Microscopic Imaging Classroom丨Widefield Fluorescence Microscope
In live cell imaging applications, widefield fluorescence microscopy facilitates observation of the dynamics of adherent cells grown in specific environmental chambers placed on the microscope stage. In its most basic configuration, a standard inverted tissue culture microscope equipped with EPI fluorescence illumination is coupled with an area array detector system (usually a CCD camera), suitable fluorescence filters, and a shutter system to limit excessive exposure of cells to harmful Excitation light. Basic fluorescence microscopy relies on carefully matched interference filters to select specific bandwidths for illumination and detection of emitted light. Light sources include mercury, xenon, and metal halide arc lamps, beam expanding laser systems, and light emitting diodes (LEDs), all of which require different filter specifications. Synthetic fluorophores used in fluorescence microscopy have emission spectra covering the near-ultraviolet, visible, and near-infrared regions. The use of genetically encoded fluorescent proteins has greatly expanded the capabilities of fluorescence microscopy in live-cell imaging, allowing researchers to precisely target subcellular regions of interest.
Nuohai LS 18, a new type of light-sheet illumination microscope independently developed by Nuohai, is specially designed for high-resolution 3D imaging of transparent large tissue samples, and is dedicated to exploring various intact tissues such as the brain, spleen, small intestine, kidney, lung, heart, and tumor. 3D precise structure of organs.
In widefield fluorescence, the full-aperture emission collected by the microscope objective maximizes the recorded signal while minimizing the required exposure time. Thus, samples can be imaged with very short light periods. The main disadvantage of widefield imaging is that fluorescence from regions far from the focal plane, as well as background signal, is unwanted light that often obscures features of interest. Therefore, widefield imaging yields the best results when the features of interest are large (such as organelles) or highly punctate in nature. A wide variety of live cell samples, including adherent cells, bacteria, yeast, and very thin tissue sections, are ideal candidates for widefield fluorescence imaging, however, thicker tissues (over 5 microns) are best used with more advanced method imaging.
Although there have been many advances in fluorescent imaging with synthetic fluorescent dyes, quantum dots, and fluorescent proteins, in some cases it is helpful to combine fluorescence with other imaging modalities. As an example, DIC can be used in conjunction with widefield fluorescence to monitor cell viability and general morphology while studying phenomena of interest for specific labeled targets. Acquiring DIC and fluorescence in a single image is usually impractical, but in a properly configured microscope the two techniques can be used sequentially. Thus, after imaging in epifluorescence mode, DIC images can be acquired from fluorescently labeled samples using transmitted light. The two images can be merged during post-analysis. Simultaneous acquisition of DIC and fluorescence images is possible in advanced widefield microscope configurations using spectrally separated illumination (such as visible and near-infrared) and dual-camera or split-view camera adapters. In most laser scanning confocal microscopes, images can be acquired in both fluorescence and DIC modes simultaneously, eliminating the need for post-acquisition processing. Phase contrast and Hoffman modulation contrast can also be used in conjunction with widefield fluorescence microscopy. However, in these techniques, the objective lens is special, whether it is a phase ring or a Hoffman modulation plate, it will cause a loss of emission intensity of 5-15%.
Article link: Instrument Equipment Network https://www.instrumentsinfo.com/technology/show-1123.html
