Glass substrate-based mid-infrared spectroscopy: Progress and applications (<italic>invited</italic>)

Significance Mid-infrared (MIR) spectroscopy has become an important tool in numerous fields, offering valuable spectroscopic information on molecular vibrations. Especially in biomedical and clinical research, with the continuous development of instrumental technologies and analytical methods, such as Fourier Transform Infrared (FTIR) spectrometers, MIR spectroscopy has demonstrated significant potential for disease identification and diagnosis, due to the unique absorption characteristics of natural tissue components—such as proteins, lipids, nucleic acids, and carbohydrates (collectively termed the disease metabolome)—in the MIR spectral region. However, its real-world clinical application has been hindered by the need for specialized substrates. Currently, commonly used infrared-transparent substrates used for transmission test, such as calcium fluoride (CaF2) or barium fluoride (BaF2), are costly and fragile, making it difficult for these substrates to be applied in real pathology. Although infrared reflective low-emission substrates are derived from glasses that are cheaper and more robust, they are incompatible with conventional visible-light microscopy due to their inability to transmit visible light. In contrast, pathological glass substrates are cost-effective, durable, and widely used in clinical settings like tissue microscopy, cell culture, and microfluidic chips. Therefore, if MIR spectroscopic analysis could be directly performed on glass substrates, it would not only significantly reduce the cost of infrared spectroscopic analysis but also enhance the compatibility of MIR spectroscopy with clinical applications, providing crucial support for its use in metabolomics and clinical pathological analysis.Progress Recent advances have enabled MIR spectral analysis on glass substrates. When maintaining the conventional transmission spectroscopy mode based on glass substrates, researchers have effectively overcome the high infrared absorption of glass materials by employing thinner glass substrates like glass coverslips and higher-intensity mid-infrared light sources like synchrotron radiation sources, enabling transmission spectroscopy detection in part of the MIR spectral region. Driven by advancements in emerging technologies such as optical photothermal infrared (O-PTIR), deep learning-based glass substrate signal restoration neural network (GLASSR-Net), and plasmonic metasurfaces, researchers have enabled MIR spectroscopic measurements across the entire fingerprint region (1 800-1000 cm−1) through modifications to the experimental setup to combine with the new technologies. These developments have significantly advanced the feasibility of MIR spectroscopy in clinical applications.Conclusions and Prospects In conclusion, this review summarizes recent advances in the measurement of MIR spectroscopy on glass substrates. MIR spectroscopy has found widespread applications across various fields, particularly in biomedical and clinical research, due to the characteristic absorption bands of multiple molecular vibrational modes in the MIR region, combined with the development of advanced technologies and analytical algorithms. As highlighted, substantial progress has been made in glass substrate-based MIR spectroscopy, paving the way for MIR spectroscopy to play an increasingly important role in broader applications. However, current research in this area remains predominantly focused on the innovation and preliminary validation of technical approaches, lacking systematic assessments of stability, applicability, and validation in real-world scenarios. Particularly in terms of effectively integrating this technology into clinical practice and broader practical applications, significant challenges and unresolved questions remain. Future research should not only continue exploring more stable and advanced technical pathways but also prioritize clinical scalability as a central objective, thereby facilitating the transition of this technology from laboratory settings to real-world applications.