How to Use Label-Free Live Cell Imaging
Introduction
Label-free live cell imaging has emerged as a powerful tool in the field of cell biology, allowing researchers to observe and analyze cellular processes without the need for external markers or dyes. This non-invasive technique provides real-time insights into the dynamic behavior of living cells, offering a more natural and holistic perspective on their functions. In this article, we'll delve into the principles and practical aspects of using label-free live cell imaging, highlighting its applications and advantages.
Figure 1. Schematic diagram of optical diffraction tomography optical diffraction tomography for label-free 3-D imaging of LDs. (Kim K, et al.; 2016)
Principles of Label-Free Live Cell Imaging
Label-free live cell imaging relies on various imaging modalities that capture cellular structures and activities without the use of exogenous labels. The most common techniques include phase contrast microscopy, differential interference contrast (DIC) microscopy, and digital holographic microscopy.
Phase Contrast Microscopy
Phase contrast microscopy enhances the contrast of transparent, unstained cells by exploiting the phase differences between the light passing through different parts of the specimen. This technique is particularly useful for observing cellular morphology, movements, and subtle changes in refractive index.
To use phase contrast microscopy, researchers need a phase contrast microscope equipped with specialized optics that convert phase variations into intensity differences. This allows for the visualization of live cells in their natural state, eliminating the need for staining or fixing.
Differential Interference Contrast (DIC) Microscopy
DIC microscopy, also known as Nomarski interference contrast microscopy, employs polarized light to generate three-dimensional images of live cells. This technique enhances the contrast by detecting variations in optical path length, providing detailed information about cell topography and internal structures.
DIC microscopy is especially effective for visualizing transparent and unstained specimens, making it an ideal choice for label-free live cell imaging. Researchers can observe cellular processes such as cell division, migration, and organelle dynamics with high resolution and minimal distortion.
Digital Holographic Microscopy
Digital holographic microscopy records and reconstructs holograms of living cells, enabling quantitative phase imaging. This technique measures the changes in optical thickness induced by cellular structures, offering precise information about cell morphology, volume, and refractive index.
Digital holographic microscopy is a powerful label-free imaging tool that provides quantitative data for studying cellular dynamics. It allows researchers to track cell movements, monitor changes in cell shape, and investigate cellular responses to external stimuli.
Practical Considerations for Label-Free Live Cell Imaging
Cell Culture Conditions:
Maintaining cells in optimal conditions is crucial for label-free live cell imaging. Researchers should use appropriate cell culture techniques, ensuring cells are healthy, adherent, and in their logarithmic growth phase. Temperature, humidity, and gas composition should be carefully controlled to mimic physiological conditions.
Microscope Setup:
Calibrating the microscope for label-free imaging is essential. Researchers must align and optimize the imaging system for their chosen modality (phase contrast, DIC, or digital holographic microscopy). Proper focus and illumination settings should be adjusted to achieve clear and high-contrast images.
Time-Lapse Imaging:
Label-free live cell imaging is particularly valuable for capturing dynamic cellular processes over time. Researchers can set up time-lapse experiments to monitor changes in cell behavior, such as migration, division, or response to stimuli. Care should be taken to minimize phototoxicity and photobleaching during prolonged imaging sessions.
Applications and Advantages of Label-Free Live Cell Imaging
Label-free live cell imaging finds applications in various research areas, including cell biology, pharmacology, and drug discovery. Its advantages include the ability to observe cells in their native state, obtain quantitative data, and reduce the potential artifacts introduced by labeling techniques.
In conclusion, label-free live cell imaging offers a non-invasive and informative approach to studying living cells. By understanding the principles and incorporating practical considerations, researchers can harness the power of label-free imaging techniques to unlock the mysteries of cellular dynamics. This technology not only enhances our understanding of fundamental biological processes but also holds great promise for advancing medical research and therapeutic development.
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- Kim K, et al.; Three-dimensional label-free imaging and quantification of lipid droplets in live hepatocytes. Sci Rep. 2016, 6:36815.
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