Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light.^[1] It enables the reconstruction of three-dimensional structures from the obtained images. This technique has gained popularity in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science.

Basic concept

The principle of confocal imaging was patented in 1957 by Marvin Minsky^[2][3] and aims to overcome some limitations of traditional wide-field fluorescence microscopes. In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen is flooded evenly in light from a light source. All parts of the specimen in the optical path are excited at the same time and the resulting fluorescence is detected by the microscope's photodetector or camera including a large unfocused background part. In contrast, a confocal microscope uses point illumination (see Point Spread Function) and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal - the name "confocal" stems from this configuration. As only light produced by fluorescence very close to the focal plane can be detected, the image's optical resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes. However, as much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of decreased signal intensity – so long exposures are often required.

As only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (i.e., a rectangular pattern of parallel scanning lines) in the specimen. The achievable thickness of the focal plane is defined mostly by the wavelength of the used light divided by the numerical aperture of the objective lens, but also by the optical properties of the specimen. The thin optical sectioning possible makes these types of microscopes particularly good at 3D imaging and surface profiling of samples.

Techniques used for horizontal scanning

Three types of confocal microscopes are commercially available:

• Confocal laser scanning microscopes use multiple mirrors (typically 2 or 3 scanning linearly along the x and the y axis) to scan the laser across the sample and "descan" the image across a fixed pinhole and detector.
• Spinning-disk (Nipkow disk) confocal microscopes use a series of moving pinholes on a disc to scan spot of light. Since a series of pinholes scans an area in parallel each pinhole is allowed to hover over a specific area for a longer amount of time thereby reducing the excitation energy needed to illuminate a sample when compared to laser scanning microscopes. Decreased excitation energy reduces photo-toxicity and photo-bleaching of a sample often making it the preferred system for imaging live cells or organisms.
• Microlens enhanced or dual spinning disk confocal microscopes work under the same principles as spinning-disk confocal microscopes except a second spinning disk containing micro-lenses is placed before the spinning disk containing the pinholes. Every pinhole has an associated micro-lens. The micro-lenses act to capture a broad band of light and focus it into each pinhole significantly increasing the amount of light directed into each pinhole and reducing the amount of light blocked by the spinning disk. Microlens enhanced confocal microscopes are therefore significantly more sensitive than standard spinning disk systems. Yokogawa Electric invented this technology in 1992.^[4]
• Programmable array microscopes (PAM) use an electronically controlled spatial light modulator (SLM) that produces a set of moving pinholes. The SLM is a device containing an array of pixels with some property (opacity, reflectivity or optical rotation) of the individual pixels that can be adjusted electronically. The SLM contains microelectromechanical mirrors or liquid crystal components. The image is usually acquired by a charge coupled device (CCD) camera.

Each of these classes of confocal microscope have particular advantages and disadvantages. Most systems are either optimized for recording speed (i.e. video capture) or high spatial resolution. Confocal laser scanning microscopes can have a programmable sampling density and very high resolutions while Nipkow and PAM use a fixed sampling density defined by the camera's resolution. Imaging frame rates are typically slower for single point laser scanning systems than spinning-disk or PAM systems. Commercial spinning-disk confocal microscopes achieve frame rates of over 50 per second^[5] – a desirable feature for dynamic observations such as live cell imaging.

In practice, Nipkow and PAM allow multiple pinholes scanning the same area in parallel^[6] as long as the pinholes are sufficiently far apart.

Cutting-edge development of confocal laser scanning microscopy now allows better than standard video rate (60 frames/second) imaging by using multiple microelectromechanical scanning mirrors.

Confocal X-ray fluorescence imaging is a newer technique that allows control over depth, in addition to horizontal and vertical aiming, for example, when analyzing buried layers in a painting.^[7]

Variants and enhancements

Improving axial resolution

The point spread function of the pinhole is an ellipsoid, several times as long as it is wide. This limits the axial resolution of the microscope. One technique of overcoming this is 4π microscopy where incident and or emitted light are allowed to interfere from both above and below the sample to reduce the volume of the ellipsoid. An alternative technique is confocal theta microscopy. In this technique the cone of illuminating light and detected light are at an angle to each other (best results when they are perpendicular). The intersection of the two point spread functions gives a much smaller effective sample volume. From this evolved the single plane illumination microscope.

Super resolution

There are confocal variants that achieve resolution below the diffraction limit such as stimulated emission depletion microscopy (STED). Besides this technique a broad variety of other (not confocal based) superresolution techniques is available like PALM, (d)STORM, SIM, and so on. They all have their own advantages like ease of use, resolution and the need for special equipment/buffers/fluorophores/... .

Low-temperature operability

To image samples at low temperature, two main approaches have been used, both based on the laser scanning confocal microscopy architecture. One approach is to use a continuous flow cryostat: only the sample is at low temperature and it is optically addressed through a transparent window.^[8] Another possible approach is to have part of the optics (especially the microscope objective) in a cryogenic storage dewar.^[9] This second approach, although more cumbersome, guarantees better mechanical stability and avoids the losses due to the window.

Images

• 
  β-tubulin in Tetrahymena (a ciliated protozoan).
• 
  Partial surface profile of a 1-Euro coin, measured with a Nipkow disk confocal microscope.
• 
  Reflection data for 1-Euro coin.
• 
  Cross section at y=200 through profile of 1-Euro coin.

References