MICROSCOPY 83
Introducing a new method for stem cell observation. By Wolfgang Hempell & Dr. Joachim Kirschner
A CLOSER LOOK at CELL CULTURE M
icroscopic observations of unstained live cells
are vital for cell cultivation applications, and a novel phase contrast technique known as inversion contrast 1
clear, 3D observation of cells such as induced pluripotent stem cells (iPS cells). Leading the way for
discoveries and applications in the area of stem cell research, the Nobel Prize in Physiology or Medicine 2012 was awarded jointly to Sir John B. Gurdon and Shinya Yamanaka “for the discovery that mature cells can be reprogrammed to become pluripotent.”2
Te scientists
found that it was possible to reprogramme mature cells to become iPS cells, which have the capacity to differentiate into any one of the diverse cells that form the body. Understandably, this has created strong hopes for truly understanding diseases through developing accurate models, and even treating diseases with regenerative medicine, growing
now enables
tissue or complete organs that match the patient and bypass the challenges associated with immune rejection. A range of tools are available to support these aims, from automated cell counters to specialised cell culture microscopes. With its speed and accessibility, light microscopy is particularly vital, since the ability to clearly observe and document stem cells as they grow, proliferate and differentiate is central to observing the progress of cell cultures. However, this approach has one important 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 specialised illumination methods – including phase contrast3 and the Hoffman modulation contrast method4
– are capable of transforming phase shifts
into a light intensity pattern to enhance image contrast. Unfortunately, both methods fall short of achieving optimum image quality, giving rise to artefacts that interfere with the detailed observation of sample structures. While the application of 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.
Fig. 1. IVC light beam for the inverted microscope. The IVC optical layout on an inverted microscope shows the placement of the annular aperture at the front focal plane of the condenser and sketches the specimen- illuminating light beam towards the image plane
A new phase in stem cell microscopy Te latest research efforts have focused on overcoming these limitations, and a new contrast technique known as inversion contrast (IVC) has been developed by Olympus. Tis novel method extends phase contrast illumination technology 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 unique setup of IVC, which is explained in greater detail in reference (1). Essentially, an annular aperture is inserted into the light path at the front focal plane of the condenser (Fig. 1). Since the aperture is larger than that used in standard phase contrast (the outer slit diameter is 10~20% larger than the pupil size and the inner slit diameter is 1~10% smaller than the pupil size), after passing through the sample, the beam illuminates the edge of the objective’s pupil. Terefore, only light passing through the sample at a small angle passes through the objective, whereas light at a large angle does not. When the sample has phase variation, the direction of the light passing through the sample changes. As a result, the image of the
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