Five-dimensional co-registered multimodal, multiscale imaging platform: reflectance, fluorescence, FLIM, and Raman

We present a co-registered microscope system that unifies macro/micro reflectance, confocal fluorescence, fluorescence lifetime (FLIM), and Raman on a shared optical path. The system acquires 5D datasets (x, y, z, λ, t) with full spectra (≤0.11 nm) and picosecond timing. A common coordinate frame preserves illumination geometry and pixel-level registration, enabling true cross-modal comparisons that are difficult on separate instruments. Capabilities include 2D FLIM up to 2048×2048, yielding per-pixel half-life maps. The Raman module uses time-gated detection to suppress fluorescence background and reject Rayleigh scattering, isolating Raman bands. A unified graphical user interface (GUI) coordinates hardware control, calibration and a custom-developed acquisition, correlation and analysis pipeline. Calibration and validation were conducted using quantum dots and plant samples, with spectral and lifetime results matching literature. The modular design enables rapid wide-field region of interest mapping and high-resolution, time-resolved detailed imaging, with wide applications in plant phenotyping and tissue characterization.