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Which two possible sources of fluorescence can be eliminated?
Background fluorescence: By thoroughly cleaning the sample, using non-fluorescent materials, and optimizing the detection settings, background fluorescence can be reduced. Autofluorescence: This can be minimized by choosing fluorophores that emit at different wavelengths than the autofluorescence or using methods such as spectral unmixing to separate the signal from autofluorescence.
Why do MCF-7 cells autofluoresce?
MCF-7 cells may autofluoresce due to the presence of intracellular compounds, such as flavins, which naturally emit fluorescence when excited by certain wavelengths of light. This autofluorescence can interfere with fluorescence imaging and should be considered when working with these cells. Special care should be taken during imaging to distinguish between autofluorescence and specific fluorescence signals.
What are the two possible sources of fluorescence within an Ecol colony when exposed to UV light?
The two possible sources of fluorescence within an E. coli colony when exposed to UV light are autofluorescence of the colony itself, caused by endogenous fluorophores present in the cells, and expression of a fluorescent protein, such as green fluorescent protein (GFP), as a result of genetic manipulation.
What is the difference between nylon membrane and nitrocellulose membrane?
Nylon membranes are less brittle and easier to handle than nitrocellulose, making them ideal for reprobing. They also respond more robustly to various environmental storage conditions than nitrocellulose. Nylon's highly hydrophilic nature makes prewetting unnecessary, and nylon membranes have much higher binding capacities than nitrocellulose for nucleic acids.
What are the limitations of microscopy imaging for cell migration?
It depends on the scope and the type of cell. The best non-electron microscope would probably be able to see some of the cell organelles. Cellular migration is a very broad statement and can mean many different things. The study of cell migration, mostly related to high-throughput live cell screen, is a major approach employed in drug discovery and gene annotation. Basically, in vitro cell population will be allocated into each well of a culture plate while images are captured from each well at a fix time resolution. By quantifying cell behavior after inducing treatment, it is possible to estimate the cell behavior when comparing to control condition. Depending on either in vivo or in vitro experiment. Here I assume you are doing in vitro high-throughput high-content screening and using either phase contrast or fluorescence microscopy imaging for live cell imaging. A short list of limitations for each microscopy imaging technology: 1. DIC or phase contrast images are difficult to process during image analysis. Also the phenotype of each cell is subjected to nonlinear light diffraction. In short word, two objects in the same size may look different under phase contrast microscope. So at least we don't use phase contrast for our experiment other than demonstration purpose. 2. Epi-fluorescence microscopy imaging is an good option for live cell imaging. However, if your sample contains nucleus staining, then phototoxicity is a serious problem. It may alter the cell migration pattern or even promote massive apoptosis. Also the strength of the laser or light source is a problem, being overexposured to laser will basically boil the cell. Autofluorescence may be a potential problem if you apply any collagen coating or introduce culture medium. But fluorescence image provides a better quality high-content data for image analysis. 3. Confocal laser is used mostly by protein and signaling process research. It is ideal for 3D imaging or visualizing a certain culture layer but image quality is highly depending on the penetration power of fluorescence staining. If the cell culture is not monolayer, you won't be able to separate each cell.
