Simple Overview

Epifluorescence microscopy

What is epifluorescence microscopy?

In epifluorescence microscopy, a parallel beam of light is passed directly upwards through the sample, maximizing the amount of illumination. This 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, when compared to other illumination modes such as TIRF and HILO.


Epifluorescence super resolution microscopy

Why is epifluorescence microscopy useful?

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 the interpretation of 3-dimensional spatial data, as any out-of-focus light will be collected. This can be resolved by using super-resolution techniques, such as dSTORM, PALM, single-particle tracking and smFRET.

Can I do epifluorescence microscopy on the Nanoimager?

As a microscope, the Nanoimager allows three different modes of imaging depending on the illumination angle: epifluorescence, TIRF or HILO. The Nanoimager takes epifluorescence microscopy to the next level by boosting its resolution to up to 20 nm, with its wide range of super-resolution techniques, such as dSTORM, PALM, single-particle tracking and smFRET. These allow a much richer visualization, with enhanced resolution and increased signal to noise ratio, when compared to standard epifluorescence microscopy. Epifluorescence imaging is often used for imaging thick samples such as whole tissues. However, HILO has become preferable to further optimise signal to noise ratio.


In all three imaging modes on the Nanoimager, two fluorophores can be captured simultaneously, improving understanding of molecular interactions 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, no longer limiting imaging to a dark room.

Learn more about the Nanoimager and its other illumination modes.

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