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'''Optical sectioning''' is the process by which a suitably designed [[microscope]] can produce clear images of a [[focal plane]]s deep within a thick sample. This is used to reduce the need for [[thin section]]ing using instruments such as the [[microtome]]. Many different techniques for optical sectioning are used and several [[microscopy]] 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 dimensions|three dimensional]] reconstruction of a sample from images captured at different focal planes.

==Optical sectioning in traditional light microscopes==
{{see also|Depth of field}}
In an ideal microscope, only light from the focal plane would be allowed to reach the [[detector]] (typically an observer or a [[Charge-coupled device|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 quality of optical sectioning is governed by the same physics as the [[depth of field]] effect in [[photography]]. For a high [[numerical aperture]] lens, equivalent to a wide [[aperture]], the depth of field is small ([[shallow focus]]) and gives good optical sectioning. High [[magnification]] 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 [[Optical resolution|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:

<math>D_z = \frac {\lambda n} {(NA)^2}</math> where '''&lambda;''' is the wavelength, '''n''' the refractive index of the objective lens immersion media and '''NA''' the numerical aperture.<ref>[http://www.microscopyu.com/articles/formulas/formulasfielddepth.html Nikon MicroscopyU - Depth of Field and Depth of Focus]</ref>

In comparison the [[diffraction limit|lateral resolution]] can be approximated as:<ref>[http://www.microscopyu.com/articles/formulas/formulasresolution.html Nikon MicroscopyU - Resolution]</ref>

<math>D_x = D_y = \frac {0.61 \lambda} {NA}</math>

==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]] limits.
{{main|differential interference contrast}}
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 difference]]s 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.
{{main|confocal microscopy}}
[[Confocal microscopy]] 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.<ref>{{cite journal |author=Conchello JA, Lichtman JW |title=Optical sectioning microscopy |journal=Nat. Methods |volume=2 |issue=12 |pages=920–31 |date=December 2005 |pmid=16299477 |doi=10.1038/nmeth815 |url=http://www.nature.com/nmeth/journal/v2/n12/abs/nmeth815.html}}</ref>
{{main|Light sheet fluorescence microscopy}}
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).<ref name="PMID15310904">J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, E. H. Stelzer: ''Optical sectioning deep inside live embryos by selective plane illumination microscopy.'' In: ''[[Science]].'' Band 305, Nr. 5686, 2004, {{ISSN|1095-9203}}, S.&nbsp;1007–1009, {{DOI|10.1126/science.1100035}}, PMID 15310904.</ref> 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.
{{main|two-photon excitation microscopy|multiphoton fluorescence microscope}}
Dual and multi-photon excitation techniques take advantage of the fact that fluorophores can be excited not just by a single [[photon]] of the correct [[energy]] but also by multiple photons, which together provide the correct energy. The additional "[[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.<ref>{{cite journal |author=Gratton E, Barry NP, Beretta S, Celli A |title=Multiphoton fluorescence microscopy |journal=Methods |volume=25 |issue=1 |pages=103–10 |date=September 2001 |pmid=11559001 |doi=10.1006/meth.2001.1219 |url=http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WN5-456JRGD-P&_user=126524&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000010360&_version=1&_urlVersion=0&_userid=126524&md5=d42b19ef62a42159c655ae6134b04164}}</ref>

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]] through two objective lenses to give extremely accurate depth information about a single fluorophore<ref>{{cite journal |author=Shtengel G, Galbraith JA, Galbraith CG, ''et al.'' |title=Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure |journal=Proc. Natl. Acad. Sci. U.S.A. |volume=106 |issue=9 |pages=3125–30 |date=March 2009 |pmid=19202073 |pmc=2637278 |doi=10.1073/pnas.0813131106|bibcode = 2009PNAS..106.3125S }}</ref> and three dimensional [[structured illumination microscopy]].<ref>{{cite journal |author=Carlton PM |title=Three-dimensional structured illumination microscopy and its application to chromosome structure |journal=Chromosome Res. |volume=16 |issue=3 |pages=351–65 |year=2008 |pmid=18461477 |doi=10.1007/s10577-008-1231-9 |url=}}</ref>

The optical sectioning of normal wide field microscopes can be improved significantly by [[deconvolution]], an image processing technique to remove blur from the image according to a measured or calculated [[point spread function]].<ref>{{cite journal |author=Sibarita JB |title=Deconvolution microscopy |journal=Adv. Biochem. Eng. Biotechnol. |volume=95 |issue= |pages=201–43 |year=2005 |pmid=16080270 |doi= |url=}}</ref>

===Other===
Optical sectioning is underdeveloped in non-light microscopes. [[X-ray microscope|X-ray]] and [[electron microscope]]s typically have a large depth of field (poor optical sectioning) and so thin sectioning of samples is still widely used.

[[Scanning probe microscope]]s and [[scanning electron microscope]]s cannot do any form of optical sectioning as these microscopes only interact with the surface of a sample. [[Total internal reflection microscopy]] 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.

==Alternatives==
The primary alternatives to optical sectioning are:
*[[Thin section]]ing of the sample, for example as used in [[histological section|histology]].
*[[Tomography]], which is particularly well developed for [[Electron tomography|transmission electron microscopy]].

==References==
{{Reflist}}

{{Optical microscopy}}

{{DEFAULTSORT:Optical Sectioning}}
[[Category:Microscopy]]