2.1.

Gating Algorithm and Circuit

The gating algorithm was designed to provide a synchronized acquisition gate, or “acquisition window,” derived from an incoming signal. Here, we describe the synchronized acquisition of images captured by a camera with respect to an incoming physiological signal. The physiological signal was assumed to be pseudo-periodic and directly linked to the motion of the image being acquired. Thus, one can use the previous period to predict the width of the following period. It was then possible to acquire the phase and period of the physiological input signal and to generate a user-controlled acquisition window in real time. Various techniques are available for performing this operation and are reviewed in Ref. 24 The most commonly used techniques are periodic threshold detection coupled with differentiation filtering, digital filters, or pattern recognition.

The following is an overview of the electronic circuit used by our gating device, with more detailed information available at www.frangionilab.org. First, the incoming analog signal ( $\pm 10\mathrm{V}$ maximum) was conditioned using a precision differential instrumentation amplifier, low-pass filter, tunable high-pass filter, and tunable gain to provide $\pm 1\mathrm{V}\text{to}\pm 4\mathrm{V}$ input to the circuit. Phase was then assessed through threshold detection and differentiation filtering. Threshold detection ensured that no other extrema were incorrectly identified as the phase of the signal. Threshold detection was also automatically scaled to the previous period’s maximum to accommodate various types of incoming signals. It is important to note that a single-threshold approach does not guarantee a completely accurate time detection of the phase due to changes in the shape of the signal. The addition of a differentiation filter ensures an accurate and repeatable detection of the signal’s phase by the means of extremum detection [in the case of ECG, the peak of the R wave in the QRS complex; Fig. 1a ]. Last, the threshold and extremum detection signals were combined together into a single transistor-transistor logic (TTL) pulse per period indicative of the incoming signal’s phase. The phase pulse was then used to reset a timing sequence, which controls the delay and width of the acquisition window [Figs. 1a and 1b].

Fig. 1

Principles of the gating circuit. (a) Representative ECG tracing showing phase detection via the R wave of the QRS complex (arrows) and generation of an acquisition window of desired delay and width. (b) Flowchart of the gating circuit algorithm starting with a pseudo-periodic signal and ending with a precise acquisition window. (c) Overview of the timing sequence embedded in the field-programmable gate array (FPGA).

The timing sequence was generated using a field-programmable gate array (FPGA; Xilinx Spartan 3AN). The schematic of the timing sequence within the FPGA (programmed by the Boston University Electronics Design Facility, Boston, Massachusetts) is shown in Fig. 1c. The phase detection pulse was used to launch a timing sequence generated by a user-controlled timing clock. In order to accommodate a wide range of signal frequencies with high timing accuracy, two $14\text{-bit}$ counters were used: one defining the start of the acquisition window (referred to as the “delay” relative to the phase signal) and the other defining the end of the acquisition window (referred to as the “width”). The desired delay and width could be entered as absolute values, representative of the units of the timing clock, or could be scaled automatically as a percentage of the previous period. The latter assumes that signal period and physical displacement are proportional, which permits compensation for variability in frequency (e.g., with respiration), as can occur with a normal ECG. The final acquisition window was a jumper-selectable TTL or complementary metal-oxide semiconductor (CMOS)-compatible pulse.

Gating device components were from DigiKey (Thief River Falls, Minnesota) and Mouser (Mansfield, Texas). Four-layer printed circuit boards (PCBs) were fabricated by Nashua Circuits (Nashua, New Hampshire) and assembled by Sure Design (Farmingdale, New Jersey). Gerber files for PCB manufacturing, a parts list, assembly instructions, and a user manual are available at www.frangionilab.org.

2.2.

In Vitro Validation Experiments

We performed two types of in vitro validation of the gating device. Electrical validation was performed to test production of a reliable acquisition window from one or more periodic input signals, while functional validation was performed to test camera acquisition of an image in cyclic motion.

Input signals were generated using a Hewlett Packard (HP) model HP3245A dual-channel signal generator. One channel was used to produce a higher frequency triangular wave (e.g., ECG-like) with frequency $f$ , $1\text{-}\mathrm{V}$ peak-to-peak (Vpp), and 10% duty cycle, while the other was used to produce a lower frequency sine wave (e.g., plethysmograph-like) with frequency $f∕10$ , $250\mathrm{mVpp}$ , and 50% duty cycle. The lower frequency signal was also added to the higher frequency signal using a model ZFRSC-42 voltage combiner (Mini-Circuits, Brooklyn, New York) to simulate the type of slow baseline drift typically seen in clinical tracings. Testing frequencies $f$ ranged from 60 to 600 beats per minute (bpm). Circuit inputs and outputs were monitored using a LeCroy WaveSurfer 44Xs, $400\text{-}\mathrm{MHz}$ $(2.5\mathrm{GS}∕\mathrm{s})$ digital oscilloscope. Power for the gating device was provided by a $\pm 12\text{-}\mathrm{V}$ , $+5\text{-}\mathrm{V}$ model WM220-1 (Elpac, Irvine, California) power supply.

To test functionality of the gating device, we constructed a motion simulator capable of moving an optical phantom using an arbitrary analog voltage input. The motion simulator consisted of a custom vertical translation stage (Microvideo Instruments, Avon, Massachusetts) that could be tilted to produce $x$ , $y$ , and $z$ motion, a model VL23-020D-ZAA DC servo motor (Applied Motion, Watsonville, California), and a model BLU-200 (Applied Motion) motor controller. Gated image acquisition was performed on a single PC with the acquisition window triggering a National Instruments (Austin, Texas) PCI-6229 multifunction board. Images were acquired, displayed, and saved using custom LabView software (National Instruments) capable of simultaneously acquiring two Firewire cameras [color and near-infrared (NIR) fluorescence] with triggering from the multifunction board. The cameras used were an Imitech (Seoul, Korea) IMC-80F for color video and a Hamamatsu (Bridgewater, New Jersey) Orca-AG for NIR fluorescence. Cameras were mounted to custom optics as described in detail previously.²⁷ Color camera exposure time was $67\mathrm{msec}$ and NIR camera exposure time was $100\mathrm{ms}$ . The optical phantom consisted of a 384-well plate $(85\mathrm{mm}\times 130\mathrm{mm})$ filled with either $10\mu \mathrm{M}$ indocyanine green (ICG; NIR fluorescent) in dimethylsulfoxide or 2% skim milk (visible with color camera). White $\left(400\text{to}650\mathrm{nm}\right)$ and $760\mathrm{nm}$ NIR fluorescence excitation light was provided by custom LED modules.²⁸

2.3.

In Vivo Validation Experiments

In vivo validation of the device was performed during NIR fluorescence angiography of the beating swine heart using intravenously injected ICG ( $0.014\mathrm{mg}∕\mathrm{kg}$ per injection) as the contrast agent. Animals were studied under the supervision of an approved institutional protocol. Yorkshire pigs $(n=3)$ weighing $30\mathrm{kg}$ and of either sex were purchased from E. M. Parsons and Sons (Hadley, Massachusetts). Anesthesia was induced using $4.4\mathrm{mg}∕\mathrm{kg}$ intramuscular Telazol (Fort Dodge Labs, Fort Dodge, Iowa), and maintained through a $7\text{-}\mathrm{mm}$ endotracheal tube with 1.5% isoflurane/balance ${\mathrm{O}}_2$ at $5\mathrm{L}∕\min$ . The heart was exposed for imaging by a midline sternotomy. After each study, anesthetized pigs were euthanized by rapid intravenous injection of $10\mathrm{ml}$ of Fatal-Plus (Vortech Pharmaceuticals, Dearborn, Michigan).

The ECG signal was obtained through standard three-lead electrodes attached to a Datex/Ohmeda model S/5 vital signs monitor. The acquisition window from the gating device was used to trigger image acquisition by custom LabView software. Color camera exposure time was $67\mathrm{msec}$ , and NIR camera exposure time was $100\mathrm{ms}$ . Color and NIR images were acquired with gating on and off, and regions of interest over the left anterior descending artery and the great cardiac vein were quantified over time for NIR fluorescence intensity.

3.1.

Gating Device

An assembled gating device is shown in Fig. 2a . The assembled PCB could be mounted in an enclosure, controlled via manual switches, and operated as a stand-alone device or operated in a fully automated fashion using the integrated USB port. Indeed, the gating device was designed with dimensions of $14.5\mathrm{cm}$ $\mathrm{W}\times 9\mathrm{cm}$ $\mathrm{D}\times 1.5\mathrm{cm}$ H, permitting up to two stacked devices to be mounted in a CA-1000 enclosure (National Instruments) and thus connected seamlessly to a multifunction board. The total price for a single prototype device was $1,000, which included $200 for board fabrication, $300 for components, and $500 for manual board assembly. When manufactured in larger quantities, the price per device fell to less than $300.

Fig. 2

Stackable gating devices. (a) Single assembled gating device showing major functions of the PCB as well as the USB control port. (b) Two stacked gating devices for use in ANDing two different, dephased input signals to provide a single image acquisition window.

To produce a single acquisition window from multiple simultaneous signals, such as from simultaneous cardiac and respiratory gating, devices were designed to be stackable [Fig. 2b]. When stacked, the acquisition window from the lowest device in the stack outputs the logical AND of the acquisition windows from all gating devices above it [Fig. 3a ], although individual acquisition windows are also available if needed. Each stacked device has its own selectable input frequency range (Table 1 ) chosen from $4\text{to}40\text{cycles}\text{per}\text{minute}$ (cpm), $25\text{to}250\mathrm{cpm}$ , and $100\text{to}1000\mathrm{cpm}$ . These frequency ranges cover the entire spectrum from mouse ECG to human plethysmograph. In addition, each board has a TTL- and CMOS-compatible input to suppress the acquisition window in the setting of nonperiodic signals, such as from involuntary or voluntary muscle movement detected using an accelerometer [Fig. 2a]. As suggested in the figure, such signals might need to be inverted depending on the sensor output.

Fig. 3

Accommodation of multiple periodic and nonperiodic input signals by the gating circuit. (a) Schematic showing $n$ pseudo-periodic input signals combined with a nonperiodic input signal to provide a single image acquisition window of any desired delay and width. Note that the nonperiodic input signal, for example, from an accelerometer detecting involuntary or voluntary motion, may have to be inverted prior to the logical AND. The image acquisition window is a TTL-or CMOS-compatible pulse. (b) Electrical validation of the gating device. Shown are a $1\text{-}\mathrm{Hz}$ , $1\text{-}\mathrm{Vpp}$ triangular wave of 10% duty cycle (ECG-like signal) with a superimposed $0.1\text{-}\mathrm{Hz}$ , $250\text{-}\mathrm{mVpp}$ sine wave (plethysmograph-like) of 50% duty cycle (top); a $0.1\text{-}\mathrm{Hz}$ , $250\text{-}\mathrm{mVpp}$ sine wave (plethysmograph-like) of 50% duty cycle (middle); and the logical AND of acquisition windows from each (bottom).

Table 1

Setting the gating circuit frequency range based on the species of interest and source of motion-induced noise.

3.2.

In Vitro Validation Experiments

By selecting the appropriate frequency range (Table 1), we tested operation of the gating device with input frequencies from $4\text{to}1000\text{cycles}\text{per}\text{minute}$ (cpm). Electrical outputs from a typical experiment are shown in Fig. 3b. Notice that a feature built into the circuit is the ability to remove slow baseline drift during signal processing. For each type of input signal, a stable and reliable acquisition window could be generated and ANDed to produce an acquisition window that fit multiple criteria. For all frequency ranges, timing resolution was $⩽0.06\%$ , and jitter was $⩽0.008\%$ of the input signal’s period. This high precision was the result of employing a $17\text{-bit}$ timing clock and $14\text{-bit}$ counters within the FPGA, which reduced synchronization jitter between the phase pulse and timing clock.

To simulate a beating heart, a motion simulator [Fig. 4a ] that moves a stage vertically and laterally in response to an analog signal was constructed. To simulate an ECG, the input signal was a $1\text{-}\mathrm{Hz}$ triangular wave of $2\mathrm{Vpp}$ and 10% duty cycle [Fig. 4b]. As shown in Fig. 4c and Videos 1 and 2 (gating off and gating on, respectively), the gating device dramatically improved both color video and NIR fluorescence image acquisition. Indeed, with gating on, the images appeared crisp, co-registered, and motionless except for an occasional slight phase shift due to software overhead.

Fig. 4

In vitro validation of the gating device using simultaneous color video and NIR fluorescence imaging. (a) DC servo motor–based motion simulator with moving stage. (b) $1\text{-}\mathrm{Hz}$ , $2\text{-}\mathrm{Vpp}$ triangular wave of 10% duty cycle (ECG-like signal) used to drive the motion simulator. Desired acquisition window is also indicated. (c) Real-time imaging of a 384-well plate having wells filled with visibly white (the word “Lab”) or NIR fluorescent (the word “JVF”) liquid and placed on the motion simulator stage. Shown are the color image (top), NIR fluorescence image (middle), and pseudo-colored (lime green) merge of the two (bottom) in the absence (left) and presence (right) of gating, as described in Fig. 4b. (Color online only.)

Video 1

Real-time imaging of a 384-well plate having wells filled with visibly white (the word “Lab”) or NIR fluorescent (the word “JVF”) liquid and placed on the motion simulator stage. Shown are the color image (top left), NIR fluorescence image (top right), and pseudo-colored (lime green) merge of the two (bottom) in the absence of gating (see Fig. 4). (Color online only.) (QuickTime, 1.81 MB). .

Video 2

Same as Video 1, but in the presence of gating (see Fig. 4). (QuickTime, 730 KB).

3.3.

In Vivo Validation Experiments

The beating heart of a $35\text{-}\mathrm{kg}$ pig is the closest approximation to a human heart and represents a worst case scenario for optical imaging since each systole results in over $3\mathrm{cm}$ of vertical, lateral, and horizontal displacement. Nevertheless, as shown in Fig. 5 (top) and Videos 3 and 4 (gating off and gating on, respectively), the gating device was able to “freeze” image acquisition during NIR fluorescence angiography such that even the smallest arteries and veins were visible and could be imaged as if they were immobile. In fact, the only blurring that occurred was the result of the $100\text{-}\mathrm{msec}$ exposure time necessary for NIR fluorescence imaging.

Fig. 5

In vivo validation of gating device performance using the beating heart of a pig. Shown is NIR fluorescence angiography using $0.014\mathrm{mg}∕\mathrm{kg}$ ICG injected intravenously with gating off (left) or gating on (right). In addition to color video images (top) and NIR fluorescence images (middle), the NIR fluorescence pixel intensity ratios of the left anterior descending coronary artery (A) to the great cardiac vein (V) are also shown (bottom). NIR fluorescence images have identical exposure times and normalizations.

Video 3

In vivo validation of gating device performance using the beating heart of a pig. Shown is NIR fluorescence angiography using $0.014\mathrm{mg}∕\mathrm{kg}$ ICG injected intravenously. Shown are the color image (top left), NIR fluorescence image (top right), and pseudo-colored (lime green) merge of the two (bottom) in the absence of gating (see Fig. 5) (Color online only.) (QuickTime, 4.98 MB). .

Video 4

Same as Video 3, but in the presence of gating (see Fig. 5) (QuickTime, 2.35 MB). .

Importantly, in addition to qualitative improvements in optical imaging, the gating device also enabled quantitation of the NIR fluorescence signal emanating from the arteries and veins of the heart. Shown in Fig. 5 (bottom) is the ratio of the NIR fluorescence signal arising from the left anterior descending coronary artery and the great cardiac vein. In the absence of gating, considerable noise exists in this measurement due to blurring and is seen as a $1.2\text{-}\mathrm{Hz}$ signal of $\approx 5\%$ standard deviation superimposed on the artery/vein ratio. However, in the presence of gating, the noise of the measurement is reduced to $<2\%$ standard deviation, and the various phases of vascular filling can be identified readily without interference from the heartbeat.