ABSTRACT Spectral imaging extends the capabilities of conventional microscopy by incorporating spectral information into spatially resolved images, enabling enhanced characterization of biological specimens. This paper presents a multimodal microscopic spectral imaging (MmSI) system that integrates spectral and temporal‐spectral scanning for comprehensive microscopic analysis. The MmSI system employs a liquid crystal tunable filter (LCTF) to achieve flexible spectral selection and high spatial resolution, facilitating transmission hyperspectral imaging (THSI), fluorescence hyperspectral imaging (FHSI), and multi‐wavelength time‐lapse imaging. A custom‐developed graphical user interface synchronizes the LCTF and CCD camera for efficient acquisition of spatial, spectral, and temporal‐spectral data. System calibration and characterization in the spatial, spectral, and temporal domains, including linearity assessment with respect to exposure time and emission intensity, confirm its quantitative accuracy. The effectiveness and reliability of the system are demonstrated through validation experiments conducted on model systems, demonstrating its versatility for advanced biomedical research, particularly in the investigation of dynamic biological processes.
Pal et al. (Sun,) studied this question.