Overview of the technique
Widefield and confocal fluorescence microscopy are techniques that illuminate the sample with lasers or LEDs and filters to excite fluorophores in the sample. The resolution is limited by the diffraction limit of light and the objective lens in use, and these techniques act as the basis for more advanced fluorescence microscopy techniques. They have both become common tools for biological imaging due to their ease of use and the robustness of the microscopes being produced by modern manufacturers.
In widefield fluorescence microscopy, there is no attempt to limit the out of focus light from the final image, and a camera can be used to collect the emitted light. Widefield is therefore most useful for thin samples where there aren’t fluorophores above or below the structures of interest, and for imaging live samples with short exposure times and low light doses. On the other hand, confocal microscopy can be used to overcome the issue of out-of-focus light in thicker samples by eliminating it using a pinhole positioned in the detection apparatus. The pinhole creates an optical section or slice through the sample, where only one plane of emission is seen at the focal plane. However, the full illumination cone above and below the focal plane is still illuminating the sample and results in photodamage and bleaching of those areas. Confocal has many more options for combinations of laser lines (including free choice of laser line using white light lasers) and detection optics with tuneable filters. Widefield microscopes usually use LEDs and physical filters that can be more limited in power, precision and flexibility.
Widefield and confocal microscopy are not offered as technologies to access at Octopus on their own, but they can be used as an accessory to other techniques.