Confocal Microscopy: Principles and Modern Practices

Abstract

In light microscopy, illuminating light is passed through the sample as uniformly as possible over the field of view. For thicker samples, where the objective lens does not have sufficient depth of focus, light from sample planes above and below the focal plane will also be detected. The out-of-focus light will add blur to the image, reducing the resolution. In fluorescence microscopy, any dye molecules in the field of view will be stimulated, including those in out-of-focus planes. Confocal microscopy provides a means of rejecting the out-of-focus light from the detector such that it does not contribute blur to the images being collected. This technique allows for high-resolution imaging in thick tissues. In a confocal microscope, the illumination and detection optics are focused on the same diffraction-limited spot in the sample, which is the only spot imaged by the detector during a confocal scan. To generate a complete image, the spot must be moved over the sample and data collected point by point. A significant advantage of the confocal microscope is the optical sectioning provided, which allows for 3D reconstruction of a sample from high-resolution stacks of images. Several types of confocal microscopes have been developed for this purpose, and each has different advantages and disadvantages. This article provides a concise introduction to confocal microscopy. © 2019 by John Wiley & Sons, Inc.

Keywords: confocal microscopy; fluorescence; laser scanning; resonant scanning; spinning disk.

Figure 1.

Components of a confocal microscope. A. Light from a laser source is passed through collimating optics to a variable dichromatic mirror or AOBS and reflected to the objective lens which focuses the beam on a point in the sample. Scanning mirrors sweep the excitation beam over the sample point by point to build the image. Emitted fluorescence passes back through the objective lens, the dichromatic mirror or AOBS, and is detected by the PMT(s). A pinhole placed in the conjugate image plane to the focal point in the sample serves to reject out-of-focus light, which does is not picked up by the detector. In this epifluorescence configuration, the illumination and emission light both pass through the same lens, thus requiring only the detector-side pinhole. Varying the size of the pinhole changes the amount of light collected and the optical section thickness. Spectral imaging can be achieved with an array of PMTs and a diffraction grating, or prism, placed in the emission light path. B. A schematic of the scanning mirrors employed by confocal microscopes to sweep the excitation light across the sample.

Figure 2.

Widefield vs confocal microscopy. One hemisegment of a Drosophila larval fillet stained with AlexaFluor 647-conjugated phalloidin to label the musculature. In the widefield image (top), data were collected on a widefield epifluorescence microscope. The confocal image was taken with the pinhole set to 1 Airy Unit. Both images were collected with 20x objective lenses. The confocal image required ~2 hours to build in a point scanning system and the widefield image was collected with an integration time of 1 second.

Figure 3.

Slices of a confocal imaging stack of microtubules. Hela cells were stained with anti-tubulin primary and AlexaFluor 488-conjugated secondary antibodies. Confocal z-stacks were collected at 40x magnification with an oil immersion objective and 1 μm slices.

Figure 4.

Maximum intensity projection of multiple fields-of-view at 40x magnification. Three fields of view of the neuromuscular junction from a Drosophila larval fillet stained with AlexaFluor 555-conjugated phalloidin (gray), AlexaFluor 488 labeling glutamatergic motor neurons (green), and AlexaFluor 647 labeling Dlg-1 expressing type 1b post-synaptic sites. Confocal z stacks were collected with an oil immersion objective and 0.8 μm slices. Each field of view required ~10 minutes to collect with a LSCM.

Figure 5.

Maximum-intensity projection of a multi-color, multi-tile confocal image stack. A single hemisegment of the sample in Figure 4 collected with a resonant scanning confocal microscope with a 20x air objective and 0.7 μm slices covering 4 tiles that were stitched together during image processing.

Figure 6.

Time-series of forward larval locomotion in Drosophila melanogaster. Larvae expressing calcium biosensor GCaMP6s (Chen, 2013) in the muscles were monitored during forward locomotion in a confocal microscope at 4x magnification with the pinhole open to collect more light.