Cells are transparent, and consequently, are difficult to observe under the microscope. Lots of work has been done in order to produce contrast inside cells to be able to observe them. One approach is to use the phase shift of the light provoked by the cells themselves. In phase contrast microscopy, the phase shift is translated into variations in intensity. Each component will have a specific intensity variation and thus a specific intensity. As they are all mixed together, it is difficult to extract quantitative information. The result can be translated into a topographic map of the cell, although there is no information on different organelles’ location in 3‐D.
Thanks to its laser inclined at 45°, Nanolive’s new microscope, the 3D Cell Explorer, is able to convert this phase shift information into comprehensive data. The localization of each cell component can therefore be determined and distinguished, making it possible to extract quantitative data.
The 3D Cell Explorer allows one to:
In the lefthand panel are the usual results of a phase imaging acquisition. The representation of the cells is a superposition of all the intensity of the pixels, ranging from violet (low intensity) to yellow (high intensity). No information about the location of the cell’s interior substructures is given by this method.
In the righthand panel is the reconstruction of the image by STEVE software, which provides all the information necessary about the location of the different structures of the cells.
On this page you can explore the difference between Fluorescence and Nanolive’s new technology.
Nanolive’s technology allows the:
Traditional microscopy is limited by the need of chemical fluorescent molecule reporters in order to visualize the different cell components. The user has to decide prior to the experiment which stains may be needed for the sample preparation. The typical time for this procedure is 1–72 hours and just a few stains are possible on the same sample. On top of that, samples need to be fixed, meaning that cells are dead, or they must be genetically modified, which can alter the behavior of the cells.
Nanolive’s technology measures the Refractive Index distribution within the cell. Therefore, one can decide after the experiment which cell parts are to be ‘digitally stained’ in a limitless number of colors, saving a lot of time and money on reagents. One can stain and re‐stain the cell over and over again without damaging it.
In the lefthand panel are the results of an immunostaining protocol and confocal microscopy acquisitions, requiring a total time in excess of four hours. Human Fibroblastic Reticular Cells (“FRC’s”) were fixed with PFA and incubated with primary and secondary antibodies in order to stain cytoskeletal proteins. After that, the nuclei were stained with DAPI.
In the righthand panel are are the 3‐D results of Nanolive’s technology, requiring a total time of less than ten minutes. The cell, without any pre‐processing step, was imaged with the 3D Cell Explorer and digitally stained with the STEVE software.
The micrograph in the lefthand panel shows an immunofluorescent staining of a 10‐micrometer cryosection of an adult zebrafish skeletal muscle. The cytoplasm of the myofibers is rich in contractile proteins such as myosins. Slow‐twitch myofibers are characterized by the presence of a particular isoform of myosin heavy chain, the myosin heavy chain 1 (green). Mitochondria (red) have been labeled with an antibody directed against Tom20, a protein specifically expressed in the mitochondrial outer membrane. Mitochondria are present in the sub-sarcolemmal compartment (red filaments) as well as between the myofibrils (little spots inside green regions). Nuclei are stained with Hoechst, showed here in blue.
The images acquired with the 3D Cell Explorer are displayed on the central and right panels: the grey scale image is a map of the tissue based on the Refractive Index of its components. The main structures were identified and reconstructed in three dimensions through STEVE’s fast, easy, inexpensive and non‐invasive digital staining procedure.