Live Cell Imaging: Understanding Real-Time Cellular Dynamics

Live cell imaging is a challenging technique for optically studying living cells in real time. Live cell imaging is applied by scientists to monitor active biological processes. Live cell imaging can be performed using different microscopies such as fluorescence and confocal microscopy, high resolution microscopy, high-throughput screening (HTS), flow cytometry and fluorescence activated cell sorter (FACS) systems. Independent of the live cell imaging microscope used, the physical condition like optimal temperature, osmolarity, pH, and humidity must be provided to keep cells healthy and alive.

Fluorescence and confocal microscopy use fluorescent markers to label and visualize specific cellular structures after excitation of these specific markers. To prevent phototoxicity and mutations of your cell culture you must choose carefully the imaging system. You need a live-cell imaging microscope with the most control of the light source so that you can minimize the wavelength range and the number of photons illuminating your cells. What you need besides the equipment is a kit live cell imaging.

The combination of automated live cell imaging microscope analysis that allows large numbers (96/384-well microplates) of samples to be analyzed simultaneously is called high-throughput screening (HTS). During the live cell imaging experiment, the cells need to be kept alive and healthy. Therefore, your HTS machine must provide consistent temperature in every well of the plate. What you need besides the special equipment is a kit live cell imaging high throughput screening.

Flow cytometry is a live cell imaging technique that detects and analyzes physical and chemical characteristics of cells or particles as they flow in a fluid stream through a laser. In this live cell imaging process, multiple parameters, such as cell size, granularity, fluorescence, can be rapidly measured to identify and analyze different cell types and states in real-time. What you need besides the equipment is a kit live cell imaging cytometry.

Live-Cell Imaging Microscopes: Key Features & Innovations

Types of Live-Cell Imaging Microscopes

Fluorescence microscopy is widespread used for live cell imaging. Live-cell imaging microscope techniques for visualizing the behavior of cells is very important in science. Almost any cell behavior can be studied under the microscope using fluorescent protein tags, live cell imaging dyes and other methods to label the proteins of interest with fluorophores.

Immunostaining, for example, is a powerful tool for tracking live cell behavior that combines antibodies coupled to a fluorophore to detect specific protein epitopes. This is very important in the world of live cell imaging for the diagnosis of disease as well as drug development.

Live-cell imaging microscopes make widespread use of fluorescent proteins. Transporting the mRNA encoding the fluorescent protein into living cells for live cell imaging allows researchers to visualize the location of proteins or organelles. Fluorescent proteins are inherited by all daughter cells of labeled parent cells, making them useful for tracking cells in real-time.

baseclick offers a range of fluorescence labelled and unlabelled mRNAs as part of its mRNA customer service. Commonly used mRNAs for live cell imaging are ones encoding luminescent proteins like GFP (green), Azurite (blue), ECFP (cyan), Topaz (yellow), mOrange (orange) and mCherry (red) fluorescent proteins.

A specialized form of standard fluorescence microscopy is the very expensive confocal microscopy. Confocal microscopy for the study of living cells imaging in real time is used in many areas of science. In medicine, confocal microscopes are used in the diagnosis of diseases by providing higher resolution imaging of tissue. Confocal microscopy allow the creation of 3D images by taking thin slices of the sample. The disadvantage of confocal microscopy for the study of living cells imaging is phototoxicity. Thereby intense light exposure can damage live cell samples.

Super-resolution microscopy is another way to study living cells. It allows researchers to see the cancer cell behavior, helping to understand how cancer grow and spread. It also allows virologists to understand the life cycle and spreading mechanism of viruses, which is important for the development of vaccines. This advanced super-resolution living cell imaging technique as well as confocal microscopy are powerful tools that allow researchers to image cells in incredible detail.

Live Cell Imaging Kits: Optimized Solutions for Cellular Studies

What is a Live Cell Imaging Kit?

If you want to perform live cell imaging you need live cell imaging kit or special markers which keeping cells alive during your experiment. A live cell imaging kit contains a set of reagents e.g. to label primary antibody marker in combination with a reduced background imaging medium or buffer and viability markers. For live cell imaging experiments it is important to use cell-permeant fluorescent dyes that stain live cells. For example, you can buy live cell imaging kits containing non-fluorescent dyes that become fluorescent after entering the cells. Calcein AM is a cell-permeant dye that can be used to determine cell viability in most eukaryotic cells. Calcein AM becomes green fluorescent calcein, after hydrolysis by intracellular enzymes. To keep cells healthy and alive stabilizing imaging buffers are used to maintain physiological conditions. This necessary reagent for live cell imaging guarantees for stable pH, osmolarity, and ion concentrations, that are necessary for cell viability during live cell imaging.

Viability markers help distinguish between live and dead cells by detecting specific cellular functions. For instance, a damaged membrane is a sign for dead. For example, propidium iodide (PI) can only enter cells with a compromised plasma membrane. The marker is slightly fluorescent active in aqueous medium, but when bound to dsDNA or RNA the dye is highly fluorescent. Further dead and live cells can be detected by enzymatic activity. E.g. the intracellular esterase activity serves as a typical indicator of live cells. Together, these components enable precise visualization and analysis of live cells, making live cell imaging kits indispensable tools in biological research.

Kit Live Cell Imaging for High-Throughput Screening

High-Throughput Screening (HTS) in combination with live-cell imaging led to an automated drug screening workflow and has crucially transformed drug discovery and development. High-throughput drug screening enables e.g. the profiling of patient’s responses in vitro and allows the repurposing of compounds currently used for other diseases, which can be immediately available for clinical application. The aim of live cell imaging using HTS machines is to identify potentially effective compounds for the treatment of patients with poor prognosis, such as patients with Down Syndrome. Live Cell Imaging allows for the rapid and efficient analysis of new genetic pathways or drug mechanisms, measuring drug cytotoxicity, efficacy, dosage, and on/off-target effects.

A typical high-throughput imaging workflow includes automated liquid handling for reagent dispensing and staining, high-throughput microscopy for capturing images, automated image analysis for cellular features, and statistical data analysis. Kit Live cell imaging High throughput Screening includes enough reagents to perform experiments in microplate formats meaning normally for 96 or more.

Patient samples, human cell lines and healthy donor samples can be screened simultaneously on a live cell imaging drug screening platform such as HTS, using a large library of compounds to identify compounds that, for example, are active against cancer but spare normal cells. This live cell imaging technology enables high throughput screening and allows millions of tests to be run.

Live Cell Imaging and Cytometry: A Powerful Combination

How Imaging Cytometry Enhances Cell Analysis

Flow cytometry provides rapid analysis of thousands of cells per second based on cell structure such as size or surface markers, and intracellular labeling. Merging real-time imaging microscopy and with flow cytometry in live cell imaging further enhances these technologies by providing the capture of high-resolution images along with quantitative data.

Application for Imaging Cytometry

· Apoptosis Detection: Live cell imaging using flow cytometry machine allows scientists to track apoptosis (form of programmed cell death) cases in real time. Each cell is individually checked for capturing processes such as membrane blebbing, cell shrinkage and chromatin condensation. Kit live cell imaging cytometry contains reagents e.g. apoptotic markers for activation of specific enzymes or phosphatidylserine externalization. This provides precise data on the extent and kinetics of apoptotic processes.

· Cell Cycle Studies: When cells are stained with a cell cycle reagent, the DNA in the cells binds the dye stoichiometrically (in proportion to the amount of DNA present in each cell. There is therefore a linearity between fluorescence and DNA distribution, and standard modelling algorithms can be used to determine the different steps of the cell cycle. Classic DNA cell cycle stains for live cell imaging are for example Hoechst 33342 and DRAQ5.

Alternatively, S phase can be detected in fixed cell applications using a baseclick’s ClickTech EdU cell proliferation assay in which the DNA of proliferating cells is labelled with a thymidine analog, namely 5-Ethynyl-2′-deoxyuridine (EdU), and then labelled with a DNA fluorophore such as 6-FAM. This method provides a more accurate quantification of S phase as the thymidine analog is selectively incorporated into the DNA of actively dividing cells.

· Immune Response Monitoring: Live cell imaging allows for the visualization the interactions signaling pathways, and the immune response between immune system cells and pathogens or cancer cells. Flow Cytometry provides rapid quantification of surface markers expression (like CD133, CD44) activated immune markers, cytokines, to improve understanding of immune responses in infection and immunotherapy.

Live cell imaging using cytometry offers innovative approaches to studying complex biological processes, driving progress in disease comprehension and therapy development.

Advantages of Live Cell Imaging Cytometry

Live cell imaging using cytometry in cellular research offers detailed insights into individual cells through high spatial and temporal resolution. The spatial organization of individual cells is a key determinant of cell state and function. For example, in human tumors, local signaling networks differentially affect individual cells and their neighbours and the surrounding microenvironment, with implications for cancer growth, progression and response to therapy. Single-cell analysis dissociates cells from their original tissue, meaning that spatial context is lost. This can now be overcome by incorporating a spatial aspect into the analysis. This allows the full heterogeneity and function of cells to be observed in their original tissue context, and data-rich, high-resolution maps to be generated from whole tissue slices.

Single-cell RNA sequencing and live cell imaging are techniques that allow researchers to observe cellular activity at the molecular and morphological level. High spatial resolution detects subtle internal changes within cells, while high temporal resolution allows dynamic processes to be observed in real time. Live Cell Imaging Cytometry improves our understanding of complex biological processes such as carcinogenesis, stem cell differentiation and immune responses.

A key advantage of Live Cell Imaging Cytometry is non-invasive monitoring of cell proliferation and differentiation without altering or damaging the cells. Live Cell Imaging Cytometry uses fluorescent markers to track cellular processes, enabling researchers to gain valuable insights into cell cycle progression, stem cell differentiation and the influence of external factors on these processes for long-term, real-time observations. This technique is essential in drug development, because it allows continuous and safe observation of cellular responses to treatment.