Confocal microscopy, or scanning laser confocal microscopy, is an optical imaging technique that increases the resolution and contrast of images. In this technique, lasers are used to illuminate the sample, as an intense and monochromatic light source is required.
A confocal microscope only illuminates one point of the sample and a pinhole is used to remove out-of-focus light. In comparison, a widefield microscope evenly illuminates the whole sample, resulting in a large, unfocused background being captured.
The illumination in a confocal microscope is achieved by scanning focused light beams across the sample, creating horizontal optical sections. These optical sections can be collected at different depths of the sample to reconstruct 3D structures - in a process known as Z-stacking. This method is direct but noninvasive, allowing imaging of thick and sometimes living samples.
Although the optical resolution is improved when using a confocal microscope, blocking the fluorescence at the pinhole leads to decreased signal intensity. This is why a laser and a photo-multiplier tube (PMT) are used. The PMT transforms the detected light into electrical energy that can be used by the computer to build the image.
Despite confocal microscopy is not a cheap technique, its benefits enable it to be widely used in biological science, ranging from cell biology and genetics to microbiology.
For a sample to be suitable for fluorescence microscopy, it must be fluorescent. This means it must contain fluorophores.