Latest Advances in Cell Culture Microscopy
Enhancing the Cell Culture Workflow
- Fig. 1: The Olympus CKX53 cell culture microscope is optimized for cell culture applications.
- Fig. 2: Integrated Phase Contrast (iPC) allows excellent phase contrast for all available objectives without needing to individually center the common annular phase ring.
- Fig. 3a: Mouse iPS cell colonies documented with InVersion Contrast (IVC).
- Fig. 3b: In comparison with standard phase contrast.
- Fig. 3c: The new contrast technique provides enhanced 3D information.
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Author: Joachim Kirschner1
Cell cultivation success depends upon insightful observation and workflow efficiency, and dedicated light microscopes such as the Olympus CKX53 have been optimized to meet these needs. The cover shows clear observation using the integrated Phase Contrast (iPC) feature, allowing fast and efficient switching from 4-40X objectives without needing to change the ring slit.
As a fast and accessible technique, light microscopy is a particularly important tool for cell cultivation, since the ability to clearly observe and document cells as they naturally grow, proliferate and differentiate is central to observing the health and progress of cultures. When it comes to cell culture microscopy, cell biologists have several priorities, including speed, efficiency and convenience during operation, and insightful high-quality imaging using brightfield, contrast and fluorescence techniques. Specialized cell
culture microscopy systems (Figure 1) support these aims through a number of features and optical innovations.
The Bigger Picture
When handling cell samples, minimizing the time each sample remains outside the incubator is important for maintaining culture health, and this can be achieved through quick observation. Microscopy optics can facilitate fast and efficient screening, and the Olympus UIS2 objectives series specialized for cell culture are designed with a field number of 22, producing images 21% larger than a field number of 20. This provides a more representative and clear sample overview, particularly when using the 2X objective, for example allowing the observation of cells or cell colonies in 96 well plates, without having to move the stage. Such specialized optics are therefore ideal for researchers working with large embryonic and induced pluripotent stem cell (iPS cell) colonies in different microplate formats. Additionally, the 2X objective together with the new LED illumination provides noticeably higher contrast, allowing transparent objects in the sample to be clearly identified. Many decisions can be reached more quickly, including whether cells are ready for assaying or passage, or if they are sufficiently dissociated following trypsinization.
Speeding up Live Cell Observation
Brightfield imaging has one significant limitation.
Known as phase objects, live, unstained cells such as those in culture do not absorb light, and many structures are therefore invisible under standard brightfield microscopy. Still, when light passes through these transparent samples, it does undergo a phase shift, and specialized illumination methods – including phase contrast  and the Hoffman modulation contrast method  – are capable of transforming phase shifts into a light intensity pattern to enhance image contrast. Phase contrast is therefore the most popular method for cell biologists, but it is time-consuming when needing to switch magnifications, and generally when preparing and centering the different objectives. Allowing fast and high contrast observation of phase objects, the integrated Phase Contrast (iPC) technology removes the need to individually change the ring slit when switching from 4X-40X objectives (Figure 2).
In addition to optical technologies, the cell culture workflow is also streamlined through the microscope’s ergonomics and versatility. For example, lightweight and compact systems fitting within a clean bench allow cell handling under completely sterile conditions, while bright LED illumination requires minimal upkeep. Since it is often the case that cells may be grown and assayed in a variety of formats, compatibility with any type of cell culture vessel from microplates to multi-layer flasks, is highly beneficial.
A Closer Look at Stem Cell Culture
Insightful imaging is also a priority throughout cell culture applications, including stem cell research. Unfortunately, phase contrast and Hoffman modulation contrast methods fall short of achieving optimum image quality during iPS cell colony observation. While phase contrast leads to a halo effect around the edge of the phase object thus obscuring the outline, the Hoffman modulation contrast method instead introduces a shadow in the direction determined by the equipment set up.
Overcoming these limitations, a new contrast technique known as inversion contrast (IVC) has been developed by Olympus. This novel method extends phase contrast illumination to create clear images with enhanced 3D information to deliver a greater level of optical information from a sample. Both halos and directional shadows are removed through the setup of IVC, which is explained in greater detail in reference . The contrast is also inverted across the focal plane and the depth of field is reduced, facilitating the morphological analysis of 3D cells such as iPS cell colonies. Clearly observing iPS cell colonies, it is possible to detect changes in morphology suggesting cellular health or differentiation. Significantly, the IVC method has been applied to visualize the outline and structure of mouse iPS cells (iPS-MEF-Ng-20D-17, Kyoto University ), capturing the 3D nature of the cells (Figure 3) . Interestingly, IVC has been found to be ideal for distinguishing iPS cells from the surrounding feeder cells, as the contrast of the feeder cells is lower due to their flatter morphology, highlighting the iPS cells against the background.
From Observation to Analysis
Extending the utility of the cell culture microscope, fluorescence imaging capabilities allow the researcher to perform functional studies, complementing brightfield and contrast observation methods. This is possible with the Olympus CKX53, which draws on the advanced fluorescence light source and filter cube technology of the high-end inverted IX3 microscopes, to enable high-contrast fluorescence imaging with a wide range of dyes.
Dedicated cell culture microscopy systems bring together a range of features to enhance cell culture applications. While advanced optical technologies and design features improve speed and efficiency to ensure precious cells are processed quickly, both novel and established imaging capabilities also maximize the quality of observation and analysis. The CKX53 completes the range of technologies centered on Olympus’ optical expertise, including the Cell Counter R1 and dedicated software solutions, which continue to drive cutting-edge discoveries in cell biology and cell cultivation techniques.
All References are available online for your
convinience at http://bit.ly/GLJ-Kirschner
1Olympus Europa SE & CO. KG, Hamburg, Germany
1. F. A. Ross, Phase Contrast and Interference Microscopy for Cell Biologists (Edward Arnold, 1967), available as antiquarian book
2. R. Hoffman, The modulation contrast microscope: principles and performance, J. Microsc. 110, 205 (1977),
3. Suzuki, Y., Kajitani, K. and Ohde, H., Method for observing phase objects without halos or directional shadows, Optics letters; Vol. 40, No. 5. pp 812-815 (2015)
4. K. Okita, T. Ichisaka, and S. Yamanaka, Generation of germline-competent induced pluripotent stem cells, Nature 448, 313 (2007), DOI:10.1038/nature05934
Phase Contrast Basics: