Digital Holography and Cell Studies (2011)

Cell imaging plays a crucial role in the understanding of cell biology. Cells are almost invisible in standard light microscopes as they do not absorb light. Cells shift the phase of the light and different light microscopy methods, such as phase contrast Holography, Research and Technologies 238 differential interference contrast (DIC) Some advantages of DIC are better contrast and sharpness of the images. These light microscopy methods can only provide qualitative information as quantitative information cannot be calculated. In a recent publication, Petibois has made a review of imaging methods for cells (Petibois, 2010). Another method that is used for measurements of unlabeled adherent cells in real-time is electric impedance. The instrument xCELLigence was developed by Roche Diagnostics (Basel, Switzerland). It is a real-time cell analyzer that measures living cells without labeling. Electric impedance allows measurements of cellular processes such as proliferation, cytotoxicity, invasion, migration and cell viability Different staining methods have been developed that enable cells to absorb light. Dyes such as methylene blue which stains e.g. nucleic acids, haematoxylin which stains cell nuclei, eosin which stains cytoplasm and silver stains which stain proteins and DNA have been used since the mid 19 th century, while fluorescent stains, such as rhodamine which stains mitochondria, neutral red which stains lysosomes, acridine orange and DAPI, which stain nucleic acids were developed and have been widely used since the beginning of the 20 th century New dyes that stain specific parts of the cells and new labels that fluoresce at certain wavelengths are developed continuously. The method to label cells with green fluorescence protein (GFP) was a major breakthrough in the 1990s. GFP is less toxic than most commonly used dyes and the DNA code of GFP can be transfected into the cell's genome When the gene contains the GFP DNA is activated, GFP will be produced by the transfected cells. As GFP affects cells less than traditional stains it gives more accurate results. However, GFP is a rather large molecule and the size probably causes steric problems. Fluorescence microscopy as well as traditional light microscopy may cause phototoxicity, and several researchers have attempted to develop non-damaging microscopy methods The search for a method to study cells accurately without labeling or staining them has resulted in several interferometric quantitative microscopy techniques utilizing the phase properties of coherent light to image a sample. One of them is digital holography. In 1948, Dennis Gabor invented a way to encode the phase of the light both as information and as a record containing all the information in a single recording, i.e. the hologram Holograms are commonly used as pieces of art and are displayed as illuminated 3-D images. Gabor's findings were the base for the development of digital holography during the 1990s (Schnars & Jueptner 1994; Cuche et al., 1999), where the information is collected on a digital sensor and then fed to a computer. Using DHM it is possible to measure cell shape, volume and dry mass without any labeling and with a very low intensity light source In Fig. The DHM images look similar to the phase contrast images. DHM is a full field imaging of the phase of the light incident on the sensor. It can be used to image solid samples in reflection, or, which is most common in the case of biological samples, to image transparent samples in transmittance. The most commonly used system for DHM today uses an optical setup common to that of a Mach-Zender interferometer, but with the reference light at a slight angle to the light passing through the sample, and the www.intechopen.