In hyperpolarized gas MRI, the accurate calibration of flip angles is important for maximizing the SNR per unit time[1]. However, the calibration of flip angles is sensitive to relaxation effects. Moreover, the spin-lattice relaxation time (T₁) of the gas can reflect the partial oxygen pressure, which is an important parameter related to the efficiency of gas exchange in the lung[2]. The measurement of T₁ also requires a pulse flip angle calibration[3]. Most previous methods generally require more than two breaths: one for the flip angle calibration and another one for the T₁ measurement. A previous study[4] by Max et al. provided a method for the simultaneous estimation of T₁ and the flip angle in a single scan through acquisition at non-regular time intervals. However, the long acquisition time needed for mounting time intervals limits its usefulness during in vivo applications. In this work, we propose a novel method to simultaneously calibrate flip angles and measure T₁ of hyperpolarized gas during a short single breath-hold in vivo.Our proposed method for calibration in a single-breath is shown in Fig. 1a. First, we set a transmitter gain TG₁ to get a small flip angle θ₁ in the range 3°-6°. After 8 excitations by θ₁, the transmitter gain is changed to a smaller value TG₂ to obtain a larger flip angle θ₂. Subsequently, we set TG₃~TG_m to obtain gradually larger flip angles θ₃~θ_m, 8 excitations for each angle. After acquiring all of the transverse signals by the N excitations, we can fit the unknown flip angles using one of two methods. The first method involves fitting the m angles to $$$S_{k}=S_{1}\cdot\left(\cos\theta\cdot\exp\left(-TR/T_{1}\right)\right)^{k-1}$$$ with k=1-8, by setting an initial T₁ value, where TR is repetition time. Once the value θ₁ is fitted, the flip angle θ₂ can be fitted to $$$\theta_{2}=\arctan\left[\tan\theta_{1}\cdot\exp\left(TR/T_{1}\right)\cdot S_{9}/S_{8}\right]$$$. We denote the flip angle θ₂ calibrated from the first method as θ₂′, and the one calibrated using the second method as θ₂″. If θ₂′ is equal to θ₂″, this means that the initial T₁ value is correct. If θ₂′ is not equal to θ₂″, this means that the initial T₁ value is incorrect, and we should change the T₁ to another value. The correct T₁ value can be derived from the m angles to minimize the measurement error. Then the correct flip angles can be fitted with the correct T₁. All experiments were performed on a 7 T animal MRI scanner with a homebuilt 8-leg rigid transmit-receive birdcage coil. The traditional multi-breath method was used for contrast. In the traditional flip angle measurement experiment, 16 different flip angles are measured with 16 breaths, and 112 excitations for each angle. T₁ is measured by varying the delay time between trigger and the 1st RF excitation, with 7 different delay times in 7 breaths. The normal breaths are ventilated with pure oxygen, where different T₁ values are acquired by using different xenon gas pre-wash times. Both the flip angle and T₁ are measured in a single breath-hold by the method described in this study. The single-breath method was repeated 5 times for testing its robustness. TR was 67 ms for balloon and 33.7 ms for rat. The breath-hold time needed was 4 seconds for rat.The result of flip angle calibration is shown in Table 1. The parameters were those in $$$TG_{\theta}=TG_{90°}+A\cdot\log_{10}{90/\theta}$$$. Xenon pre-wash time was 0 in all measurements. T₁ was set to 14 s in balloon, and 5 s in rat, when calibrated by multi-breath method. The result of T₁ measurement is shown in Table 2, where the traditional method was repeated twice for the 0-time pre-wash in balloon, and once for others.The calibration results of both flip angles and T₁ by the single-breath method described in this study are accurate. As shown in Table 1, the TG_90° in rat lung is smaller than that in balloon, which is because of the RF stacking in rat tissues. The parameter A is close to 20, but not exactly equal to 20, which is an interesting phenomenon. The measured T₁ values with 0-time pre-wash are shorter than those with the 1-time pre-wash, which reflects different oxygen pressures. The deviations of 5 measurements are small, thus confirming the robustness of this method.This study proposed a novel method to simultaneously calibrate flip angles and measure T₁ of hyperpolarized gas during a short single breath-hold in vivo.