Efficient Long-Term Time-Lapse Microscopy

Studying the formation of spheroids

When doing time-lapse microscopy experiments with spheroids, there are certain challenges which can arise. As the experiments can last for several days, prolonged sample survival must be achieved which requires that near physiological conditions are ensured. The long-term time-lapse study described in this article involves the use of Mica - the world’s first Microhub, to investigate the formation of spheroids from U343 and MDCK cells. Growing spheroids requires optimal conditions to ensure undisturbed cell cycles and proliferation.

Image: Formation of 3D spheroids from 1000 MDCK cells per well where cells were stably transfected with MX1 GFP (green). Time lapse acquisition over 72 hours with 30 minutes interval. Gray is integrated modulation contrast.

Time-lapse Microscopy

Time-lapse microscopy involves capturing images of a specimen via a microscop e at a specific rate, usually mentioned as frames per second (fps), over a certain period of time. If the image-capture rate is lower than the viewing rate used to observe the recorded images, then time appears to be moving faster when observing the sequence of images like a video. Similarly, if the image-capture rate is higher than the viewing rate, then time seems to be moving slower. Therefore, time-lapse microscopy enables the observation of long microscale events, i.e., which occur over minutes, hours, or days, to be viewed within seconds, minutes, or hours. Time-lapse microscopy is used for the study of live specimens, such as cell cultures, acute tissue samples, and model organisms as they grow and develop over time, to gain more insights about biological processes. For instance, cell migration is important for multicellular organisms during embryonic development, tissue repair, functioning of the immune system, and tumor invasion. In order to accurately follow the changes of cells and organisms over time, then “longitudinal” studies are used where the same sample or specimen is observed under specific conditions over a defined period of time.

Spheroids

Spheroids are a type of 3D cell culture, like organoids, which mimic the physiological functions of living tissues and organs . Monolayer 2D cell cultures are normally cells simply growing flat on a substrate. Spheroids and other 3D cell cultures can grow into large volumes that enable 3-dimensional cell-cell interactions which is more like native tissue in an organism. These 3D cell cultures can be used for research to help obtain a better understanding of cells in a more real-life microenvironment. Spheroids are useful for applications in neuroscience, regenerative medicine, as well as cancer and cardiovascular research.

Challenges

When doing time-lapse microscopy experiments with spheroids, there are certain challenges which can arise. As the experiments can last for several days or even weeks, prolonged sample survival must be achieved which requires that near physiological conditions are ensured. Expression of fluorescent markers must be kept at endogenous levels to prevent impairing cell homeostasis. There must be a stable supply of nutrients and an unchanged concentration in the medium which can be impaired by evaporation. Proper imaging over long times requires that microscopeimaging stay in focus and that it is adapted to changing sample characteristics, e.g., lateral and axial growth.

Research labs may not have the instrumentation needed for time-lapse microscopy work involving 3D cell cultures. In this case, normally researchers must have access to shared facilities utilized by multiple users to get around this problem, but that can mean long waiting times before the needed instruments are available. These challenges can lead to delays in acquiring the important, quantifiable results that lead to fundamental breakthroughs and the gaining of new insights.

Introducing Mica

Mica, the world's first imaging Microhub, unifies everything a researcher needs in one completely controlled, highly flexible environment, supercharging microscopy workflows to get meaningful scientific results faster. With Mica, users can benefit from:

• Access for all: Easy setting up of controlled environmental conditions, matching focus strategy, and setting up the imaging conditions

• Radically simplified workflows: On screen annotation and extraction of multiple parameters

• No constraints: Optimal environmental conditions and multiple imaging modalities (brightfield, widefield, and confocal) to match experimental needs and intelligent automation for water immersion and focus strategies

Methods

This long-term time-lapse study involves the use of Mica to investigate the formation of spheroids starting from either U343 cells or MDCK cells stably transfected with MX1-GFP. Growing spheroids requires optimal physiological conditions which ensure undisturbed cell cycles and proliferation. Previous results show that the growth itself is often correlated with the expression of specific proteins or markers for certain hallmarks of cell-states and differentiation ​​​​​​​.

Fig. 1: Diagram showing the formation of spheroids from cells over time.

Mica is used for this key application as an incubator to maintain 3D cell cultures and spheroids under near physiological conditions and with minimized media evaporation. Mica allows users to measure spheroid growth and to analyze protein-expression levels.

Images and data acquired during the long-term time-lapse study of the formation of 3D spheroids and their growth are shown in figures 2 and 3.

Fig. 2: Analysis of the 60 wells over time: red indicates the detected area by the trained pixel classifier at a time point after 60 hours of imaging.

Fig. 3: Upper row: selected images from a time-lapse series of a forming 3D spheroid after plating 1000 MX1-GFP stably transfected MDCK cells. The images were taken at timepoints 0, 11, 20, 40 and 61 hours after seeding the cells. The corresponding measurement of the size of the forming MDCK-cell spheroid is the red curve shown in the graph. Lower row: selected images from a time-lapse series of a forming 3D spheroid after plating 1000 U343 cells. The images were also taken at timepoints 0, 11, 20, 40 and 61 hours after seeding the cells. The corresponding measurement of the size of the forming U343-cell spheroid is the green curve shown in the graph.

References

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2. Time-Lapse Microscopy: Technique and Significance, Looking at Cell Migration: What is Time-Lapse Microscopy (TLM)? Microscope Master.
3. Live-Cell Imaging Techniques Visualizing the Molecular Dynamics of Life, Science Lab (2022) Leica Microsystems.
4. T. Veitinger, Introduction to Live-Cell Imaging, Science Lab (2012) Leica Microsystems.
5. R.R. Shields-Cutler, G.A. Al-Ghalith, M. Yassour, D. Knights, SplinectomeR Enables Group Comparisons in Longitudinal Microbiome Studies, Front. Microbiol. (2018) vol. 9, DOI: 10.3389/fmicb.2018.00785.
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The original article was published byLeica Microsystems