Label-free imaging of intracellular organelle dynamics using flat-fielding quantitative phase contrast microscopy (FF-QPCM)

Abstract

Panoramic and long-term observation of nanosized organelle dynamics and interactions with high spatiotemporal resolution still hold great challenge for current imaging platforms. In this study, we propose a live-organelle imaging platform, where a flat-fielding quantitative phase contrast microscope (FF-QPCM) visualizes all the membrane-bound subcellular organelles, and an intermittent fluorescence channel assists in specific organelle identification. FF-QPCM features a high spatiotemporal resolution of 245 nm and 250 Hz and strong immunity against external disturbance. Thus, we could investigate several important dynamic processes of intracellular organelles from direct perspectives, including chromosome duplication in mitosis, mitochondrial fusion and fission, filaments, and vesicles’ morphologies in apoptosis. Of note, we have captured, for the first time, a new type of mitochondrial fission (entitled mitochondrial disintegration), the generation and fusion process of vesicle-like organelles, as well as the mitochondrial vacuolization during necrosis. All these results bring us new insights into spatiotemporal dynamics and interactions among organelles, and hence aid us in understanding the real behaviors and functional implications of the organelles in cellular activities.

Label-free imaging of cellular organization in living mammalian cells via external apodization phase-contrast microscopy

“…The results showed that the nucleus, nucleolus, and mitochondria, but not the entire endoplasmic reticulum or Golgi apparatus, were clearly visualized by ExAPC microscopy, similar to conventional label-free techniques (Fig. 1c) 10,15 .…”

Molecularly Specific and Functional Live Cell Imaging by Label-Free Interference Microscopy

“…This is most likely due to the small RI contrast of these organelles. Interestingly, the ER network has been visualized by other QPI method . Visualization of these weakly scattering organelles calls for a higher imaging sensitivity and other analytical strategies.…”

“…The microscope technology based on interference imaging of complex linear scattered fields has made tremendous progress in the past two decades. Several schemes have been demonstrated for phase-sensitive imaging and detection, including but not limited to the interferometric scattering microscopy (iSCAT), − coherent brightfield microscopy (COBRI), − spatial light interference microscopy (SLIM), − interferometric reflectance imaging sensor (IRIS), − rotating coherent scattering microscopy (ROCS), , digital holographic microscopy (DHM), diffraction phase microscopy (DPM), , optical diffraction tomography (ODT, also known as tomographic phase microscopy), − Fourier ptychographic microscopy (FPM), , partial-wave spectroscopic microscopy (PWS), , and quantitative DIC microscopy . These various techniques share a common working principle, that is to detect the complex signal fields through interference.…”

Label-Free Analysis of Organelle Interactions Using Organelle-Specific Phase Contrast Microscopy (OS-PCM)

“…Moreover, multilabel fluorescence staining is typically required, which increases the requirement of sample preparation and the microscope system’s complexity. Alternatively, label-free methods such as quantitative phase imaging hold great advantages where long-term observation of various organelles in an unlabeled cell is possible. − …”