confocal scanning microscopes (original) (raw)
Acronym: CLSM = confocal laser scanning microscope
Definition: optical microscopes with enhanced depth resolution based on the confocal measurement principle
Alternative terms: confocal microscopes, confocal laser scanning microscopes
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- imaging systems
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* confocal scanning microscopes
* fluorescence microscopes
* laser microscopes
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Related: laser microscopyfluorescence microscopyoptical profilometers
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Contents
What are Confocal Scanning Microscopes?
Confocal microscopes are scanning microscopes, the primary feature of which is a substantially improved longitudinal (axial) resolution by suppressing image contributions from light outside a rather thin plane. This allows one to acquire three-dimensional images e.g. of transparent biological and medical samples — effectively applying a kind of “optical sectioning” instead of physical sectioning of samples. With such microscopes, one can even observe tiny details within living cells, for example. Therefore, such instruments have become important for biological and medical research and various other areas of research and industrial inspection.
The principle of confocal microscopy is also applied in some optical profilometers.
Operation Principle of Confocal Microscopy
In a conventional optical microscope, a quite high lateral image resolution (well below 1 ÎĽm) can be achieved by using a microscope objective with short focal length and a high numerical aperture. A remaining problem, however, is that light from different longitudinal positions in a transparent sample can contribute to an image, leading to a limited longitudinal resolution. Some limited amount of suppression is obtained by the fact that contributions from light outside the object plane (selected with the focus adjustment) are out of focus, i.e., strongly blurred.
Substantially enhanced longitudinal (axial) resolution can be obtained by utilizing the principle of confocal microscopy, which was invented and patented by Marvin Lee Minsky in the 1950s [2], i.e., before the invention of the laser. (The confocal principle was actually recognized even earlier [1], but apparently the term has been introduced only much later, in 1977 [5], in the context of a thorough theoretical analysis of the operation principle of confocal microscopy.)
Figure 1: Setup of a confocal laser microscope.
A confocal scanning microscope (see Figure 1) in a modern form essentially works as follows:
- A diffraction-limited collimated laser beam is tightly focused onto the sample, using a microscope objective (point illumination instead of illumination of the whole object).
- The light coming back from the focus point (e.g. through scattering in the sample, or fluorescence light) is imaged onto a small pinhole and transmitted to a photodetector.
- The recorded intensity conveys information on just a single object point. A complete two-dimensional or three-dimensional image is obtained by systematically translating either the beam focus (with some kind of laser scanner, e.g. with oscillating mirrors) or alternatively the sample, scanning a certain area or volume in the sample (flying spot microscopy).
Light coming from other longitudinal or transverse positions in the sample is largely suppressed:
- At other lateral positions, there is hardly any incident laser light, except somewhat before and after the beam focus due to the substantial beam divergence. Also, such light could not get through the pinhole, since it would be focused on points away from the hole.
- Light from positions in the sample above or below the beam focus can also not well get through the pinhole because its focus position is not in the plane of the pinhole (the selected optically conjugate plane). In addition, the laser intensity quickly gets smaller due to the substantial beam divergence.
It is essential to ensure that the pinhole is adjusted such that one collects light exactly from the laser beam focus.
The obtained optical power registered by the photodetector is usually rather small. Therefore, sensitive detectors such as photomultiplier tubes or avalanche photodiodes are normally used to limit the required exposure time.
The principle of confocal microscopy can be modified in various ways:
- For operation in transmission, laser illumination may be done through a second microscope objective on the other side of the sample.
- One may use an angular separation of the beam paths for illumination and detection, enhancing the imaging of weakly scattering objects, e.g. nanoparticles. This idea of “angular gating” led to the selected plane illumination microscope (SPIM), also called orthogonal-plane fluorescence optical sectioning (OPFOS).
- The pinhole can be replaced with the end of a single-mode fiber, acting as an optical aperture both for illumination and detection [10, 11]. This principle led to a particularly flexible microscope.
- One may employ second harmonic generation [7] or third harmonic generation [13] in certain samples (SHG or THG microscopy). In that case, detection must occur in the direction of the laser beam.
- The lateral resolution of confocal microscopes is usually similar to that of conventional optical microscopes — limited by diffraction to the order of half the optical wavelength. However, sub-diffraction resolution (super-resolution leading to nanoscopy) can be realized in some fluorescence microscopes based on the principle of stimulated emission depletion microscopy (STED microscopy); see the article on fluorescence microscopy for more details.
Scanning with a Nipkow Disk
In the 1960s, a new scanning technique for confocal microscopes (tandem scanning microscopy) was invented by Mojmir Petran and Milan Hadravsky. They used a Nipkow disk, which is a disk having a spiral pattern of thousands of pinholes. During operation, that disk is rotated, so that many moving points are simultaneously illuminated in the sample, which helps to substantially decrease the overall exposure time. The collected light also goes through the disk (or alternatively through a second, synchronously rotating disk) and gets to an electronic image sensor, which can simultaneously measure intensities corresponding to multiple pinholes.
The first commercially available confocal scanning microscope was based on the principle of the Nipkow disk.
The method has been developed further, replacing the spinning disk with an electronically controlled spatial light modulator (programmable array microscopes, PAM).
Light Sources for Confocal Microscopes
Modern confocal microscopes usually use some kind of laser, since its high spatial coherence is ideal for tightly focusing to a small spot in the sample. In principle, however, one can also use a non-coherent light source, where the radiation first needs to be focused on an additional pinhole and then collimated; that was done in early confocal microscopes. That way, however, one loses most of the optical power. The first scanning laser microscope (with a helium–neon laser) was demonstrated in 1969 at Yale University [3].
Many confocal microscopes use a continuous-wave laser, but some fluorescence microscopes require a mode-locked laser (ultrafast laser), emitting ultrashort pulses for two-photon excitation or multi-photon excitation of fluorescence.
Frequently Asked Questions
What is a confocal scanning microscope?
A confocal scanning microscope is an optical instrument that creates sharp images by using a pinhole to block out-of-focus light. This technique provides a substantially improved longitudinal (axial) resolution, enabling 'optical sectioning' to build three-dimensional images of a sample.
How does a confocal microscope achieve optical sectioning?
It uses a laser beam focused to a single point within the sample. Light returning from this focal point is imaged onto a small pinhole that blocks light coming from above or below the focal plane. An image is then constructed by scanning this point across the sample.
What is the main advantage of confocal microscopy over conventional microscopy?
The primary advantage is the significantly enhanced longitudinal resolution, which allows for the rejection of out-of-focus blur. This enables the acquisition of clear, optically-sectioned images from deep within thick or transparent samples.
Why are lasers the preferred light source for modern confocal microscopes?
Lasers are ideal because their high spatial coherence allows the beam to be focused down to a very small, diffraction-limited spot. This tight focusing is essential for the point-by-point illumination and scanning principle of confocal microscopy.
What are typical applications of confocal microscopes?
These microscopes are vital in biological and medical research for tasks like observing fine details within living cells and acquiring 3D images of tissues. They are also used in industrial inspection and for creating detailed surface profiles with optical profilometers.
What is a Nipkow disk used for in some confocal microscopes?
A Nipkow disk is a rotating disk with a spiral pattern of thousands of pinholes. It enables the simultaneous illumination and imaging of many points in the sample, which substantially decreases the time needed to acquire a full image.
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Bibliography
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|---|---|
| [2] | Marvin Minsky, “Microscopy Apparatus”, US Patent 3.013.467, granted in 1961 |
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(Suggest additional literature!)
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