period in the life cycle of fish, during which rapid morphological and physiological changes occur. Subtle shifts in environmental conditions--such as temperature, pH, or exposure to pollutants--can significantly affect cardiac performance during this stage, potentially leading to developmental abnormalities or increased mortality [9; 10; 11; 12; 13; 14; 15]. As such, heart rate measurements serve as an early and sensitive biomarker for detecting physiological stress and sublethal toxicological effects [16; 17; 18; 19; 20; 21; 22]. Second, in the context of comparative physiology and developmental biology, heart rate data provide key insights into the evolution and function of the vertebrate cardiovascular system. Transparent fish models, like Danionella or zebrafish, are widely used in genetics, neurobiology, and pharmacological research. These species allow researchers to correlate physiological readouts such as heart rate with molecular or genetic manipulations, enhancing our understanding of gene function and cardiac regulation. Furthermore, developmental studies often require high-throughput screening methods, and heart rate monitoring is an effective metric for evaluating the impact of genetic mutations, drugs, or environmental exposures.  
Traditionally, heart rate in larval fish is monitored using video imaging methods. These techniques involve recording high-frame-rate video of the fish and analyzing changes in pixel intensity within the heart region to detect rhythmic movements associated with cardiac contractions. While this approach is non-invasive and provides accurate results, it typically requires manual pre-processing steps, including the selection and definition of the heart region (the area of interest) in each video. This manual annotation is time-consuming, subjective, and limits scalability, especially in high-throughput experiments. Moreover, many of the existing software solutions for video-based heart rate analysis are commercially available and expensive. Their high cost and complexity can be barriers for small laboratories or educational institutions, and their lack of adaptability may hinder their application across different species or experimental settings.  
To overcome these limitations, this study introduces a novel heart rate monitoring system based on the analysis of interframe luminance differences. By measuring frame-to-frame changes in brightness within video sequences of Danionella larvae, our system detects the rhythmic movements of the beating heart of the region of interest. This approach enables semi-automated, low-cost, and accessible heart rate monitoring suitable for a wide range of applications in developmental biology, ecophysiology, and toxicology research.  
The aim of this study is to develop and evaluate the feasibility and accuracy of this interframe luminance difference-based method for heart rate monitoring in fish larvae. We test our system using transparent Danionella larvae and compare the heart rate measurements obtained through our method with values reported in previous studies. By validating our system, we demonstrate its potential as a reliable and efficient tool for non-invasive physiological monitoring in aquatic research. The ability to monitor heart rate accurately and continuously at early developmental stages holds significant implications for understanding larval development, environmental adaptation, and stress physiology in fish.