Epifluorescence microscopy is a mode of fluorescence imaging where light passes through the sample in a straight angle, maximizing the amount of illumination (it is also referred to as widefield microscopy). Like in any fluorescence microscope, a high-intensity light source is used. In epifluorescence microscopes, both the illuminated and emitted light travel through the same objective lens - hence the term "epi" from Greek, meaning "same".
Epifluorescence microscopy is particularly useful when imaging thick samples over 10µm deep. However, the intense illumination and excitation of molecules outside the focal plane can produce images with a high background signal in contrast to illumination modes like TIRF and HILO.
Epifluorescence microscopy is widely used in cell biology as the illumination beam penetrates the full depth of the sample, allowing easy imaging of intense signals and co-localization studies with multi-colored labeling on the same sample. Epifluorescence imaging can, however, limit the precise localization of fluorescence molecules and does not allow to interpret 3-dimensional spatial data, as any out-of-focus light will be collected. This can be resolved by using super-resolution techniques, which circumvent the main limitation of fluorescence microscopy in general, a limited resolution power that cannot distinguish objects that are less than 200nm apart.
As a microscope, the Nanoimager allows three different modes of imaging depending on the illumination angle: epifluorescence, TIRF or HILO. With thick samples, such as whole tissues, epifluorescence can be employed but frequently HILO would be preferable to optimise signal to noise ratio. The Nanoimager takes epifluorescence microscopy to the next level by boosting its resolution to up to 20nm, with its wide range of super-resolution techniques, such as dSTORM, PALM, single particle tracking, smFRET and SIM microscopy. These allow a much richer visualisation, with enhanced resolution and increased signal to noise ratio, when compared to standard epifluorescence microscopy.
With the Nanoimager, two fluorophores can be captured simultaneously to improve understanding of how different molecules interact and with a total of four laser colors, four different fluorophores can be used in a single sample. Its small, compact design also provides unrivalled stability, allowing the microscope to be used in any lab environment, not just in a dark room.
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