Widefield microscopy is a fundamental imaging technique that illuminates and captures images of an entire sample area simultaneously. This method provides a direct view of a broad field, making it a common tool in various scientific disciplines.
How Widefield Microscopy Operates
Widefield microscopy illuminates the entire specimen at once, collecting light from the whole field of view. Light originates from a source such as a mercury arc lamp, xenon arc lamp, or LED. This light then passes through various optical components, including filters and mirrors, before reaching the sample.
For fluorescence widefield microscopy, the process involves specific light wavelengths. An excitation filter allows a narrow range of light to pass through, which reflects off a dichroic mirror and travels through the objective lens to illuminate the sample. Fluorescent molecules within the specimen absorb this excitation light and emit light at a longer wavelength. This emitted light travels back through the objective lens, passes through the dichroic mirror, and then goes through an emission filter that blocks any remaining excitation light. The emitted fluorescent light then reaches the observer’s eyepieces or a digital camera, forming the image.
Primary Uses and Advantages
Widefield microscopy finds extensive use in biology and medicine due to its ability to quickly capture images of large areas. Researchers employ it for observing live cell dynamics, analyzing tissue sections, and examining general biological samples. This technique is well-suited for thin specimens like adherent cells or cryosections, typically less than 10 microns thick, where high contrast and clear images can be obtained.
Advantages of widefield microscopy include its relative simplicity and cost-effectiveness compared to more complex imaging systems. Its ability to image the entire field of view at once allows for rapid data acquisition, which is beneficial for observing fast cellular events. This speed and ease of use make it a popular choice for initial screening of experimental protocols and for real-time live-cell imaging applications.
Considerations and Drawbacks
Despite its benefits, widefield microscopy has limitations, particularly when imaging thicker samples. A challenge is the presence of out-of-focus light, which originates from planes above and below the focal plane but is still collected by the detector. This extraneous light reduces image clarity and contrast, making it difficult to discern fine details in samples thicker than approximately 10-30 micrometers.
Prolonged observation of live samples using widefield microscopy can lead to issues like photobleaching and phototoxicity. Photobleaching refers to the irreversible fading of fluorescent signals due to chemical damage to fluorophores caused by light exposure. Phototoxicity involves light-induced damage to living cells, potentially altering their behavior or leading to cell death, often due to the generation of reactive oxygen species. Researchers minimize these effects by optimizing illumination settings and using specific fluorophores.