There have been significant advancements in microscopy and camera technology, as well as advancements in technologies for labeling molecules of interest over the past decade. These advancements have led to the increased prevalence of live cell imaging in a variety of fields, from basic research, to advanced studies in neurobiology, developmental biology, cancer research, and drug development. Across these fields, live cell imaging can be used to study cellular processes that take place over a period of time from the whole organism level down to the molecular level.

Although there are many applications for live cell imaging and many different microscopy methods that can be used, a common challenge is maintaining sample integrity during the course of the experiment while acquiring images with sufficient resolution. These two factors are critical for obtaining relevant and reproducible data from your live cell imaging experiments.

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Best practices to obtain relevant and reproducible data

Here are some tips and techniques to help you maintain the health of your sample and run a successful live cell imaging experiment.

1. Use the correct media formulation when preparing your samples

In addition to ensuring that media formulations contain the appropriate nutrients to nurture the growth and development of cells, accounting for factors such as pH, buffering capacity, and osmolarity are critical to running a successful live cell imaging experiment. Changes in media pH and osmolarity (i.e., the amount of solute inside the media solution) can affect the expression of cells, their phenotype, and ultimately affect how they look and behave. Regulating and maintaining optimal environmental conditions will help to prevent unwanted or harmful changes to the media.

Additionally, there are a lot of factors within media that can contribute to autofluorescence our high background signal within your images. Consider using phenol red-free media as well as reducing serum concentration. This will greatly reduce background signal.

Left: 40X Plan Apo image of HeLa cells treated with straurosporine for 18 hours and then stained with the EarlyTox™ Live Dead Assay – calcein AM (green) and ethidium homodimer-III (red)

Right: 100X Plan Apo (air) images of Transfluor U2OS treated with isoproterenol to stimulate receptor internalization, which is visualized by GFP-tagged ß-arrestin (green). The cells were counterstained with Hoechst 33342 nuclear stain.

2. Control and maintain proper environmental conditions when imaging your samples

It is important to maintain proper environmental conditions for your samples throughout the entirety of the live cell imaging experiment. Environmental controls includes temperature, humidity, and gas (i.e., oxygen and carbon dioxide).

The ability to regulate and preserve a stable temperature not only maintains cellular health, but also reduces the risk of focus drift. Focus drift is the inability to maintain focus over time. It is typically due to temperature variations, which cause expansion and contraction of the materials used to make the microplate or other culture vessels. To further reduce the risk of encountering focus drift due to thermal fluctuations, ensure that the microplate is seated on the instrument stage or in the environmental control chamber to allow for thermal equilibration before configuring acquisition settings. Also, if media must be added to wells during the time-lapse experiment, ensure that it is the same temperature as the media in the imaging vessel. On-board fluidics control options, or automation integration with liquid handlers for media and reagent additions, increases throughput and minimizes the risk of contaminating or disturbing the cells through manual intervention.

Maintaining proper humidity helps to prevent media evaporation especially for long-term live cell experiments. Evaporation causes osmolarity changes which can negatively impact the behavior of your cells and affect your assay. A hypotonic (low solute) solution can be used if humidity control is not an option.

Controlling carbon dioxide levels is also important, as it helps to regulate pH within your sample. If you are not able to control carbon dioxide levels, there are different synthetic buffers, such as HEPES that can be used to maintain pH. However, compatibility between the cells of interest and synthetic buffer should be verified before use, as synthetic buffers can be harmful to many cell types. If the cells are compatible, HEPES should be utilized for short-term studies that last a few hours, as prolonged maintenance in HEPES buffered media could be harmful to the cells.

Maintaining proper oxygen levels is also critical, as oxygen requirements can vary widely depending on the cell type or the requirements of the particular live cell experiment.

Imagers such as the ImageXpress® Pico Automated Cell Imaging System and ImageXpress® Confocal HT.ai High-Content Imaging System include a variety of environmental control options for temperature, humidity, oxygen, and carbon dioxide. Because these imagers can be fully integrated with a closed environmental control system, plates are not exposed to ambient light and other external laboratory conditions that could compromise the integrity of the sample. Additionally, the ImageXpress systems’ software allows you to accurately monitor cellular environment during the course of the experiment via environmental control sensor readings.

3. Use robust autofocus modes during image acquisition

Our ImageXpress systems are designed with a variety of hardware (laser and LED) and software (image-based) autofocus modes that allow you to find and preserve in focus images between experiments and minimize focus drift. The autofocus modes can accommodate a wide range of samples, culture vessel thicknesses, objectives, and imaging parameters.

Hardware autofocus methods work for most samples and are independent of sample quality and brightness. Hardware autofocus allows for faster acquisition speeds, thereby minimizing photobleaching of samples. When the Z-position of the sample varies over time or across a plate, adding software autofocus methods can help in providing reliable focus across the entire sample and labware. For most cases when software-based focus is required, both hardware and software autofocus should be enabled. Hardware autofocus will find the plate bottom, well bottom, or both surfaces. Then software autofocus will utilize image contrast to find the ideal focal plane. Thus, samples should be bright and free of debris for software autofocus to work optimally.

In contrast to hardware autofocus, software autofocus decreases acquisition speeds and runs the risk of photobleaching samples. To alleviate this, software autofocus should be enabled for the first channel being acquired, and transmitted light can be utilized as this first channel instead of a fluorescent channel. Additionally, reduce your exposure time and use brighter, more stable fluorophores. Tools such as binning (see #6) can also be used to help reduce exposure times.

Software autofocus methods should be used alone only when hardware autofocus has not been configured for your plate, the hardware autofocus fails due to plate imperfections, low volume in the wells diminish the reliability of the hardware autofocus, or when oil immersion objectives are being used.

MetaXpress® High-Content Image Acquisition and Analysis Software enables users to configure autofocus settings to meet the needs of a particular assay. When conducting fast kinetic time-lapse experiments in a single well, autofocus can be applied to the first time point only to increase acquisition rate. For long-term time-lapse experiments or when the imaging rate is not critical, autofocus can be set on all time points to reduce focus drift.

In the CellReporterXpress® Image Acquisition and Analysis Software a variety of hardware and software autofocus routines can be enabled. Each are designed for specific use cases, providing unparalleled focusing options for a wide variety of samples. For example, the Well Insert hardware autofocus routine detects three peaks (plate bottom, well bottom, and well insert) for rapid detection of and optimal focusing for labware that contain three distinct surfaces. For acquisitions that require increased speed at low magnification, the Anchor Focus Position routine retains the selected in-focus Z-position, disables the autofocus, and acquires images with this saved focus position. This is particularly useful when working with macroscopic samples like whole organisms or tissues.

Left: Representative 20X confocal, 2D projection image from an immune-oncology assay showing a mouse colon cancer spheroid; made from GFP-expressing MC38 cells, treated with T-cells expressing RFP.

Right: 4X wound healing assay image of Sigma U2OS cells that were stably transfected to express RFP.

4. Minimize the power of the illuminating light source as well as your exposure time during image acquisition

The power of your illuminating light source and exposure time can cause phototoxicity of your cells. Even small changes to the cells can affect their behavior or gene expression. Hence, you need to find the right balance, where you are able to acquire a quality image without exposing your sample to too much light.

In terms of the illumination, when a fluorophore or fluorescent molecule is put into an exited state, it generates free radicals which can cause DNA damage and stress upon the cells. The higher the intensity of light, the higher the excitation state, which can result in phototoxicity or damage to the cells. Ultraviolet (UV) light is known to be more phototoxic, so using excitable fluorophores such as DAPI can yield more phototoxicity, as compared to using green or red fluorophores.

When it comes to fluorescence imaging or multi-color imaging, it is best to use photostable, and very bright, high signal-to-noise fluorophores with distinct emission peaks. This enables you to reduce your exposure time. The ImageXpress system includes narrow band pass filter cubes to help eliminate crosstalk. It also includes high-power light sources that can be attenuated and controlled. The ImageXpress Confocal HT.ai system for example, includes a seven-channel laser light source with eight imaging channels to accommodate multi-color labeling experiments, like Cell Painting applications.

High numerical aperture (NA) objectives, such as water immersion objectives enable you to generate brighter, high-resolution images at lower exposure times. When using high NA objectives, it is preferable to use thin plastic microplates or culture dishes (i.e., those that have the thickness of a coverslip). The use of black wall, clear-bottom microplates is also preferable, as they generate less autofluorescence.

5. If appropriate, employ image deconvolution algorithms when using widefield microscopy systems

Widefield microscopy systems, such as the ImageXpress Pico system and ImageXpress® Micro Confocal High-Content Imaging System are optimal for running live cell imaging experiments. To increase resolution or sensitivity with these systems, image deconvolution algorithms can be used. Running image deconvolution software reduces out-of-focus light during acquisition, thereby allowing you to decrease exposure times and maintain assay quality. However, deconvolution should be applied accurately and carefully as to not generate artifacts.

6. Use confocal imaging systems that allow for the use of high-power laser light sources and binning

Confocal microscopy systems such as the ImageXpress Confocal HT.ai system include laser light sources that are best used for imaging 3D samples, thick samples, or dim fluorophores (e.g., fluorescently tagged proteins). The confocal instruments allow you to regulate and control laser and LED light intensity power.

Representative 10X Plan Apo images of HCT116 spheroids grown in Corning Elplasia micro-cavity plates and treated with varying concentrations of staurosporine for six days total. The spheroids were stained with Hoechst 33342 (blue, nuclei), calcein AM (green, live cells), and ethidium homodimer-III (red, dead cells).

Binning is another tool that can be used when imaging in widefield or confocal, yet the effects of binning are more apparent when imaging with confocal instruments. Because the spinning disk configuration of the confocal system limits the amount of light that hits the sample and blocks out-of-focus light, you typically have to use higher exposure times or higher intensity light. Binning alleviates the need to do this by combining the electric charge or intensity signal from adjacent pixels and generating a summation of pixel intensities in a certain region. This summation gives you a higher intensity. Essentially, binning increases signal to noise in the image, thus allowing you to use a lower exposure time and lower light intensity. This does, however, decrease spatial resolution. Again, it is a matter of finding the balance between acquiring a quality image and maintaining the health of your sample.

7. Utilize powerful analysis software to obtain reproducible and meaningful data

Live cell imaging applications require the ability to extract relevant and vast amounts of quantitative data from large and sometimes complex images and data sets.

High content analysis (HCA) solutions like Our MetaXpress and CellReporterXpress® software include robust analysis algorithms to accurately segment images and generate reproducible data. They include preconfigured analysis modules for a variety of live cell imaging applications. More customized analysis can be performed using the Customer Module Editor within MetaXpress software.

Left: 20X Plan Apo image of HeLa cells transfected with the Fucci Cell Cycle sensors (GFP - geminin and RFP-Cdt-1). This image was taken 13 hours post-treatement with the cell cycle inhibitor noccodazole.

Right: Analysis segmentation masks generated from a MetaXpress Custom Module analysis.

If you are looking to obtain more in-depth information about the cells being studied, machine learning algorithms with advanced data analytics capabilities can be used. Our IN Carta™ Image Analysis Software package contains guided machine learning workflows to improve analysis accuracy and to sort and compare vast amounts of heterogenous imaging data.

Because live cell imaging experiments generate a lot of data, it is critical to have adequate storage capacity as well as processing power for data analysis. As mentioned previously, binning can reduce the image data size and allow for faster data transfer speed so that images can be analyzed quicker. The ImageXpress system also includes MetaXpress® PowerCore™ High-Content Distributed Image Analysis Software which utilizes parallel processing to dramatically increase analysis speed.

To learn more about the various applications and techniques used for live cell imaging, visit our Live Cell Imaging page.

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