# Non-Invasive PPG-Based System for Continuous Heart Rate Monitoring of Incubated Avian Embryo

^{*}

## Abstract

**:**

## 1. Introduction

^{2}) in the eggshell. Although the technique developed by Youssef et al. [22] provided continuous and real-time measurements of the developing embryo during incubation, it is an invasive technique that requires careful handling to prevent contamination from taking place. Yu et al. [23] used the PPG technology to non-invasively distinguish between live and dead chicken embryos. In a recent study [24], Phuphanin et al. developed a smartphone-based algorithm for non-invasive detection of the embryonic heart rate.

## 2. Photoplethysmographic (PPG)-Based Embryonic Heart Rate Extraction

^{−1}), respectively. The penetration depth depends mainly on the extinction coefficient of the tissue or the material through which the light passes. This extinction coefficient is defined as the sum the absorption and scatter coefficients (i.e., $\left({\mu}_{a}\left(\lambda \right)+{\mu}_{s}\left(\lambda \right)\right)$). As known fact, light travels further through tissue with low absorptive and scatter properties [26]. Because wavelengths such as red and infrared have low coefficients in most biological tissue, these wavelengths are used in the transmittance mode.

#### 2.1. Hardware Design and Prototype (Egg-PPG)

#### 2.1.1. Light Source System

- (a)
- The light emitting diodes (LEDs)

_{0}, L

_{1}, and L

_{2}(Figure 1) with emitting peak wavelength (${\lambda}_{peak}$) of 945 nm and maximum radiant intensity of 300 mW·Sr

^{−1}(at ~1 A) are used as the light source in the Egg-PPG.

- (b)
- The LED control board

^{−1}.

#### 2.1.2. Photodiode System

- (a)
- Photodiode light sensor

^{2}, is used in the Egg-PPG.

- (b)
- Amplification board

- -
- Transimpedance amplifier

- -
- Analog filters

- -
- Programmable gain amplifier

#### 2.2. Embrypnic Cardiac Wave Extraction Algorithm and Heart Rate Calculation

#### 2.2.1. Pre-Processing of PPG Signals

#### 2.2.2. Wavelet Analysis and Peak Detection

- -
- Continuous Wavelet Transform method

- Compute the discrete Fourier transform (DFT) of the analysed signal $x\left(n\right)$, including $N$ samples, using Fast Fourier Transform (FFT) as follows:$$\widehat{x}\left(k\right)={{\displaystyle \sum}}_{n=0}^{N-1}x\left(n\right){e}^{-i\frac{2\pi}{N}nk},\text{}k=0,1,2\cdots N-1$$
- Obtain the DFT ($\widehat{\psi}$) of the analysed wavelet ($\psi $) at the appropriate angular frequencies as follows:$$\widehat{\psi}\left(k\right)={{\displaystyle \sum}}_{n=0}^{N-1}\psi \left(n\right){e}^{-i\frac{2\pi}{N}nk},\text{}k=0,1,2\cdots N-1$$
- Scale the DFT of the analysed wavelet at different scales to ensure different scales are directly comparable.To obtain the unit energy for each scale $s$, the wavelet function is normalized using the following formula:$$\widehat{\psi}\left(s{\omega}_{k}\right)=\sqrt{\frac{2\pi s}{\mathsf{\Delta}t}}\widehat{\psi}\left(s{\omega}_{k}\right),$$
- Compute the product of the signal DFT and the wavelet DFT over all of the scales. Invert the DFT to obtain the CWT coefficients as follows:$${W}_{s}\left(b\right)=\frac{1}{N}\sqrt{\frac{2\pi s}{\mathsf{\Delta}t}}{{\displaystyle \sum}}_{k=0}^{N-1}\widehat{x}\left(\frac{2\pi}{N\mathsf{\Delta}t}k\right){\widehat{\psi}}^{\ast}\left(s\frac{2\pi}{N\mathsf{\Delta}t}k\right){e}^{i\frac{2\pi}{N}kb}.$$

#### 2.2.3. Power Spectral Entropy and Embryonic Cardiac Wave Recognition

- -
- The probability density function (PDF) of the spectrum of the PPG signal can be estimated by normalisation over all of the frequency components:$${P}_{i}=\frac{S\left({f}_{i}\right)}{{{\displaystyle \sum}}_{k=1}^{{N}_{f}}S\left({f}_{k}\right)},\text{}i=1,2,\cdots {N}_{f}$$
- -
- Then, the spectral entropy (${H}_{n}$) of the PPG $n$th segment is calculated as follows:$${H}_{n}=-{\displaystyle \sum}_{i=1}^{{N}_{f}}{P}_{i}{\mathrm{log}}_{2}{P}_{i}$$

#### 2.2.4. Peak Detection and Heart Rate Calculation

## 3. Experiment and Measurements

#### 3.1. Incubation and Incbuated Eggs

#### 3.2. Data Acquesition and PPG Measurments

_{0}, L

_{1}, and L

_{2}) and the photodiode (P) around the egg (see Figure 2). The housing system (HS) consists of two sections: the largest (M

_{1}) was designed to hold the three LEDs around the egg with the possibility of placing each LED around the vertical axis of the egg (see Figure 2); the small section (M

_{2}) held the photodiode (P) tightly around the egg.

^{®}USB-6009 multifunction data-acquisition (DAQ) device as an interface between the Egg-PPG and the computer. A customized MATLAB script was developed to interface/communicate with the Egg-PPG prototype through the National Instrument

^{®}USB-6009. Using the developed MATLAB interface script, the PPG signal was acquired at a sampling rate of 128 Hz, and the interface controlled both the light intensity of the three LEDs and amplification gain of the acquired PPG signal.

#### 3.3. Detection of Embryonic Cardiac Wave

^{−1}in 50 mW·Sr

^{−1}dB steps) levels and 10 levels of gain (from −10 to −100 dB in 10 dB steps) to search for the ECW. For each combination, a segment of 60 s of PPG measurement was recorded. After performing the pre-processing step, spectral analysis was performed on the recorded PPG segment. Together with the visual inspection, the spectral analysis results were used to detect the presence of the ECW.

## 4. Results and Discussion

#### 4.1. Detection of Embyonic Cardiac Wave and Signal Quality

^{−1}and amplification gain range within $G$ = −50 to −100 dB.

#### 4.2. Embryonic Cadiac Wave Extraction and Heart Rate Calculation

#### 4.3. Real-Time Heart Rate Monitoring Algorithm

PPG_signal (G_{i}, I_{j}) INPUT: G_{i}∈ {−50, −60, −70, −80, −90, −100} dB; and I_{j}∈ {150, 200, 250, 300} mW·Sr^{−1}LOOP initiation: i = 0 and j = 0; i ∈ {0, 1, 2, 3, 4, 5}; j ∈ {0, 1, 2, 3} FOR: G_{i}and I_{j}DO: record 15 s of PPG_signal (G_{i}, I_{j}) DO: calculate E_{S}IF: lower_threshold ≤ E_{S}≤ upper_threshold DO: PPG_signal (G_{i}, I_{j}) contains ECW DO: continue recording ELSE: DO: continue LOOP END

## 5. Conclusions

## Author Contributions

## Funding

## Acknowledgments

## Conflicts of Interest

## Abbreviations

AB | Amplification circuit board |

ACG | Acoustocardiogram |

APG | Acceleration Plethysmogram |

BCG | Ballistocardiogram |

CAM | Chorioallantoic Membrane |

CWT | Continuous Wavelet Transform |

DFT | Discrete Fourier Transform |

DOG | Derivative of Gaussian |

ECG | Electrocardiogram |

ECW | Embryonic Cardiac Wave |

ED | Embryonic Day |

FFT | Fast Fourier Transform |

HPF | High-Pass Filter |

HR | Heart Rate |

ICG | Impedance Cardiogram |

iPPG | image Photoplethysmographic |

LB | LED’s Control Circuit Board |

LED | Light-Emitting Diode |

LPF | Low-Pass Filter |

LVR | Linear Voltage Regulator |

PGA | Programmable Gain Amplifier |

PPG | Photoplethysmography |

SNR | Signal-to-Noise Ratio |

TIA | Transimpedance Amplifier |

WT | Wavelet Transform |

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**Figure 1.**Main hardware components of the developed prototype (Egg-PPG) shows the LED control circuit board (

**LB**) for the infrared LEDs (

**L**,

_{0}**L**, and

_{1}**L**), photodiode (

_{2}**P**), and the photodiode and amplification circuit board (

**AB**) includes a transimpedance amplifier (

**TIA**), low-pass filter (

**LPF**), high-pass filter (

**HPF**), and programmable gain amplifier (

**PGA**).

**Figure 2.**(left graph) a top-view schematic representation of the developed Egg-PPG prototype shows the relative positions of the three infrared LEDs (

**L**,

_{0}**L**and

_{1}**L**), photodiode (

_{2}**P**) and the two sections

**(M**and

_{1}**M**) of the Egg-PPG housing system (

_{2}**HS**), (right graph) the corresponding photographic picture of the Egg-PPG prototype shows the photodiode and amplification circuit board (

**AB**).

**Figure 3.**Block diagram showing the main signal processing steps to extract the embryonic heart rate using the Egg-PPG prototype. The block diagram includes the pre-processing, continuous wavelet transform Fourier transform (

**CWTFT**) algorithm, inverse continuous wavelet transform (

**invCWT**), and peak detection algorithm blocks.

**Figure 4.**The acceleration plethysmogram (APG) signal (red line) resulting from pre-processing compared to the raw PPG signal (blue line), obtained from an incubated fertile egg at embryonic day ED09.

**Figure 5.**The scalogram shows the distribution of the calculated energy using the CWTFT algorithm for the PPG segment, obtained from an incubated fertile egg at embryonic day ED09; most of the energy dominated the scale range from 0.05 to 0.10.

**Figure 6.**Detected cardiac peaks based on the reconstructed embryonic cardiac wave (ECW) in comparison to a pre-processed PPG segment obtained from an incubated fertile egg at embryonic day ED09. The magnified graph shows that the developed ECW extraction algorithm is not susceptible to false readings of the dicrotic notch.

**Figure 7.**Average ± standard deviation (error bars) of estimated embryonic daily heart rate based on the acquired PPG segments using the developed Egg-PPG prototype and embryonic cardiac wave extraction algorithm.

**Figure 8.**Normalized PPG signal with and without the embryonic cardiac wave (ECW) and the corresponding normalised spectral entropy (${E}_{S}$).

**Figure 9.**Flow chart shows the main steps of the developed cardiac signal detection algorithm based on spectral entropy (${E}_{S}$).

**Figure 10.**Raw PPG signal (upper graph) showing signal regions including cardiac waves (shaded regions) and regions with no cardiac waves. Additionally, the corresponding calculated spectral entropy, ${E}_{S}$, (lower graph) of the raw PPG signal shows the defined ${E}_{S}$ thresholds for the cardiac signal.

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## Share and Cite

**MDPI and ACS Style**

Youssef, A.; Berckmans, D.; Norton, T. Non-Invasive PPG-Based System for Continuous Heart Rate Monitoring of Incubated Avian Embryo. *Sensors* **2020**, *20*, 4560.
https://doi.org/10.3390/s20164560

**AMA Style**

Youssef A, Berckmans D, Norton T. Non-Invasive PPG-Based System for Continuous Heart Rate Monitoring of Incubated Avian Embryo. *Sensors*. 2020; 20(16):4560.
https://doi.org/10.3390/s20164560

**Chicago/Turabian Style**

Youssef, Ali, Daniel Berckmans, and Tomas Norton. 2020. "Non-Invasive PPG-Based System for Continuous Heart Rate Monitoring of Incubated Avian Embryo" *Sensors* 20, no. 16: 4560.
https://doi.org/10.3390/s20164560