excite individual metabolites which are then typically imaged with fast k-space trajectories such as echo planar imaging (EPI) or spirals (86).

Their Application To Different

organ systems is described below. The image reconstruction methods used in human [1-13C]pyruvate studies have typically been conventional methods (e.g. FFT, non-uniform FFT, or equivalent). The incorporation of accelerated imaging and advanced reconstruction methods including parallel imaging (4,57,87) and compressed sensing (7) has also been applied in human studies for improved spatial resolution, temporal resolution and coverage, but have the potential for additional artifacts as well as SNR losses due to ill-conditioning of the reconstruction (e.g. g-factor).

The Majority Of

published studies do not use accelerated imaging indicating the resolution and coverage achievable without acceleration is currently adequate for successful data collection. Performing coil combination, even with fully sampled data has also been shown to have specific challenges for HP human images: using naive sum-of-squares methods suffer from high noise amplification in the relatively low SNR regime of HP [1-13C]pyruvate (compared to 1H), motivating several HP 13C-specific methods that include data-driven coil sensitivity estimation which have shown obvious improvements over sum-of-squares (11).

More recently denoising techniques have been applied as post-processing of human HP data(41,42,44). The techniques applied are based on spatial-temporal singular value decomposition for unsupervised estimation of signal and noise components. They have shown improvements in apparent SNR in the brain and liver, while care must be taken to choose parameters such as the rank threshold to avoid oversmoothing and overfitting to the estimated signal components.

Prostate Studies

Prostate cancer was the first human application of HP [1-13C]pyruvate (1), and data was acquired with MRS/I methods: 1D dynamic MRS, single-slice 2D dynamic echo-planar spectroscopic imaging (EPSI), and single time point 3D EPSI. Advances in imaging strategies led to the development and application of new acquisition schemes, including undersampled 3D EPSI with compressed-sensing (7), model-based chemical shift encoding methods that use a priori information (47,59), and metabolite-specific EPI (10), all of which can provide volumetric whole-organ coverage and dynamic acquisitions.

The pyruvate bolus arrival in the prostate can vary by ± 10 s between patients, necessitating dynamic imaging to reliably and consistently capture the pyruvate bolus (18). For this reason, all currently ongoing studies acquire dynamic data. While MRS/I, chemical shift encoding, and metabolite-specific imaging can all achieve dynamic imaging, chemical shift encoding and metabolite-specific imaging provide greater dynamic and volumetric coverage (85). For scan prescriptions, the FOV is designed to provide full prostate coverage and typically to match the orientation of the anatomic imaging used for registration. Flip angles used in current studies are constant through time, as quantification with a variable-through-time flip scheme is highly sensitive to bolus timing (8) and errors in the RF transmit (B1 +) field (76).

Heart Studies

Data acquisition methods for 13C imaging in the heart must be designed to meet the demands of significant cardiac motion and blood flow. To cope with the periodic cardiac motion, most human heart studies to date used gating to the diastolic window, the longest cardiac cycle interval, which has reduced motion (2,22,28,30,35,36,38,45,52). The duration of the diastolic window limits the available data sampling time, making cardiac acquisitions the most time-constrained of the HP 13C MRI applications. The most common acquisition approach is metabolite-specific imaging with spiral k-space trajectories (2). Their single-shot imaging capability makes these methods particularly robust to motion effects. Furthermore, spiral k-space trajectories provide rapid k-space coverage and relatively benign flow and motion artifacts. The majority of studies have used 2D multi-slice acquisitions, but 3D encoding has also been used successfully (35).

Brain Studies

For HP 13C MRI of the human brain, the majority of studies have also used 2D (slice selective) acquisitions (10–12,14,16,28,33,40,41,44,51,53,60), with a trend toward volumetric coverage using 2D multi-slice metabolite-specific imaging. 3D metabolite-specific imaging of the whole brain, with phase encoding of the slice direction (34,57), has been shown to provide similar SNR efficiency (88) compared with multislice imaging. A number of studies have employed MRS/I (5,6,29,31–33,50,55) resulting in a spectrum from each voxel, which has the advantage of not requiring a priori information about which peaks to encode. This was important in early brain studies when it was not known which peaks would be detectable. Chemical shift encoding, using a set of images with different echo times and an iterative reconstruction of the individual resonances (i.e. the IDEAL approach (85)), has also been used (12,49,54), with the drawback that coverage in the slice direction was limited due to the time required to acquire multiple echo time images.

Abdomen And Breast Studies

The fundamental approaches to data acquisition and reconstruction in the abdomen and breast are largely similar to the aforementioned applications, but demand attention to particular challenges associated with these anatomic regions, especially relating to respiratory motion.

Although it has been shown that a basic 2D MRSI approach based on phase encoding and FID readout can be successfully applied for HP 13C imaging in breast (15) and kidney (13), major advantages in terms of spatiotemporal resolution and coverage have been realized using tailored approaches based on metabolite-specific imaging (43,62) and chemical shift encoding (43), which have facilitated multi-slice or 3D dynamic acquisitions over large FOVs in the abdomen (4,37,46).

The significant respiratory motion encountered in these regions can directly blur 13C images, and has further favored these rapid acquisition strategies. Motion also degrades B0 homogeneity, which can shift frequency-selective excitation profiles and introduce artifacts into rapid imaging readouts. This makes accurate determination of the acquisition center frequency and shimming essential in these regions which often cover large FOVs. (See “Prescan Calibration” section for more information). In some studies, breath-holding was used to minimize motion effects and enforce frame-to-frame data consistency (42). A pragmatic and reasonably effective approach for dealing with respiratory motion during 13C data acquisition is an initial breath-hold (as long as can be tolerated), followed by free-breathing (46,62).

1H Imaging

Collection of 1H imaging data is essential both for prescribing the 13C acquisition and for interpretation of the resulting 13C data. Multi-planar 1H scouts are acquired prior to 13C acquisition to enable graphical prescription of the 13C imaging region. All human HP 13C-pyruvate imaging studies acquire conventional MRI scans (e.g. T1- and T2-weighted volumes) for anatomic reference, aiming to cover at least the full 13C FOV. Acquiring these anatomic scans as close as possible to the time of 13C imaging (immediately before or after) minimizes potential misregistration between the data sets. Depending on the application, other advanced 1H sequences are also acquired (e.g. diffusion-weighted imaging for cancer imaging).

When contrast-enhanced data is acquired, it is done after 13C imaging, as paramagnetic contrast agents will accelerate 13C relaxation.

Reported Study Parameters

Figures 5 and 6, and Supporting Table S2 shows the reported acquisition study parameters for human HP [1-13C]pyruvate studies published as of September 2022. Figure 5 shows a mixture of MRS/I, metabolite-specific imaging, and chemical shift encoding methods have been successfully used, where spectroscopy-based methods have become less prevalent in recent studies. Figure 6 shows the acquisition timing, including the important start time and interval/temporal resolution, is quite variable across studies.

Figure 5: Acquisition methods used in published HP [1-13C]pyruvate human studies published up to September 2022, classified into: MR spectroscopy and spectroscopy imaging (MRS/I); chemical shift encoding methods, such as IDEAL, that use multiple TEs and model-based reconstructions; and metabolite-specific imaging methods that use spectrally-selective excitation to image a single resonance at a time.

Figure 6: Temporal acquisition characteristics reported in HP [1-13C]pyruvate human studies published up to September 2022. (a) Reported referencing of acquisition start times.

(B)

Acquisition start times reported when using dynamic imaging and when timing was reported relative to the end of the injection. (c) Temporal resolutions. “Not Applicable” indicates dynamic imaging was not used.

Summary

Three general categories of acquisition strategies have been used successfully for human HP 13C-pyruvate studies: MRS/I, model-based chemical shift encoding (e.g. IDEAL) methods, and metabolite-specific imaging methods. These have enabled successful studies in the prostate, heart, brain, abdomen, and breast. Recent studies increasingly have used the imaging-based strategies of metabolite-specific imaging and chemical shift encoding which are the fastest methods, although a heads-to–head comparison between techniques has not been performed.

Metabolite-specific imaging is quite popular because of its speed and compatibility with single-shot imaging, but is sensitive to B0 field variations and thus requires careful calibrations. Nearly all studies surveyed acquired data dynamically, allowing measurement of the bolus and metabolite kinetics. The exact timings and associated flip angles vary quite widely across reported studies, with no consensus yet as to how to choose these parameters. Image reconstruction is typically done directly using Fourier Transform methods, and accelerated imaging strategies are uncommon.

Data Analysis And Quantification

This section covers the analysis of data from human HP [1-13C]pyruvate studies, including modeling and metrics, visualization, as well as considerations for how to store data and metadata. Depending on study design, the analysis may need to give quantitative or semi-quantitative output reflecting a biological process or may just reflect a contrast between different regions of interest for quantitative evaluation.

Metrics

Figure 7: HP [1-13C]pyruvate raw data (A) have typically been quantified using four categories of metrics depending on the acquisition. Data acquired as a single time point are often quantified using normalized metabolite images or metabolite ratios (B). Dynamic data can be quantified using normalized metabolite images or metabolite ratios (B), or with metabolite timings such as time-to-peak (TTP) or pharmacokinetic (PK) models (C). The latter two require the data to be time-resolved. [1-13C]alanine and 13C-bicarbonate are analyzed similarly to [1-13C]lactate but omitted here for display.

Metabolite images are commonly used as summary metrics for HP MRI data, often including some form of normalization as well as summed over time as an area under the time curve (AUC) (17). These are analogous to the visual evaluation that is most used for routine clinical work (89,90). In these metabolite images, we expect that the [1-13C]pyruvate AUC signal is predominantly weighted towards perfusion and uptake, while [1-13C]lactate, [1-13C]alanine and 13C-bicarbonate AUCs represent metabolic conversion. The strength of this approach lies in its simplicity and relatively few underlying assumptions. Limitations to the use of single-metabolite images or AUCs include sensitivity to inhomogeneous coil profiles (57,87,91), the acquisition strategy and acquisition parameters, pyruvate polarization and concentration level, and signal relaxation rates (92). Further, the reader must be careful to interpret all the images in conjunction to better understand the underlying biology; for example, increased [1-13C]lactate in the presence of decreased [1-13C]pyruvate delivery can have a very different meaning compared to increased [1-13C]lactate with increased [1-13C]pyruvate delivery.

In an attempt to address variations in coil sensitivity, polarization level, and pyruvate delivery, AUC images are often computed by normalizing to a specified parameter, such as the maximum pyruvate or average lactate signals, or presented as a ratio such as lactate/pyruvate or divided by “total Carbon” - the sum total of HP 13C signal observed across all metabolites. The AUC ratios between metabolites and pyruvate are proportional to the corresponding forward kinetic rates (81,93), but are not directly comparable to rate constants when magnetization loss rates (e.g. relaxation and losses due to signal excitation) differ between studies. Similarly, the ratios between the produced metabolites (e.g. bicarbonate/lactate) can reflect the balance between downstream metabolic pathways (12,55). Care must be taken to consider how AUC images are calculated and normalized before comparing values between studies.

To further quantify the interpretation, pharmacokinetic (PK) modeling approaches were developed to compute the apparent kinetics of pyruvate-to-metabolite exchange (92,94–99). These yield semi-quantitative to quantitative apparent rate constants, given in s-1. Some models require a vascular input function, while others avoid this requirement (95). PK models can explicitly account for acquisition-specific details such as excitation angle and repetition time, and thus may reduce the effects of these details on quantification. An input-less model, provided in the Hyperpolarized-MRI-Toolbox (https://github.com/LarsonLab/hyperpolarized-mri-toolbox) (100) and thus frequently employed for human data, has been shown to fit well and robustly to prostate and brain data (8,20). PK models are quantitative in nature, arguably provide more relevant biological information (8,20), and appear to be reproducible across sites (51). However, rate constants derived from PK models are still apparent rates, and likely do not reflect a single biological characteristic.

Some additional considerations include whether complex or magnitude data is used, as the noise behaviors will impact the analysis differently. Additionally, cut-off thresholds or other criteria may be used to identify and avoid voxels with insufficient SNR before analysis to improve robustness (20,41).

Regardless of the analysis approach, the underlying biology is not always clearly represented by the data; instead, the metrics may be influenced by perfusion, barrier permeability, intercellular shuttles, enzyme activities, co-substrate concentrations, or combinations thereof, depending on the organ and disease of interest (19,43,94,101–103). This may be addressed by incorporating complementary information. As an example, HP 13C pyruvate data is influenced by perfusion, and thus addition of perfusion MRI could be important for interpretation (98,104,105).

All the methods outlined above have been explored in clinical studies, described in Supporting Table 3 and summarized in Figure 8. As of September 2022, approximately 52% of studies involving human subjects report rate constants derived from a PK model with a few different models reported. A nearly equal fraction (51%) of the studies report AUC ratio values.

Approximately 66% of these studies report metabolite-specific images or AUC values. About 40% report SNR values; this metric is particularly frequent in manuscripts that describe technical developments for clinical HP MRI. Approximately 16% of these studies summarize model-free metrics, and 10% report measurements from a single timepoint. Most studies report a combination of quantities.

Figure 8: Reported metrics used for analysis in HP [1-13C]pyruvate human studies published up to September 2022.

Visualization

A wide variety of approaches have been used for visualizing data from human HP 13C-MRI studies. The challenges and practical considerations are: 1) choosing the appropriate metrics to display, 2) how to encode the parameters (e.g. the colormap), and 3) choosing how to provide anatomical context and other multi-parametric data. The choice of visualization also depends on the goal which could be for diagnostic interpretation, but also quality control, reproducibility among readers and publication.

Metrics

The choice of HP 13C metrics is described in detail above. At this stage in HP 13C development where there is no standardized metric, often a combination of metabolite images and ratios or PK model parameters are shown.

Parameter Encoding

The mapping function chosen should provide an adequate, often quantitative, impression of the parameter mapped. There is a consensus in the visualization field that perceptually uniform maps are best suited to visualize continuous parameters, like the greyscale typically used by radiologists as well as other monochrome (black to blue) and color ranges (fire-type, rainbow-type) (106,107). Multi-color heatmaps have been the most frequently employed method for HP 13C data, while greyscale has infrequently been used but it ensures there is no coloring-based bias as well as facilitating later reuse (Fig. 9a). Among the color schemes employed in the clinical HP 13C literature, fire-type scheme seems to be the most common [similar to “Plasma” or “Inferno” in matplotlib.org]. Next most commonly employed is the rainbow-type scheme [similar to “Rainbow” in matplotlib.org].

Anatomical Context

HP MRI faces the challenge that it does not necessarily depict the anatomical features, similar to PET, and thus requires an anatomical reference. Most often, a grayscale anatomical image is overlaid with a HP colormap (Fig. 9c,d). This approach is very intuitive, but can skew perception as the grey-scale anatomical reference may affect the brightness of the HP data (e.g. signal in the skull). This bias does not occur when showing adjacent maps (Fig. 9a, b). Here, anatomical outlines may help to provide reference (Fig. 9b).

Related Journal Articles & DOI Links

Selected peer-reviewed publications relevant to 12 Lead ECG Acquisition. Click the DOI to access the full paper (may require institutional access).

Why Choose Us?

Bangalore guidance for robotics, Spectre and autonomous systems projects.