Optical sectioning is the process by which a suitably designed
microscope
A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisi ...
can produce clear images of
focal plane
In Gaussian optics, the cardinal points consist of three pairs of points located on the optical axis of a rotationally symmetric, focal, optical system. These are the '' focal points'', the principal points, and the nodal points. For ''ideal'' ...
s deep within a thick sample. This is used to reduce the need for
thin section
In optical mineralogy and petrography, a thin section (or petrographic thin section) is a thin slice of a rock or mineral sample, prepared in a laboratory, for use with a polarizing petrographic microscope, electron microscope and electron ...
ing using instruments such as the
microtome
A microtome (from the Greek ''mikros'', meaning "small", and ''temnein'', meaning "to cut") is a cutting tool used to produce extremely thin slices of material known as ''sections''. Important in science, microtomes are used in microscopy, all ...
. Many different techniques for optical sectioning are used and several
microscopy
Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of micr ...
techniques are specifically designed to improve the quality of optical sectioning.
Good optical sectioning, often referred to as good depth or z resolution, is popular in modern microscopy as it allows the
three-dimensional
Three-dimensional space (also: 3D space, 3-space or, rarely, tri-dimensional space) is a geometric setting in which three values (called '' parameters'') are required to determine the position of an element (i.e., point). This is the inform ...
reconstruction of a sample from images captured at different focal planes.
Optical sectioning in traditional light microscopes
In an ideal microscope, only light from the focal plane would be allowed to reach the
detector (typically an observer or a
CCD) producing a clear image of the plane of the sample the microscope is focused on. Unfortunately a microscope is not this specific and light from sources outside the focal plane also reaches the detector; in a thick sample there may be a significant amount of material, and so spurious signal, between the focal plane and the
objective lens.
With no modification to the microscope, i.e. with a simple wide field
light microscope
The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microsc ...
, the quality of optical sectioning is governed by the same physics as the
depth of field
The depth of field (DOF) is the distance between the nearest and the furthest objects that are in acceptably sharp focus in an image captured with a camera.
Factors affecting depth of field
For cameras that can only focus on one object dis ...
effect in
photography
Photography is the art, application, and practice of creating durable images by recording light, either electronically by means of an image sensor, or chemically by means of a light-sensitive material such as photographic film. It is emplo ...
. For a high
numerical aperture
In optics, the numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. By incorporating index of refraction in its definition, NA has the proper ...
lens, equivalent to a wide
aperture
In optics, an aperture is a hole or an opening through which light travels. More specifically, the aperture and focal length of an optical system determine the cone angle of a bundle of rays that come to a focus in the image plane.
An ...
, the depth of field is small (
shallow focus) and gives good optical sectioning. High
magnification
Magnification is the process of enlarging the apparent size, not physical size, of something. This enlargement is quantified by a calculated number also called "magnification". When this number is less than one, it refers to a reduction in si ...
objective lenses typically have higher numerical apertures (and so better optical sectioning) than low magnification objectives.
Oil immersion objectives typically have even larger numerical apertures so improved optical sectioning.
The
resolution in the depth direction (the "z resolution") of a standard wide field microscope depends on the numerical aperture and the wavelength of the light and can be approximated as:
where λ is the wavelength, n the refractive index of the objective lens immersion media and NA the numerical aperture.
In comparison, the
lateral resolution can be approximated as:
Techniques for improving optical sectioning
Bright-field light microscopy
Beyond increasing numerical aperture, there are few techniques available to improve optical sectioning in bright-field light microscopy. Most microscopes with oil immersion objectives are reaching the limits of numerical aperture possible due to
refraction
In physics, refraction is the redirection of a wave as it passes from one medium to another. The redirection can be caused by the wave's change in speed or by a change in the medium. Refraction of light is the most commonly observed phenomen ...
limits.
Differential interference contrast (DIC) provides modest improvements to optical sectioning. In DIC the sample is effectively illuminated by two slightly offset light sources which then interfere to produce an image resulting from the
phase differences of the two sources. As the offset in the light sources is small the only difference in phase results from the material close to the focal plane.
Fluorescence microscopy
In
fluorescence microscopy objects out of the focal plane only interfere with the image if they are illuminated and fluoresce. This adds an extra way in which optical sectioning can be improved by making illumination specific to only the focal plane.
Confocal microscopy
Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a ...
uses a scanning point or points of light to illuminate the sample. In conjunction with a pinhole at a
conjugate focal plane this acts to filter out light from sources outside the focal plane to improve optical sectioning.
Lightsheet based fluorescence microscopy illuminates the sample with excitation light under an angle of 90° to the direction of observation, i.e. only the focal plane is illuminated using a laser that is only focused in one direction (lightsheet).
This method effectively reduces out-of focus light and may in addition lead to a modest improvement in longitudinal resolution, compared to epi fluorescence microscopy.
Dual and multi-photon excitation techniques take advantage of the fact that fluorophores can be excited not just by a single
photon
A photon () is an elementary particle that is a quantum of the electromagnetic field, including electromagnetic radiation such as light and radio waves, and the force carrier for the electromagnetic force. Photons are massless, so they alwa ...
of the correct
energy
In physics, energy (from Ancient Greek: ἐνέργεια, ''enérgeia'', “activity”) is the quantitative property that is transferred to a body or to a physical system, recognizable in the performance of work and in the form of ...
but also by multiple photons, which together provide the correct energy. The additional "
concentration
In chemistry, concentration is the abundance of a constituent divided by the total volume of a mixture. Several types of mathematical description can be distinguished: '' mass concentration'', '' molar concentration'', ''number concentration'', ...
"-dependent effect of requiring multiple photons to simultaneously interact with a fluorophore gives stimulation only very close to the focal plane. These techniques are normally used in conjunction with confocal microscopy.
Further improvements in optical sectioning are under active development, these principally work through methods to circumvent the diffraction limit of light. Examples include single photon
interferometry
Interferometry is a technique which uses the '' interference'' of superimposed waves to extract information. Interferometry typically uses electromagnetic waves and is an important investigative technique in the fields of astronomy, fiber o ...
through two objective lenses to give extremely accurate depth information about a single fluorophore and three-dimensional
structured illumination microscopy.
The optical sectioning of normal wide field microscopes can be improved significantly by
deconvolution
In mathematics, deconvolution is the operation inverse to convolution. Both operations are used in signal processing and image processing. For example, it may be possible to recover the original signal after a filter (convolution) by using a deco ...
, an image processing technique to remove blur from the image according to a measured or calculated
point spread function.
Clearing agents
Optical sectioning can be enhanced by the use of clearing agents possessing a high refractive index (>1.4) such as Benzyl-Alcohol/Benzyl Benzoate (BABB) or Benzyl-ether which render specimens transparent and therefore allow for observation of internal structures.
Other
Optical sectioning is underdeveloped in non-light microscopes.
X-ray
An X-ray, or, much less commonly, X-radiation, is a penetrating form of high-energy electromagnetic radiation. Most X-rays have a wavelength ranging from 10 picometers to 10 nanometers, corresponding to frequencies in the range 30&nb ...
and
electron microscope
An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a hi ...
s typically have a large depth of field (poor optical sectioning), and thus thin sectioning of samples is still widely used.
Although similar physics guides the focusing process,
Scanning probe microscope
Scan may refer to:
Acronyms
* Schedules for Clinical Assessment in Neuropsychiatry (SCAN), a psychiatric diagnostic tool developed by WHO
* Shared Check Authorization Network (SCAN), a database of bad check writers and collection agency for bad ...
s and
scanning electron microscopes are not typically discussed in the context of optical sectioning as these microscopes only interact with the surface of the sample.
Total internal reflection microscopy
Total internal reflection microscopy is a specialized optical imaging technique for object tracking and detection utilizing the light scattered from an evanescent field in the vicinity of a dielectric interface. Its advantages are a high signal-to ...
is a fluorescent microscopy technique, which intentionally restricts observation to either the top or bottom surfaces of a sample, but with extremely high depth resolution.
3D imaging using a combination of focal sectioning and tilting has been demonstrated theoretically and experimentally in order to provide exceptional 3D resolution over large fields of view.
Alternatives
The primary alternatives to optical sectioning are:
*
Thin section
In optical mineralogy and petrography, a thin section (or petrographic thin section) is a thin slice of a rock or mineral sample, prepared in a laboratory, for use with a polarizing petrographic microscope, electron microscope and electron ...
ing of the sample, for example as used in
histology
Histology,
also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures v ...
.
*
Tomography
Tomography is imaging by sections or sectioning that uses any kind of penetrating wave. The method is used in radiology, archaeology, biology, atmospheric science, geophysics, oceanography, plasma physics, materials science, astrophysics, ...
, which is particularly well developed for
transmission electron microscopy
Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a ...
.
References
{{DEFAULTSORT:Optical Sectioning
Microscopy