Asymmetric cell division in a live zebrafish embryo. Credit: Paula Alexandre via Wellcome Collection
Live cell imaging is an important analytical tool in many laboratories. The ability to visualize protein dynamics in vesicles, organelles, cells, and tissues has provided new insights into how cells function in healthy and disease states. These insights include the spatiotemporal dynamics of processes like mitosis, embryonic development, and cytoskeleton changes.
But, biological imaging of sensitive samples is complex. The microscopy method utilized should minimize photobleaching, phototoxicity and other potentially harmful outcomes that can irrevocably damage a sample. At the same time, the technique must deliver ultra-sharp resolution and allow researchers to peer deep into specimens.
Tip No. 1: Consider Sensitivity, Speed and Sample
When choosing an optical microscopy system, there are three important variables: sensitivity of the detector (signal-to-noise), the speed required for image acquisition, and type of specimen being analyzed.
To optimize signal-to-noise, the combination of filters selected for imaging of live cells should closely match the spectral profiles of the fluorophores used for experiments. Signal-to-background ratio can be optimized by using reagents that reduce extracellular fluorescence and increase fluorophore photostability.1 It is important to image in media that have been specifically designed for maintaining cell health while reducing or eliminating background fluorescence. The addition of a background suppressor compatible with live cells can also help reduce extracellular background fluorescence and eliminate the need for a wash step. Antifade mounting media for live cells can be applied to samples to reduce photobleaching of fluorophores, preventing signal loss with multiple or long exposures.1
With regard to speed of acquisition, particularly with simultaneous imaging of multiple fluorophores, switching between filters can reduce acquisition time.2
Additionally, the type of sample being analyzed is important. For example, for a thick dynamic sample, researchers should use spinning disk confocal, multi-photon or light sheet microscopy. Meanwhile, for thin dynamic samples, consider fluorescence widefield, total internal reflection or light sheet microscopy.
For static thick samples, use point scanning confocal, multi-photon or light sheet microscopy. But for thin static samples, it is best to use widefield or super-resolution microscopy.
For thick cleared tissues, meanwhile, researchers recommend the use of multi-photon or light sheet microscopy. For thin cleared tissue samples, confocal, multi-photon and light sheet microscopy techniques are the most appropriate.
As shown, light sheet microscopy is suitable for the analysis of most sample types. In fact, in the last decade, light sheet microscopy has been a game-changer for many fields of biological research. Previously, scientists had to compromise between capturing lower resolution video—which reveals dynamic processes like shape changes or movement—or higher definition still images, which provide more precise detail about how cells and molecules function. With light sheet microscopy, however, researchers can obtain high-resolution videos of cells while simultaneously decreasing phototoxicity damage.
Tip No. 2: Control the Environment
One of the biggest challenges during live cell imaging is how to keep samples healthy and normal on the microscope stage—outside of their warm and comfortable incubator. While it’s not a necessarily easy task, microscope-dedicated stage-top incubators or enclosed incubators can provide a stable temperature, humidity, and CO2 for cells.When working with stage-top incubators, it’s critical to heat the objective—this is a common source of cold contact to the samples that can harm their health. 3
In addition to the environmental conditions of the cells, temperature stability for the microscope is especially critical for high magnification observation of live cells. Since high-numerical aperture observation has a shallow field depth, the system can lose focus even with a small Z-drift caused by temperature change.To optimize the environment, ensure any air conditioners are working and the room temperature is stable before starting an experiment. Additionally, position your microscope/sample to avoid direct air flow from any units.
Keep in mind that the peripheral region of microplates might have a higher risk of environmental fluctuation than the central region, so be sure to use the central wells and avoid the wells around the edge to improve stability. For example, if you’re using a 96-well plate (12x8 wells), then only use the central 60 wells (10x6 wells) for the experiment.3
Tip No. 3: Mount Correctly
When imaging live cells, there are multiple mounting options available—some better suited for the job than others.
Tissue culture plates, for example, are not the best imaging option because the bottom of the plates typically comprise 1 to 2 millimeters of plastic that can exceed the working distance of some high-resolution objectives. Additionally, light has a hard time passing through the plates, so it is best to avoid these.
On the other hand, a common mounting option is 35-mm-round dishes that have a glass coverslip inserted in the bottom. Or, some manufacturers offer glass bottom dishes that combine the convenience of a standard 35mm dish with the imaging benefits of cover glass. This option is also available in 6-wellplates. It is recommended by many researchers as it allows you to grow your sample directly on the plate, avoiding disturbances. If you need more than 6-wells, multi-well plates are also available—but they need to have thin bottoms. The bottom of the glass, plastic or polymer film plates should be #1.5 thickness, or 170 µm. There can be a lot of variation on these plates, but the closer to 170 µm, the better.
Some researchers use pulp oil to help maintain humidity inside their samples. Pulp oil allows gasses like CO2 and oxygen to exchange through it, but water will not get through. This keeps the sample nice and humid and won’t dry it out—all while allowing gas exchange.
References
1. 5 Steps to Live Cell Imaging. Thermo Fisher Scientific. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/fluorescence-microscopy-and-immunofluorescence-if/microscopy-reagents-and-media/live-cell-imaging-reagents.html
2. Jensen, E.C. Overview of live-cell imaging: Requirements and methods used. Anat. Rec. Adv. Integr. Anat. Evol. Biol. 2013, 296, 1–8.
3. Takeo Ogama. 4 Tips to Achieve Longer Live Cell Imaging with Less Time in the Lab. Evident Discovery Blog. https://www.olympus-lifescience.com/en/discovery/4-tips-to-achieve-longer-live-cell-imaging-with-less-time-in-the-lab/