What is widefield microscopy?
Similar to brightfield microscopy, widefield fluorescence microscopy illuminates the sample from below. The difference is in the light. Instead of a white light, fluorescence microscopy uses light of frequencies that can excite fluorescent molecules associated to your sample. With the widefield fluorescence microscopy technique, you illuminate the whole sample with light of a specific wavelength. You can then visualize or record the emitted light through the microscope’s eyepieces or camera.
The image shows multiple fluorescently-labeled microtubules anchored at a glass surface, visualized using widefield microscopy
How does a widefield microscope works?
- A light source emits an excitation light, which passes through an excitation filter that only allows light of specific wavelengths to pass. The excitation light is reflected off a dichroic mirror, a filter that selectively lets certain colors pass while reflecting the rest.
- Once the excitation light hits the sample, it temporarily excites fluorescent molecules associated to the sample to a higher energy state. Once they relax back to their ground-state, light of a longer wavelength is emitted.
- The objective lens collects some of the emitted light. The dichroic mirror transmits the rest through an emission filter.
- The emission filter blocks excitation light and transmits emission light to the eyepiece or camera.
Explore other imaging techniques
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