Standard fluorescence microscopy illuminates the entire volume of the sample (or as far as the excitation light can penetrate). This generates out-of-focus fluorescent light, reducing resolution and obscuring fine details when captured by the detector. Confocal microscopy removes this light and significantly improves the imaging resolution.
Confocal microscopy is achieved by eliminating fluorescence from outside the plane of interest. One method for achieving this in practice is by adding two pinhole apertures to a standard fluorescence microscope. The first pinhole is placed in front of the excitation source to allow a very specific optical path. This excitation light is reflected from a dichroic mirror towards an objective lens which focusses the light to a spot in the sample. Fluorophores within the sample absorb the light and fluoresce.
The objective lens collects the resulting fluorescent light which then passes through the dichroic mirror. As a small amount of fluorescence is also generated along the excitation path, a second pinhole in front of the detector is used to block this out-of-focus light from reaching the detector.
Two-dimensional slices are captured by scanning the excitation light in X and Y using one or more motorised oscillating mirrors (“galvanometer mirror scanners”), or by moving the sample under a stationary beam using a motorised translation stage. Successive slices are then computationally stacked to produce a three-dimensional image.
Spinning disk confocal microscopy
Spinning disk confocal microscopy is an imaging method that uses an array of rotating pinholes to speed up standard confocal microscopy.
In standard confocal microscopy, the excitation light is passed through a single pinhole in front of the source to generate a single spot. The spot is then scanned in a raster across the sample. This is a relatively slow technique as fluorescence is only generated in a single spot at a time.
Spinning disk microscopy instead uses an array of rotating pinholes. They are often combined with an array of lenses, providing each pinhole with a dedicated focussing component. This creates dozens of spots that scan across the sample and generate fluorescence simultaneously, increasing the rate of emission.
The fluorescent light returns through the same confocal pinholes, thus blocking out-of-focus light. A dichroic mirror is positioned between the lenses and the pinholes to preferentially reflect the emitted wavelengths onto a CCD camera.