Label-free subcellular imaging with dynamic spectrally encoded confocal microscopy (D-SECM) with subpixel jitter correction

We present dynamic spectrally encoded confocal microscopy (D-SECM), a high-speed, label-free imaging modality for real-time visualization of subcellular metabolic activity. Sequential, optically sectioned SECM images, with a lateral resolution of 0.62 µm-0.88 µm, were acquired at 100 fps by a modified swept source SECM system, stabilized by fiber-Bragg grating swept source synchronization. D-SECM images are created through real-time pixelwise power spectra mapping into frequency-dependent RGB channels, enabled by GPU-accelerated parallel processing. Validation on freshly excised mouse liver tissue demonstrates that metabolic contrast is detectable with exposure durations as short as 40 ms in the liver. Moreover, volume-prioritized scanning enables three-dimensional D-SECM images to be acquired within a second. In addition to the liver, D-SECM has also been demonstrated in multiple organs, including the lung, thyroid, kidney, and pancreas. These capabilities position D-SECM as a powerful and scalable platform for high-speed, label-free imaging of metabolic dynamics, with unique potential for both basic research and real-time clinical applications.