Studying individual cells offers deep insights into biological processes, but the long-term observation of non-adherent, or suspension, cells presents a challenge. These cells float in their culture medium, making them difficult to track and image over time. To solve this, researchers developed microfluidics-assisted platforms that use microscopic channels to immobilize individual suspension cells. This stability allows for high-resolution, time-lapse imaging to capture cellular events as they unfold, overcoming the problem of cell drift.
Principles of Microfluidic Cell Trapping and Manipulation
This technology relies on microfluidic chips, small devices made from a flexible polymer like polydimethylsiloxane (PDMS). These chips contain a network of microscopic channels, inlets for introducing cell suspensions, and outlets for waste. The channel dimensions are on the scale of micrometers, allowing for precise control over the cellular environment.
The primary function of these chips is to isolate and hold single cells for observation using various trapping mechanisms. These methods are categorized as either passive, relying on the chip’s geometry and fluid dynamics, or active, using external energy fields. The choice of method depends on the cell type and experimental goals.
A common passive method is hydrodynamic trapping, which uses the physical structure of the microchannels to capture cells. Designs may include an array of cell-sized constrictions or U-shaped pockets that act as filters. As fluid flows through the chip, cells are guided into these traps and held by fluid pressure while the medium continues to flow past.
Active trapping methods offer more dynamic control. Optical trapping, or optical tweezers, uses a focused laser beam to hold a single cell in a specific location. Acoustic trapping uses sound waves to generate pressure fields that guide cells to the nodes of the sound waves, immobilizing them without physical contact.
Precise fluid control is paramount regardless of the trapping method. Syringe pumps or pressure controllers introduce the cell suspension into the chip at a controlled flow rate. Once cells are trapped, these systems allow for the sequential introduction of other solutions, such as growth media or stains, to perform multi-step experiments on the same cell.
High-Resolution Imaging on a Microfluidic Platform
The integration of microfluidic chips with powerful microscopes enables the acquisition of detailed visual data. Because materials like PDMS and glass are transparent, chips can be placed on an inverted microscope stage, allowing light to pass through the cell for clear observation. The thin profile of many devices is designed to accommodate high-resolution microscope objectives.
Different imaging modalities provide unique information. Brightfield and phase-contrast microscopy are used to observe basic morphology, size, and cell division in real-time without stains. Fluorescence microscopy is a powerful tool that uses fluorescent probes to visualize specific components or processes, such as tracking a protein of interest using Green Fluorescent Protein (GFP).
For greater detail, confocal microscopy is employed. This technique uses a pinhole to block out-of-focus light, resulting in sharper images of specific focal planes. By taking a series of images at different depths, a 3D reconstruction of the cell and its internal structures can be created, which is useful for understanding the spatial organization of organelles.
A primary advantage of this setup is the ability to perform time-lapse imaging. Because a cell is held securely, it can be monitored continuously over hours or days. This longitudinal tracking reveals dynamic processes like the stages of apoptosis or the interaction between two different cell types, providing a movie-like view of cellular life.
Applications in Cellular Screening and Analysis
Observing individual suspension cells over time has direct applications in drug discovery. Researchers can trap single cancer cells, like those from leukemia, and expose them to various drug candidates. By monitoring markers for cell viability or apoptosis in real-time, it is possible to determine a drug’s efficacy and generate precise dose-response curves at the single-cell level. This approach can also identify drug-resistant subpopulations missed in bulk analyses.
This technology is also instrumental in studying cellular heterogeneity. Within a genetically identical population of cells, there can be significant variations in how individual cells behave. A microfluidic platform can trap hundreds of individual cells and expose them to the same signal, revealing that some cells may respond quickly, others slowly, and some not at all. This variability is a fundamental aspect of biology often obscured in traditional experiments.
In immunology, these platforms provide a window into the dynamic interactions between immune cells. For example, a T-cell and an antigen-presenting cell can be trapped in close proximity to visualize the formation of an immunological synapse. Researchers can also measure the secretion of signaling molecules, called cytokines, from a single trapped immune cell to get quantitative data on its activation state.
Another application is in the screening of high-producing cells for biotechnology. In producing therapeutic antibodies, it is necessary to identify cells that secrete the desired antibody at the highest rate. Microfluidic devices can co-encapsulate a single antibody-secreting cell with a detection system, allowing for the rapid screening of thousands of individual cells to isolate the most productive clones.
From Image Acquisition to Quantitative Data
Raw images and videos from a microfluidic experiment must be converted into quantitative data through a computational workflow. This process transforms pixels into measurable biological insights for objective analysis. The first step is image processing, which begins with cell segmentation—the process of automatically identifying the precise boundaries of each cell and its components.
Once cells are segmented, the next step is feature extraction, which involves quantifying parameters from the segmented regions. These features can include morphological characteristics like cell size and shape, as well as fluorescence intensity. For time-lapse data, motility features such as the cell’s displacement or changes in shape over time can also be extracted.
These extracted features are then used to generate quantitative metrics that describe the biological process. For example, measuring the average fluorescence intensity within the nucleus over time can plot the rate of a signaling event. By counting cells that exhibit specific features, one can determine the percentage of a population that undergoes apoptosis after a drug treatment.
This process is facilitated by specialized software. Open-source platforms like ImageJ/Fiji and CellProfiler are widely used for their flexibility. Commercial software packages are also available, sometimes accompanying specific microfluidic systems, which offer more streamlined interfaces for these analysis tasks.
Comparative Analysis and System Limitations
Microfluidic microscopy offers distinct advantages over other single-cell analysis techniques. Flow cytometry, for example, has extremely high throughput but provides only a single snapshot in time for each cell, losing all spatial and historical information. In contrast, microfluidic platforms allow for dynamic, longitudinal analysis, enabling researchers to track the history and fate of the same cell over extended periods.
Compared to traditional well plates, microfluidic systems provide a more controlled environment for suspension cells. Well plates consume significantly larger volumes of expensive reagents and cells, and it is challenging to ensure only a single cell is being analyzed. Microfluidics overcomes these issues by isolating individual cells in precisely controlled microenvironments.
Despite its strengths, the technology has notable limitations:
- The throughput is significantly lower than that of flow cytometry, making it better for in-depth studies rather than large-scale population screening.
- Cell health can be affected by shear stress as they are forced through narrow micro-channels.
- The setup and operation of these experiments can be complex, requiring expertise in both microfluidics and advanced microscopy.
- During long-term experiments, biofouling can occur, where proteins or cells stick to channel walls, potentially interfering with fluid flow and imaging.