Live-cell imaging
Live-cell imaging is the study of living cells using time-lapse microscopy. It is used by scientists to obtain a better understanding of biological function through the study of cellular dynamics. Live-cell imaging was pioneered in the first decade of the 21st century. One of the first time-lapse microcinematographic films of cells ever made was made by Julius Ries, showing the fertilization and development of the sea urchin egg. Since then, several microscopy methods have been developed to study living cells in greater detail with less effort. A newer type of imaging using quantum dots have been used, as they are shown to be more stable. The development of holotomographic microscopy has disregarded phototoxicity and other staining-derived disadvantages by implementing digital staining based on cells’ refractive index.
Overview
s exist as a complex interplay of countless cellular components interacting across four dimensions to produce the phenomenon called life. While it is common to reduce living organisms to non-living samples to accommodate traditional static imaging tools, the further the sample deviates from the native conditions, the more likely the delicate processes in question will exhibit perturbations. The onerous task of capturing the true physiological identity of living tissue, therefore, requires high-resolution visualization across both space and time within the parent organism. The technological advances of live-cell imaging, designed to provide spatiotemporal images of subcellular events in real time, serves an important role for corroborating the biological relevance of physiological changes observed during experimentation. Due to their contiguous relationship with physiological conditions, live-cell assays are considered the standard for probing complex and dynamic cellular events. As dynamic processes such as migration, cell development, and intracellular trafficking increasingly become the focus of biological research, techniques capable of capturing 3-dimensional data in real time for cellular networks and entire organisms will become indispensable tools in understanding biological systems. The general acceptance of live-cell imaging has led to a rapid expansion in the number of practitioners and established a need for increased spatial and temporal resolution without compromising the health of the cell.Types of microscopy used
Phase contrast
Before the introduction of the phase-contrast microscope, it was difficult to observe living cells. As living cells are translucent, they must be stained to be visible in a traditional light microscope. Unfortunately, the process of staining cells generally kills them. With the invention of the phase-contrast microscopy it became possible to observe unstained living cells in detail. After its introduction in the 1940s, live-cell imaging rapidly became popular using phase-contrast microscopy. The phase-contrast microscope was popularized through a series of time-lapse movies, recorded using a photographic film camera. Its inventor, Frits Zernike, was awarded the Nobel Prize in 1953. Other later phase-contrast techniques used to observe unstained cells are Hoffman modulation and differential interference contrast microscopy.Fluorescent
Phase-contrast microscopy does not have the capacity to observe specific proteins or other organic chemical compounds which form the complex machinery of a cell. Synthetic and organic fluorescent stains have therefore been developed to label such compounds, making them observable by fluorescent microscopy. Fluorescent stains are, however, phototoxic, invasive and bleach when observed. This limits their use when observing living cells over extended periods of time. Non-invasive phase-contrast techniques are therefore often used as a vital complement to fluorescent microscopy in live-cell imaging applications. Deep learning-assisted fluorescence microscopy methods, however, help to reduced light burden and phototoxicity and allow even repeated high resolution live imaging.Quantitative phase contrast
As a result of the rapid increase in pixel density of digital image sensors, quantitative phase-contrast microscopy has emerged as an alternative microscopy method for live-cell imaging. Quantitative phase-contrast microscopy has an advantage over fluorescent and phase-contrast microscopy in that it is both non-invasive and quantitative in its nature.Due to the narrow focal depth of conventional microscopy, live-cell imaging is to a large extent currently limited to observing cells on a single plane. Most implementations of quantitative phase-contrast microscopy allow creating and focusing images at different focal planes from a single exposure. This opens up the future possibility of 3-dimensional live-cell imaging by means of fluorescence techniques. Quantitative phase-contrast microscopy with rotational scanning allow 3D time-lapse images of living cells to be acquired at high resolution.
Holotomography
is a laser technique to measure three-dimensional refractive index tomogram of a microscopic sample such as biological cells and tissues. Because the RI can serve as an intrinsic imaging contrast for transparent or phase objects, measurements of RI tomograms can provide label-free quantitative imaging of microscopic phase objects. In order to measure 3D RI tomogram of samples, HT employs the principle of holographic imaging and inverse scattering. Typically, multiple 2D holographic images of a sample are measured at various illumination angles, employing the principle of interferometric imaging. Then, a 3D RI tomogram of the sample is reconstructed from these multiple 2D holographic images by inversely solving light scattering in the sample.The principle of HT is very similar to X-ray computed tomography, or CT scan. CT scan measures multiple 2D X-ray images of a human body at various illumination angles, and a 3D tomogram is then retrieved using the inverse scattering theory. Both the X-ray CT and laser HT shares the same governing equation – Helmholtz equation, the wave equation for a monochromatic wavelength. HT is also known as optical diffraction tomography.
The combination of holography and rotational scanning allows long-term, label-free, live-cell recordings.
Non-invasive optical nanoscopy can achieve such a lateral resolution by using a quasi-2π-holographic detection scheme and complex deconvolution. The spatial frequencies of the imaged cell do not make any sense to the human eye. But these scattered frequencies are converted into a hologram and synthesize a bandpass, which has a resolution double the one normally available. Holograms are recorded from different illumination directions on the sample plane and observe sub-wavelength tomographic variations of the specimen. Nanoscale apertures serve to calibrate the tomographic reconstruction and to characterize the imaging system by means of the coherent transfer function. This gives rise to realistic inverse filtering and guarantees true complex field reconstruction.
In conclusion, the 2 terminologies of optical resolution and sampling resolution are separated for 3D holotomographic microscopy.