Objective-Launched TIRF Causes Reduced Field-of-View

To induce fluorescence in a fluoresence microscope, excitation light is directed onto the labeled specimen. The emitted fluorescence provides valuable information, e.g.  indicating a specific biological reaction or structure. Therefore, ensuring uniform illumination of the specimen by the excitation light is crucial. This ensures that any subtle fluorescence signals originating from distinct areas of the sample are attributed to biological phenomena rather than uneven light distribution.

Most microscopes use lenses to shine light on the sample, but this often results in uneven illumination. The light forms a Gaussian shape, meaning it’s brightest at the center and fades towards the edges of the field-of-view. To get clear images, only the central area with manageable light drop-off is used, especially important for super-resolution microscopes. This limits the field-of-view, resulting in clear but small images.