Phase Contrast
Applications:
Phase contrast is by far the most frequently used method in biological light microscopy. It is an established microscopy technique in cell culture and live cell imaging. When using this inexpensive technique, living cells can be observed in their natural state without previous fixation or labeling.
Principle:
Unstained living cells absorb practically no light. Poor light absorption results in extremely small differences in the intensity distribution in the image. This makes the cells barely, or not at all, visible in a brightfield microscope. When light passes through cells, small phase shifts occur, which are invisible to the human eye. In a phase contrast microscope, these phase shifts are converted into changes in amplitude, which can be observed as differences in image contrast. However, this label-free technique is strongly dependent on the correct alignment of components in the optical pathway. This alignment can be disturbed by the naturally occurring meniscus effect, causing weak phase contrast.
Phase contrast microscopy of Rat1 cells.
F. Zernike. "Phase contrast, a new method for the microscopic observation of transparent objects". Physica, 1942, part I: 10.1016/S0031-8914(42)80035-X, part II: 10.1016/S0031-8914(42)80079-8.
read abstract part I / part II
E. Horn, R Zantl. Phase-Contrast Light Microscopy of Living Cells Cultured in Small Volumes. Microsc Anal, 2006, 20(3):5–7
read abstract
ibidi Solutions:
An important issue to be considered in phase contrast microscopy is the meniscus, which is naturally formed at the air-liquid interface. This phenomenon can significantly reduce the image quality, especially in small culture wells like the standard 96 well plates. The diffraction, due to the meniscus, disarranges the correct alignment of the phase ring and phase plate inside the optical pathway.
ibidi has developed several solutions to overcome this problem—and guarantee excellent phase contrast images:
For detailed information, please refer to "Phase Contrast in Channel Slides" or read our Application Note 03 (PDF).
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Beam path with meniscus |
Beam path without meniscus |
- The µ-Slide Angiogenesis and µ-Plate Angiogenesis 96 Well are not only designed for angiogenesis and tube formation assays, they also provide the ideal cell culture vessel for brilliant phase contrast images. A geometrical trick, the "well in a well" technology, inhibits the meniscus formation and results in good phase contrast over the entire observation area.
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Standard well 1) Meniscus on air-liquid interface: poor phase contrast in most of the observation area. 2) Meniscus on the gel surface: |
µ-Slide / Plate Angiogenesis 1) Planar air-liquid interface: 2) Planar gel surface: |
- The ibidi channel μ-Slides provide ideal optical conditions for phase contrast microscopy. When culturing cells, the channel is filled with medium from bottom to top. This geometrically inhibits meniscus formation and allows for excellent phase contrast across the whole channel.
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96 well plate or small open well |
Channel or parallel plates |
- The ibidi µ-Slides Ph+ are specifically designed for phase contrast microscopy. A special intermediate plate in each well avoids meniscus formation and guarantees brilliant phase contrast—no matter which part of the well is being imaged.
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Standard well |
Ph+ well |