Live cell imaging is a specialized field of microscopy focused on observing dynamic biological processes within living cells or tissues over a period of time. Unlike traditional methods that use chemical stains and fixatives, which permanently preserve and kill the cell, live cell imaging allows researchers to capture real-time events such as cell division, migration, and the movement of internal organelles. The primary challenge in this type of imaging is that living cells are mostly transparent and composed of water, offering very little inherent contrast under a standard brightfield microscope. Successfully visualizing these transparent, moving specimens requires advanced optical techniques that generate contrast without compromising the cell’s delicate physiological state.
Visualizing Unstained Cells: Contrast-Based Microscopy
The most straightforward approaches to live cell observation rely on manipulating light waves to make the cell’s internal structures visible without using any toxic labels. This category of label-free techniques converts the subtle differences in light speed caused by various cellular components into differences in image brightness. The cell’s density and thickness affect the light’s path, causing small phase shifts that the human eye cannot normally detect.
Phase Contrast Microscopy, developed by Frits Zernike, transforms these invisible phase shifts into visible intensity variations. It employs a special annular ring in the condenser and a phase plate in the objective lens to separate and manipulate the light that passes through the cell from the light that passes around it. This interference creates a high-contrast image, typically appearing as bright structures on a dark background or vice versa, allowing the observation of basic cell morphology and movement.
Differential Interference Contrast (DIC) Microscopy, often called Nomarski microscopy, provides a detailed, three-dimensional-like relief of the cell. DIC works by splitting polarized light into two rays that pass through adjacent points in the sample. These rays are recombined, and their interference reveals the local gradient in the refractive index, which is perceived as a shadow-cast image. DIC offers superior optical sectioning capability and avoids the distracting “halo” artifact found in phase contrast images, making it suitable for slightly thicker specimens.
Tracking Specific Molecules: Fluorescence and Confocal Imaging
While contrast methods reveal general cellular structure, observing the behavior of specific proteins or molecules requires fluorescence imaging, which relies on adding a fluorescent tag. This is achieved by introducing fluorescent proteins, such as Green Fluorescent Protein (GFP), or specific fluorescent dyes that bind to the target molecule. Standard widefield fluorescence microscopy illuminates the entire sample at once, exciting all fluorophores along the light path.
Whole-volume illumination creates two major problems when imaging living samples. First, light emitted from out-of-focus fluorophores results in a blurred image with low contrast, making clear three-dimensional data impossible to obtain. Second, intense light exposure generates highly reactive oxygen species, leading to phototoxicity (cellular damage) and photobleaching (destruction of the fluorescent tag), which limits observation time.
Confocal Microscopy largely solves the blurring problem by employing a spatial filter, or pinhole, placed in front of the detector. This pinhole is precisely positioned to block any light that originates from above or below the focal plane, ensuring only light from a single, thin optical section reaches the sensor. By collecting a series of these sharp optical sections, a high-resolution, three-dimensional image of the cell can be reconstructed.
The conventional laser-scanning confocal system, however, can still be relatively slow, and the focused laser spot can deliver a high light dose to the scanned area, risking phototoxicity. A faster variant, the Spinning Disk Confocal Microscope, uses a Nipkow disk containing thousands of tiny pinholes arranged in a spiral pattern. This parallel illumination and detection significantly increases the imaging speed and spreads the light dose across a larger area, making it a more gentle tool for live cell time-lapse experiments.
Viewing Deep Tissues and Extended Processes
Imaging samples significantly thicker than a single cell layer, such as embryos or organoids, presents unique challenges due to light scattering and the increased risk of phototoxicity from deep penetration. Two-Photon Microscopy is specifically designed for deep tissue imaging by utilizing the principle of two-photon excitation. This technique uses two low-energy, long-wavelength photons, typically in the near-infrared range (700–1100 nm), to excite a single fluorophore.
Since excitation depends on the square of the photon density, fluorescence emission only occurs at the high-intensity focal point where the two photons converge. This non-linear excitation eliminates out-of-focus background and phototoxicity outside of the focal plane. Furthermore, near-infrared light penetrates biological tissue much deeper than visible light, allowing clear imaging up to one millimeter deep.
For long-term, low-damage imaging of large specimens, Light-Sheet Microscopy (Selective Plane Illumination Microscopy or SPIM) is often preferred. This technique employs a thin sheet of light to illuminate only the single plane of the sample being viewed, perpendicular to the detection objective. Because the remaining volume of the sample remains unexposed, total phototoxicity and photobleaching are drastically minimized. This unique illumination geometry allows for rapid image acquisition and enables the observation of sensitive biological processes, like embryonic development, over extended periods.
Maintaining Cell Health During Imaging
Regardless of the microscopy technique used, maintaining the physiological health of the cells on the microscope stage is paramount for successful live imaging experiments. A dedicated environmental control system, often a stage-top or full enclosure incubator, is necessary to mimic the natural biological environment. Temperature must be precisely maintained at \(37^\circ\text{C}\) for mammalian cells, with stability often required to within \(\pm 0.1^\circ\text{C}\) to prevent thermal shock or drift.
The culture medium’s pH must also be regulated, typically between 7.2 and 7.4, by supplying a controlled concentration of carbon dioxide. For bicarbonate-buffered media, this usually involves maintaining a 5% to 7.5% \(\text{CO}_2\) atmosphere. High humidity (90% to 95%) is also maintained to prevent the culture medium from evaporating and altering the concentration of nutrients. Finally, the practical challenge of phototoxicity requires balancing sufficient image brightness with the lowest possible light dose to ensure the cell remains viable throughout the experiment.