Phase Contrast

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 and Applications of Phase Contrast Microscopy:
Simply Explained

Phase Contrast Principle: How It Works

Unstained living cells absorb practically no light. Poor light absorption results in extremely low contrast between the cells and the surrounding media. This makes unstained cells barely visible in a brightfield microscope. However, when light passes through cells, small phase shifts occur as well, which are invisible to the human eye. A phase contrast microscope transfers these small phase shifts into intensity shifts to enhance the contrast of non-pigmented cells. The optical key components of a phase contrast microscope are an annular ring located in the condenser and a phase ring located in the back focal plane of the objective. The annular ring creates a tube-shaped illumination that is matched with the phase ring in the detection path, to apply an ±90° (ʎ/4) phase shift. However, scattered light from the sample mostly bypasses the phase ring. Now, interference between the scattered light and the tube-shaped and phase-shifted light changes the detection lights amplitude and by this creates a good contrast.

Phase Contrast: Optimize Your Contrast

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.

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

Choosing the right labware can help achieve optimal imaging results using phase contrast microscopy. One frequently occurring problem is the meniscus formation at the air-liquid interface in the cell culture vessel. The meniscus acts as a lens and disarranges the correct alignment of the phase ring and phase plate inside the optical pathway. This effect significantly reduces the image quality, especially in small culture wells such as in standard 96 well plates.

ibidi has developed several solutions to overcome this problem—and guarantee excellent phase contrast images:

• ibidi µ-Slide 15 Well 3D and µ-Plate 96 Well 3D
• ibidi Channel µ-Slides
• ibidi µ-Slides Ph+

For detailed information, please refer to "Phase Contrast in Channel Slides" or read our Application Note 03 (PDF).

ibidi µ-Slide 15 Well 3D and µ-Plate 96 Well 3D

The µ-Slide 15 Well 3D and µ-Plate 96 Well 3D feature the "well-in-a-well" technology that prevents gel meniscus formation. This ensures a flat gel surface with all cells on a single focal plane without image-distorting meniscus effects—ideal for phase contrast imaging.

ibidi Channel µ-Slides

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, and by this avoids a meniscus formation and allows for excellent phase contrast across the entire channel.

ibidi µ-Slides Ph+

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.