Editing Microscopy (section)

=== Techniques ===
{{Main|Optical microscope}}
To improve specimen [[contrast (vision)|contrast]] or highlight structures in a sample, special techniques must be used. A huge selection of microscopy techniques are available to increase contrast or label a sample.

<gallery caption="Four examples of transillumination techniques used to generate contrast in a sample of [[tissue paper]]. 1.559 μm/pixel." align="center">
Image:Paper_Micrograph_Bright.png|[[Bright field microscopy|Bright field]] illumination, sample contrast comes from [[absorbance]] of light in the sample
Image:Paper_Micrograph_Cross-Polarised.png|[[Polarized light microscopy|Cross-polarized light]] illumination, sample contrast comes from rotation of [[Polarization (waves)|polarized]] light through the sample
Image:Paper_Micrograph_Dark.png|[[Dark field]] illumination, sample contrast comes from light [[scattered radiation|scattered]] by the sample
Image:Paper_Micrograph_Phase.png|[[Phase contrast]] illumination, sample contrast comes from [[Interference (wave propagation)|interference]] of different path lengths of light through the sample
</gallery>

==== Bright field ====
{{Main|Bright field microscopy}}
Bright field microscopy is the simplest of all the light microscopy techniques. Sample illumination is via transmitted white light, i.e. illuminated from below and observed from above. Limitations include low contrast of most biological samples and low apparent resolution due to the blur of out-of-focus material. The simplicity of the technique and the minimal sample preparation required are significant advantages.{{cn|date=June 2022}}

==== Oblique illumination ====
The use of oblique (from the side) illumination gives the image a three-dimensional appearance and can highlight otherwise invisible features. A more recent technique based on this method is ''Hoffmann's modulation contrast'', a system found on inverted microscopes for use in cell culture. Oblique illumination enhances contrast even in clear specimens; however, because light enters off-axis, the position of an object will appear to shift as the focus is changed. This limitation makes techniques like optical sectioning or accurate measurement on the z-axis impossible.

==== Dark field ====
{{Main|Dark field microscopy}}
Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens.<ref>{{cite web | vauthors = Abramowitz M, Davidson MW | title = Darkfield Illumination | date = 2003-08-01 | url = http://micro.magnet.fsu.edu/primer/techniques/darkfield.html | access-date = 2008-10-21 | archive-date = 2015-11-23 | archive-url = https://web.archive.org/web/20151123031042/http://micro.magnet.fsu.edu/primer/techniques/darkfield.html | url-status = live }}</ref> Dark field illumination uses a carefully aligned light source to minimize the quantity of directly transmitted (unscattered) light entering the image plane, collecting only the light scattered by the sample. Dark field can dramatically improve image contrast – especially of transparent objects – while requiring little equipment setup or sample preparation. However, the technique suffers from low light intensity in the final image of many biological samples and continues to be affected by low apparent resolution.

[[File:Rheinberg 6.jpg|thumb|250px|A [[diatom]] under Rheinberg illumination]]
''Rheinberg illumination'' is a variant of dark field illumination in which transparent, colored filters are inserted just before the [[Condenser (microscope)|condenser]] so that light rays at high aperture are differently colored than those at low aperture (i.e., the background to the specimen may be blue while the object appears self-luminous red). Other color combinations are possible, but their effectiveness is quite variable.<ref>{{cite web | vauthors = Abramowitz M, Davidson MW | title = Rheinberg Illumination | date = 2003-08-01 | url = http://micro.magnet.fsu.edu/primer/techniques/rheinberg.html | access-date = 2008-10-21 | archive-date = 2008-09-29 | archive-url = https://web.archive.org/web/20080929144548/http://micro.magnet.fsu.edu/primer/techniques/rheinberg.html | url-status = live }}</ref>

==== Dispersion staining ====
{{Main|Dispersion staining}}
Dispersion staining is an optical technique that results in a colored image of a colorless object. This is an optical staining technique and requires no stains or dyes to produce a color effect. There are five different microscope configurations used in the broader technique of dispersion staining. They include brightfield Becke line, oblique, darkfield, phase contrast, and objective stop dispersion staining.

==== Phase contrast ====
[[File:Hypertrophic Zone of Epiphyseal Plate.jpg|thumb|250px| Phase-contrast [[light micrograph]] of undecalcified [[hyaline cartilage]] showing [[chondrocyte]]s and [[organelle]]s, [[Lacuna (histology)|lacunae]] and [[extracellular matrix]]]]
{{Main|Phase contrast microscopy}}
: ''In [[electron microscopy]]: [[Phase-contrast imaging]]''
More sophisticated techniques will show proportional differences in optical density. '''Phase contrast''' is a widely used technique that shows differences in [[refractive index]] as difference in contrast. It was developed by the Dutch physicist [[Frits Zernike]] in the 1930s (for which he was awarded the Nobel Prize in 1953). The nucleus in a cell for example will show up darkly against the surrounding cytoplasm. Contrast is excellent; however it is not for use with thick objects. Frequently, a halo is formed even around small objects, which obscures detail. The system consists of a circular annulus in the condenser, which produces a cone of light. This cone is superimposed on a similar sized ring within the phase-objective. Every objective has a different size ring, so for every objective another condenser setting has to be chosen. The ring in the objective has special optical properties: it, first of all, reduces the direct light in intensity, but more importantly, it creates an artificial phase difference of about a quarter wavelength. As the physical properties of this direct light have changed, interference with the diffracted light occurs, resulting in the phase contrast image. One disadvantage of phase-contrast microscopy is halo formation (halo-light ring).

==== Differential interference contrast ====
{{Main|Differential interference contrast microscopy}}
Superior and much more expensive is the use of '''interference contrast'''. Differences in optical density will show up as differences in relief. A nucleus within a cell will actually show up as a globule in the most often used '''differential interference contrast''' system according to [[Georges Nomarski]]. However, it has to be kept in mind that this is an ''optical effect'', and the relief does not necessarily resemble the true shape. Contrast is very good and the condenser aperture can be used fully open, thereby reducing the depth of field and maximizing resolution.

The system consists of a special prism ([[Nomarski prism]], [[Wollaston prism]]) in the condenser that splits light in an ordinary and an extraordinary beam. The spatial difference between the two beams is minimal (less than the maximum resolution of the objective). After passage through the specimen, the beams are reunited by a similar prism in the objective.

In a homogeneous specimen, there is no difference between the two beams, and no contrast is being generated. However, near a refractive boundary (say a nucleus within the cytoplasm), the difference between the ordinary and the extraordinary beam will generate a relief in the image. Differential interference contrast requires a [[polarized light]] source to function; two polarizing filters have to be fitted in the light path, one below the condenser (the polarizer), and the other above the objective (the analyzer).

Note: In cases where the optical design of a microscope produces an appreciable lateral separation of the two beams we have the case of [[classical interference microscopy]], which does not result in relief images, but can nevertheless be used for the quantitative determination of mass-thicknesses of microscopic objects.

==== Interference reflection ====
{{Main|Interference reflection microscopy}}
An additional technique using interference is '''interference reflection microscopy''' (also known as reflected interference contrast, or RIC). It relies on cell adhesion to the slide to produce an interference signal. If there is no cell attached to the glass, there will be no interference.

Interference reflection microscopy can be obtained by using the same elements used by DIC, but without the prisms. Also, the light that is being detected is reflected and not transmitted as it is when DIC is employed.

==== Fluorescence ====
[[File:Anther of thale cress (Arabidopsis thaliana), an artefact.jpg|thumb|Images may also contain [[Artifact (error)|artifacts]]. This is a [[Confocal laser scanning microscopy|confocal laser scanning]] [[Fluorescence microscopy|fluorescence]] [[micrograph]] of [[Arabidopsis thaliana|thale cress]] anther (part of [[stamen]]). The picture shows among other things a nice red flowing collar-like structure just below the anther. However, an intact thale cress stamen does not have such collar, this is a fixation artifact: the stamen has been cut below the picture frame, and [[Epidermis (botany)|epidermis]] (upper layer of cells) of stamen stalk has peeled off, forming a non-characteristic structure. Photo: Heiti Paves from [[Tallinn University of Technology]].]]
{{Main|Fluorescence microscopy}}

When certain compounds are illuminated with high energy light, they emit light of a lower frequency. This effect is known as [[fluorescence]]. Often specimens show their characteristic [[autofluorescence]] image, based on their chemical makeup.

This method is of critical importance in the modern life sciences, as it can be extremely sensitive, allowing the detection of single molecules. Many fluorescent [[dye]]s can be used to stain structures or chemical compounds. One powerful method is the combination of [[antibody|antibodies]] coupled to a fluorophore as in [[immunostaining]]. Examples of commonly used fluorophores are [[fluorescein]] or [[rhodamine]].

The antibodies can be tailor-made for a chemical compound. For example, one strategy often in use is the artificial production of proteins, based on the genetic code (DNA). These proteins can then be used to immunize rabbits, forming antibodies which bind to the protein. The antibodies are then coupled chemically to a fluorophore and used to trace the proteins in the cells under study.

Highly efficient fluorescent [[protein]]s such as the [[green fluorescent protein]] (GFP) have been developed using the [[molecular biology]] technique of [[fusion gene|gene fusion]], a process that links the [[gene expression|expression]] of the fluorescent compound to that of the target protein. This combined fluorescent protein is, in general, non-toxic to the organism and rarely interferes with the function of the protein under study. Genetically modified cells or organisms directly express the fluorescently tagged proteins, which enables the study of the function of the original protein [[in vivo]].

Growth of [[Protein crystallization|protein crystals]] results in both protein and salt crystals. Both are colorless and microscopic. Recovery of the protein crystals requires imaging which can be done by the intrinsic fluorescence of the protein or by using transmission microscopy. Both methods require an ultraviolet microscope as proteins absorbs light at 280&nbsp;nm. Protein will also fluorescence at approximately 353&nbsp;nm when excited with 280&nbsp;nm light.<ref name="Laboratory Communications">{{cite journal|last=Gill|first=Harindarpal|title=Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals|journal=Acta Crystallographica|date=January 2010|volume=F66|issue=Pt 3|pages=364–372 |pmid=20208182|pmc=2833058|doi=10.1107/S1744309110002022|bibcode=2010AcCrF..66..364G }}</ref>

Since [[fluorescence|fluorescence emission]] differs in [[wavelength]] (color) from the excitation light, an ideal fluorescent image shows only the structure of interest that was labeled with the fluorescent dye. This high specificity led to the widespread use of fluorescence light microscopy in biomedical research. Different fluorescent dyes can be used to stain different biological structures, which can then be detected simultaneously, while still being specific due to the individual color of the dye.

To block the excitation light from reaching the observer or the detector, [[filter (optics)|filter sets]] of high quality are needed. These typically consist of an [[excitation filter]] selecting the range of excitation [[wavelength]]s, a [[dichroism|dichroic]] mirror, and an [[Emission (electromagnetic radiation)|emission]] filter blocking the excitation light. Most fluorescence [[microscope]]s are operated in the Epi-illumination mode (illumination and detection from one side of the sample) to further decrease the amount of excitation light entering the detector.

See also:
[[total internal reflection fluorescence microscope]]
[[Neuroscience]]

==== Confocal ====
{{Main|Confocal microscopy}}
Confocal laser scanning microscopy uses a focused [[laser]] beam (e.g. 488&nbsp;nm) that is scanned across the sample to excite [[fluorescence]] in a point-by-point fashion. The emitted light is directed through a pinhole to prevent out-of-focus light from reaching the detector, typically a [[photomultiplier tube]]. The image is constructed in a computer, plotting the measured fluorescence intensities according to the position of the excitation laser. Compared to full sample illumination, confocal microscopy gives slightly higher lateral resolution and significantly improves [[optical sectioning]] (axial resolution). Confocal microscopy is, therefore, commonly used where 3D structure is important.

A subclass of confocal microscopes are '''spinning disc microscopes''' which are able to scan multiple points simultaneously across the sample. A corresponding disc with pinholes rejects out-of-focus light. The light detector in a spinning disc microscope is a digital camera, typically [[Charge-coupled device|EM-CCD]] or [[sCMOS]].

==== Two-photon microscopy ====
A two-photon microscope is also a laser-scanning microscope, but instead of UV, blue or green laser light, a [[Ultrashort pulse|pulsed infrared laser]] is used for excitation. Only in the tiny focus of the laser is the intensity high enough to generate fluorescence by [[Two-photon excitation microscopy|two-photon excitation]], which means that no out-of-focus fluorescence is generated, and no pinhole is necessary to clean up the image.<ref>{{Cite journal|last1=Denk|first1=Winfried|last2=Svoboda|first2=Karel|date=March 1997|title=Photon Upmanship: Why Multiphoton Imaging Is More than a Gimmick|journal=Neuron|language=en|volume=18|issue=3|pages=351–357|doi=10.1016/S0896-6273(00)81237-4|pmid=9115730|s2cid=2414593|doi-access=free}}</ref> This allows imaging deep in scattering tissue, where a confocal microscope would not be able to collect photons efficiently.<ref>{{Cite journal|last1=Denk|first1=W.|last2=Delaney|first2=K.R.|last3=Gelperin|first3=A.|last4=Kleinfeld|first4=D.|last5=Strowbridge|first5=B.W.|last6=Tank|first6=D.W.|last7=Yuste|first7=R.|date=1994|title=Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy|journal=Journal of Neuroscience Methods|language=en|volume=54|issue=2|pages=151–162|doi=10.1016/0165-0270(94)90189-9|pmid=7869748|s2cid=3772937}}</ref> Two-photon microscopes with wide-field detection are frequently used for functional imaging, e.g. [[calcium imaging]], in brain tissue.<ref>{{Cite journal|last1=Svoboda|first1=Karel|last2=Denk|first2=Winfried|last3=Kleinfeld|first3=David|last4=Tank|first4=David W.|date=1997|title=In vivo dendritic calcium dynamics in neocortical pyramidal neurons|url=http://www.nature.com/articles/385161a0|journal=Nature|language=en|volume=385|issue=6612|pages=161–165|doi=10.1038/385161a0|pmid=8990119|bibcode=1997Natur.385..161S|s2cid=4251386|issn=0028-0836|access-date=2020-05-20|archive-date=2021-05-25|archive-url=https://web.archive.org/web/20210525175417/https://www.nature.com/articles/385161a0|url-status=live|url-access=subscription}}</ref> They are marketed as '''Multiphoton microscopes''' by several companies, although the gains of using 3-photon instead of 2-photon excitation are marginal.

==== Single plane illumination microscopy and light sheet fluorescence microscopy ====
{{Main|Light sheet fluorescence microscopy}}
Using a plane of light formed by focusing light through a cylindrical lens at a narrow angle or by scanning a line of light in a plane perpendicular to the axis of objective, high resolution optical sections can be taken.<ref>{{Cite journal| doi = 10.1016/S0378-5955(02)00493-8| pmid = 12204356| issn = 0378-5955| volume = 71| pages = 119–128| last = Voie| first = A.H.| title = Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy| journal = Hearing Research| year = 1993| issue = 1–2| s2cid = 12775304}}</ref><ref>{{Cite journal| doi = 10.1063/1.2428277| issn = 0034-6748| volume = 78| last = Greger| first = K.| author2 = J. Swoger| author3 = E. H. K. Stelzer| title = Basic building units and properties of a fluorescence single plane illumination microscope| journal = Review of Scientific Instruments| year = 2007| issue = 2| pages = 023705–023705–7| pmid = 17578115| bibcode = 2007RScI...78b3705G| doi-access = free}}</ref><ref>{{Cite journal| doi = 10.1155/2012/206238| pmid = 22567307| pmc = 3335623| issn = 2090-2743| volume = 2012| page = 206238| last = Buytaert| first = J.A.N. |author2=E. Descamps |author3=D. Adriaens |author4=J.J.J. Dirckx| title = The OPFOS Microscopy Family: High-Resolution Optical Sectioning of Biomedical Specimens| journal = Anatomy Research International| year = 2012| arxiv = 1106.3162| doi-access = free}}</ref> Single plane illumination, or light sheet illumination, is also accomplished using [[Beam expander#Extra-cavity beam shaping|beam shaping techniques incorporating multiple-prism beam expanders]].<ref>F. J. Duarte, in ''High Power Dye Lasers'' (Springer-Verlag, Berlin,1991) Chapter 2.</ref><ref>[[F. J. Duarte|Duarte FJ]] (1993), Electro-optical interferometric microdensitometer system, [http://www.patentgenius.com/patent/5255069.html ''US Patent'' 5255069] {{Webarchive|url=https://web.archive.org/web/20171013042159/http://www.patentgenius.com/patent/5255069.html |date=2017-10-13 }}.</ref> The images are captured by CCDs. These variants allow very fast and high signal to noise ratio image capture.

==== Wide-field multiphoton microscopy ====
{{Main|Wide-field multiphoton microscopy}}Wide-field multiphoton microscopy<ref name=":1">{{Cite journal|last1=Peterson|first1=Mark D.|last2=Hayes|first2=Patrick L.|last3=Martinez|first3=Imee Su|last4=Cass|first4=Laura C.|last5=Achtyl|first5=Jennifer L.|last6=Weiss|first6=Emily A.|last7=Geiger|first7=Franz M.|date=2011-05-01|title=Second harmonic generation imaging with a kHz amplifier [Invited]|journal=Optical Materials Express|language=EN|volume=1|issue=1|doi=10.1364/ome.1.000057|page=57|bibcode=2011OMExp...1...57P}}</ref><ref name=":0">{{Cite journal|last1=Macias-Romero|first1=Carlos|last2=Didier|first2=Marie E. P.|last3=Jourdain|first3=Pascal|last4=Marquet|first4=Pierre|last5=Magistretti|first5=Pierre|last6=Tarun|first6=Orly B.|last7=Zubkovs|first7=Vitalijs|author8-link=Aleksandra Radenovic|last8=Radenovic|first8=Aleksandra|last9=Roke|first9=Sylvie|date=2014-12-15|title=High throughput second harmonic imaging for label-free biological applications|journal=Optics Express|language=EN|volume=22|issue=25|pages=31102–12|doi=10.1364/oe.22.031102|pmid=25607059|bibcode=2014OExpr..2231102M|url=http://infoscience.epfl.ch/record/203737|doi-access=free|access-date=2019-12-11|archive-date=2020-06-20|archive-url=https://web.archive.org/web/20200620070847/https://infoscience.epfl.ch/record/203737|url-status=live|hdl=10754/563269|hdl-access=free}}</ref><ref name=":2">{{Cite journal|last1=Cheng|first1=Li-Chung|last2=Chang|first2=Chia-Yuan|last3=Lin|first3=Chun-Yu|last4=Cho|first4=Keng-Chi|last5=Yen|first5=Wei-Chung|last6=Chang|first6=Nan-Shan|last7=Xu|first7=Chris|last8=Dong|first8=Chen Yuan|last9=Chen|first9=Shean-Jen|date=2012-04-09|title=Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning|journal=Optics Express|language=EN|volume=20|issue=8|pages=8939–48|doi=10.1364/oe.20.008939|pmid=22513605|bibcode=2012OExpr..20.8939C|doi-access=free}}</ref><ref name=":3">{{Cite journal|last1=Oron|first1=Dan|last2=Tal|first2=Eran|last3=Silberberg|first3=Yaron|date=2005-03-07|title=Scanningless depth-resolved microscopy|journal=Optics Express|language=EN|volume=13|issue=5|pages=1468–76|doi=10.1364/opex.13.001468|pmid=19495022|bibcode=2005OExpr..13.1468O|doi-access=free}}</ref> refers to an [[Nonlinear optics|optical non-linear imaging technique]] in which a large area of the object is illuminated and imaged without the need for scanning. High intensities are required to induce non-linear optical processes such as [[Two-photon excitation microscopy|two-photon fluorescence]] or [[Second-harmonic generation|second harmonic generation]]. In [[Multiphoton fluorescence microscope|scanning multiphoton microscopes]] the high intensities are achieved by tightly focusing the light, and the image is obtained by beam scanning. In '''wide-field multiphoton microscopy''' the high intensities are best achieved using an [[Optical amplifier|optically amplified]] pulsed laser source to attain a large field of view (~100&nbsp;μm).<ref name=":1" /><ref name=":0" /><ref name=":2" /> The image in this case is obtained as a single frame with a CCD camera without the need of scanning, making the technique particularly useful to visualize dynamic processes simultaneously across the object of interest. With wide-field multiphoton microscopy the frame rate can be increased up to a 1000-fold compared to [[Multiphoton fluorescence microscopy|multiphoton scanning microscopy]].<ref name=":0" /> In scattering tissue, however, image quality rapidly degrades with increasing depth.

==== Deconvolution ====
Fluorescence microscopy is a powerful technique to show specifically labeled structures within a complex environment and to provide three-dimensional information of biological structures. However, this information is blurred by the fact that, upon illumination, all fluorescently labeled structures emit light, irrespective of whether they are in focus or not. So an image of a certain structure is always blurred by the contribution of light from structures that are out of focus. This phenomenon results in a loss of contrast especially when using objectives with a high resolving power, typically oil immersion objectives with a high numerical aperture.
[[File:THZPSF.jpg|thumb|247x247px|Mathematically modeled Point Spread Function of a pulsed THz laser imaging system.<ref>{{Cite book |last1=Ahi |first1=Kiarash |first2=Mehdi |last2=Anwar |title=Terahertz Physics, Devices, and Systems X: Advanced Applications in Industry and Defense |editor3-first=Tariq |editor3-last=Manzur |editor2-first=Thomas W |editor2-last=Crowe |editor1-first=Mehdi F |editor1-last=Anwar |date=2016-05-26 |chapter=Modeling of terahertz images based on x-ray images: a novel approach for verification of terahertz images and identification of objects with fine details beyond terahertz resolution |chapter-url=https://www.researchgate.net/publication/303563365  |volume=9856 |pages=985610 |doi=10.1117/12.2228685|series=Proceedings of SPIE |bibcode=2016SPIE.9856E..10A |s2cid=124315172 }}</ref>]]
However, blurring is not caused by random processes, such as light scattering, but can be well defined by the optical properties of the image formation in the microscope imaging system. If one considers a small fluorescent light source (essentially a bright spot), light coming from this spot spreads out further from our perspective as the spot becomes more out of focus. Under ideal conditions, this produces an "hourglass" shape of this [[point source]] in the third (axial) dimension. This shape is called the [[point spread function]] (PSF) of the microscope imaging system. Since any fluorescence image is made up of a large number of such small fluorescent light sources, the image is said to be "convolved by the point spread function". The mathematically modeled PSF of a terahertz laser pulsed imaging system is shown on the right.

The output of an imaging system can be described using the equation:

<math>s(x,y) = PSF(x,y) * o(x,y) + n</math>

Where {{math|<var>n</var>}} is the additive noise.<ref>{{Cite book|title=Fundamentals of Digital Image Processing|last=Solomon|first=Chris|publisher=John Wiley & Sons, Ltd|year=2010|isbn=978-0-470-84473-1}}</ref> Knowing this point spread function<ref>{{cite journal |author1=Nasse M. J. |author2=Woehl J. C. |title=Realistic modeling of the illumination point spread function in confocal scanning optical microscopy |journal=J. Opt. Soc. Am. A |volume=27 |issue=2 |pages=295–302 |year=2010 |doi=10.1364/JOSAA.27.000295 |pmid=20126241|bibcode=2010JOSAA..27..295N }}</ref> means that it is possible to reverse this process to a certain extent by computer-based methods commonly known as [[deconvolution]] microscopy.<ref>{{cite journal |vauthors=Wallace W, Schaefer LH, Swedlow JR |title=A workingperson's guide to deconvolution in light microscopy |journal=BioTechniques |volume=31 |issue=5 |pages=1076–8, 1080, 1082 passim |year=2001 |pmid=11730015 |doi=10.2144/01315bi01|doi-access=free }}</ref> There are various algorithms available for 2D or 3D deconvolution. They can be roughly classified in ''nonrestorative'' and ''restorative'' methods. While the nonrestorative methods can improve contrast by removing out-of-focus light from focal planes, only the restorative methods can actually reassign light to its proper place of origin. Processing fluorescent images in this manner can be an advantage over directly acquiring images without out-of-focus light, such as images from [[confocal microscopy]], because light signals otherwise eliminated become useful information. For 3D deconvolution, one typically provides a series of images taken from different focal planes (called a Z-stack) plus the knowledge of the PSF, which can be derived either experimentally or theoretically from knowing all contributing parameters of the microscope.