Long-term cellular imaging and analysis are pivotal for biomedical research and clinical diagnosis. Effective and continuous nanoscale imaging of living cells reveals natural, dynamic, and subcellular alterations at the microscale, which are crucial for understanding long-term cellular morphology and metabolism. Interferometric spectroscopy, which has garnered considerable attention for molecular detection, elucidates nanoscale fluctuations on the sample surfaces. This technology dispenses with the need for precise instruments for generating analytically coherent signals or costly optical setups. The algorithmic reconstruction of the collected interferometric spectral data yields quantitative imaging outcomes. Furthermore, it obviates the reliance on the lens selection ability of the microscope objective, thereby allowing space for the integration of microcell culture devices. Consequently, interferometric spectroscopy analysis has emerged as a highly promising method for addressing the extant challenges in the in situ analysis of unlabeled live cells. A hyperspectral interferometric imaging system for long-term in situ analysis of unlabeled live cells is introduced in this paper, facilitating the quantitative imaging of nanostructures and measurements of the dry mass. Moreover, the system is employed in investigating the quantitative nanostructure and dry mass dynamics of various cells throughout the entire cell cycle, which showcases a potential application of the proposed method and system in the biomedical realm.

The coherent signals generated by light reflected from a substrate and the scattered light within a cell provide valuable insights into the three-dimensional structure and dry mass distribution of the sample. A pivotal innovation in this study is the proposal for a label-free quantitative microscopy imaging technique for hyperspectral interferometric reconstruction. By formulating a mathematical model to characterize the interferometric signal, a sample quantitative reconstruction algorithm was devised, enabling the acquisition of quantitative nanostructures and the dry mass distribution of live cells. In the methodology section, we first establish a hyperspectral interference model for adherent cells on silicon wafers, and subsequently propose a label-free quantitative imaging approach based on this model. The phase distribution in the spatial domain, derived from the reflection spectrum containing the interference information, was converted into refractive index and dry mass information. Subsequently, a microscopic imaging system for hyperspectral interference is introduced. Unlike conventional wide-field optical microscopes, this system features a fiber-optic spectrometer structure for hyperspectral imaging. Additionally, a two-dimensional scanning platform with sub-nanometer-level step accuracy was positioned beneath the sample, facilitating point-by-point scanning with a minimum step of 0.16 nm. An integrated live-cell culture incubator was incorporated into this system. Unlike conventional microscope-equipped incubators, the incubator in this system included a liquid delivery function. Finally, a comprehensive overview of the microscale in situ live cell culture device is provided and the performance of the incubator is demonstrated.

Microscopic imaging and hyperspectral interferometric reconstruction imaging of processed three-dimensional silica photon and individually cultured HeLa cells (Fig.5) are demonstrated in this study. The interferometric reconstruction exhibits a thickness error of merely 1.27 nm, accurately restoring the three-dimensional structure of the phantom. Each pixel achieves a dry mass measurement accuracy of 25.75×10^-18 g. Moreover, quantitative imaging of the entire cell cycle of HeLa cells and HCerEpiC cells is conducted with a comparison of the stem mass changes between the two cell types (Fig.6). Subsequently, the nuclear-to-cytoplasmic ratios of the two different cell types throughout the cell cycle are determined. Notably, the nuclear-to-cytoplasmic ratio of HeLa cells increases to 24.45% compared with 16.27% in HCerEpiC cells, indicating a relatively higher proportion of nuclear dry mass. The lateral resolution of the imaging system is 708.1 nm, and the axial resolution can achieve 91.89 nm. The complete spectral data of a single cell can be obtained in approximately 1.5 min.

As the fundamental building blocks of life, cells hold significant importance across various application domains, including in clinical medical diagnosis, life science research, and basic medical investigations. However, high-resolution measurement techniques such as electron microscopy, near-field optics, and stochastic optical reconstruction microscopy, as well as separation and purification methods such as chromatography, mass spectrometry, and spectrophotometry, are not conducive to in situ real-time measurements of live cell cultures. The novel hyperspectral white-light interference live-cell microscopy imaging technology proposed in this study captures interferometric hyperspectral data from samples, accomplishes three-dimensional reconstruction using an algorithmic design, and constructs a corresponding automated integrated instrument. This instrument comprises a reflective optical microscopy imaging setup, an interference spectrum acquisition and processing module, a sample nanoscale precision scanning module, and a live cell culture module. To some extent, it addresses the limitations of current measurement techniques and achieves the objective of simultaneously acquiring the quantitative nanostructures and dry mass distribution of live cells. The self-reflective interference structure employed by the system eliminates the need for intricate optical modulation components, exhibiting a straightforward design and convenient operation, thereby introducing a novel imaging approach to the biomedical field.