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A '''4Pi microscope''' is a laser scanning [[fluorescence microscope]] with an improved [[optical axis|axial]] [[Optical resolution|resolution]]. The typical value of 500–700 nm can be improved to 100–150 nm which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard [[confocal microscopy]]. | |||
==Working principle== | |||
[[Image:Scheme-4PiMicroscope.jpg|thumb|Scheme of a 4Pi microscope.]] | |||
The improvement in resolution is achieved by using two opposing objective lenses which both are focused to the same geometrical location. Also the difference in [[optical path length]] through each of the two objective lenses is carefully aligned to be minimal. By this, molecules residing in the common focal area of both objectives can be illuminated coherently from both sides and also the reflected or emitted light can be collected coherently, i.e. coherent superposition of emitted light on the detector is possible. The [[solid angle]] <math>\Omega</math> that is used for illumination and detection is increased and approaches the ideal case. In this case the sample is illuminated and detected from all sides simultaneously. | |||
The operation mode of a 4Pi microscope is shown in the figure. The laser light is divided by a beam splitter (BS) and directed by mirrors towards the two opposing objective lenses. At the common focal point superposition of both focused light beams occurs. Excited molecules at this position emit fluorescence light which is collected by both objective lenses, combined by the same beam splitter and deflected by a dichroic mirror (DM) onto a detector. There superposition of both emitted light pathways can take place again. | |||
In the ideal case each objective lens can collect light from a solid angle of <math>\Omega=2\pi</math>. So, with two objective lenses one can collect from every direction (solid angle <math>\Omega=4\pi</math>). The name of this type of microscopy is derived from the maximal possible solid angle for excitation and detection. Practically one can achieve only aperture angles of about 140° for an objective lens, which corresponds to <math>\Omega</math> about <math>1.3\pi</math>. | |||
It can be operated in three different ways: In a 4Pi microscope of type A, the coherent superposition of excitation light is used to generate the increased resolution. The emission light is either detected from one side only or in an incoherent superposition from both sides. In a 4Pi microscope of type B, only the emission light is interfering. When operated in the type C mode, both excitation and emission light are allowed to interfere leading to the highest possible resolution increase (~7 fold along the optic axis as compared to confocal microscopy). | |||
In a real 4Pi microscope light is not applied from all sides, leading to so-called side lobes in the [[point spread function]]. Typically (but not always) [[two-photon excitation microscopy]] is used in a 4Pi microscope in combination with an emission pinhole to lower these side lobes to a tolerable level. | |||
==History== | |||
In 1971, [[Christoph Cremer]] and [[Thomas Cremer]] proposed the creation of a perfect [[hologram]], i.e. one that carries the whole field information of the emission of a point source in all directions, a so-called <math>4\pi</math> hologram.<ref>Cremer C, Cremer T (1971) <math>4\pi</math> Punkthologramme: Physikalische Grundlagen und mögliche Anwendungen. Enclosure to Patent application DE 2116521 „Verfahren zur Darstellung bzw. Modifikation von Objekt-Details, deren Abmessungen außerhalb der sichtbaren Wellenlängen liegen" (Procedure for the imaging and modification of object details with dimensions beyond the visible wavelengths). Filed April 5, 1971; publication date October 12, 1972.Deutsches Patentamt, Berlin. http://depatisnet.dpma.de/DepatisNet/depatisnet?action=pdf&docid=DE000002116521A</ref><ref>Considerations on a laser-scanning-microscope with high resolution and depth of field: C. Cremer and T. Cremer in M1CROSCOPICA ACTA VOL. 81 NUMBER 1 September, pp. 31—44 (1978)<br /> [http://www.kip.uni-heidelberg.de/AG_Cremer/pdf-files/Cremer_Micros_Acta_1978.pdf Basic design of a confocal laser scanning fluorescence microscope & principle of a confocal laser scanning 4Pi fluorescence microscope, 1978]</ref> | |||
The first description of a practicable system of 4Pi microscopy, i.e. the setup with two opposing, interfering lenses, was invented by [[Stefan Hell]] in 1991.<ref>European patent [http://v3.espacenet.com/publicationDetails/biblio?CC=EP&NR=0491289 EP 0491289]</ref> He could demonstrate it experimentally also in 1994.<ref>{{cite journal |author=S.W. Hell, E.H.K. Stelzer, S. Lindek, C. Cremer |title=Confocal microscopy with an increased detection aperture: type-B 4Pi confocal microscopy |journal=Optics Letters |volume=19 |issue=3 |year=1994 |pages=222–224 |url=http://www.opticsinfobase.org/viewmedia.cfm?uri=ol-19-3-222&seq=0 |doi=10.1364/OL.19.000222 | pmid = 19829598 |bibcode = 1994OptL...19..222H }}</ref> | |||
In the following years the number of applications for this microscope have grown. Parallel excitation and detection with 64 spots in the sample simultaneously combined with the improved spatial resolution resulted in the successful recording of the dynamics of [[mitochondria]] in yeast cells with a 4Pi microscope in 2002.<ref>{{cite journal |author=A. Egner, S. Jakobs, S.W. Hell | |||
|title=Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast |journal=PNAS | |||
|volume= 99 |issue=6 |year= 2002 |pages= 3370–3375 |pmid=11904401 |url=http://www.pnas.org/content/99/6/3370.full.pdf |pmc=122530 |doi=10.1073/pnas.052545099|bibcode = 2002PNAS...99.3370E }}</ref> | |||
A commercial version was launched by microscope manufacturer [[Leica Microsystems]] in 2004.<ref>Review article [http://www.kompetenznetze.de/netzwerke/opti-photonicnet/innovationshighlights/en/4Pi_Mikroskop 4Pi microscopy]</ref> | |||
Up to now, the best quality in a 4Pi microscope was reached in conjunction with the [[Stimulated Emission Depletion Microscope|STED]] principle.<ref>{{cite journal |author= R. Schmidt, C.A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, S.W. Hell |title=Spherical nanosized focal spot unravels the interior of cells |journal=Nature Methods |volume=5 |issue= 6 |year= 2008 |pages= 539–544 |pmid= 18488034 |url=http://www.nature.com/nmeth/journal/v5/n6/pdf/nmeth.1214.pdf |doi= 10.1038/nmeth.1214}}</ref> Using a 4Pi microscope with appropriate excitation and de-excitation beams it was possible to create a uniformly 50 nm sized spot which corresponds to a decreased focal volume compared to confocal microscopy by a factor of 150–200. | |||
==See also== | |||
* [[Stimulated emission depletion microscope]] (STED) | |||
* [[Multifocal plane microscopy]] (MUM) | |||
==References== | |||
{{reflist}} | |||
{{Optical microscopy}} | |||
{{DEFAULTSORT:4pi Microscope}} | |||
[[Category:Microscopes]] | |||
[[Category:Fluorescence]] | |||
Revision as of 23:55, 25 August 2013
A 4Pi microscope is a laser scanning fluorescence microscope with an improved axial resolution. The typical value of 500–700 nm can be improved to 100–150 nm which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard confocal microscopy.
Working principle
The improvement in resolution is achieved by using two opposing objective lenses which both are focused to the same geometrical location. Also the difference in optical path length through each of the two objective lenses is carefully aligned to be minimal. By this, molecules residing in the common focal area of both objectives can be illuminated coherently from both sides and also the reflected or emitted light can be collected coherently, i.e. coherent superposition of emitted light on the detector is possible. The solid angle that is used for illumination and detection is increased and approaches the ideal case. In this case the sample is illuminated and detected from all sides simultaneously.
The operation mode of a 4Pi microscope is shown in the figure. The laser light is divided by a beam splitter (BS) and directed by mirrors towards the two opposing objective lenses. At the common focal point superposition of both focused light beams occurs. Excited molecules at this position emit fluorescence light which is collected by both objective lenses, combined by the same beam splitter and deflected by a dichroic mirror (DM) onto a detector. There superposition of both emitted light pathways can take place again.
In the ideal case each objective lens can collect light from a solid angle of . So, with two objective lenses one can collect from every direction (solid angle ). The name of this type of microscopy is derived from the maximal possible solid angle for excitation and detection. Practically one can achieve only aperture angles of about 140° for an objective lens, which corresponds to about .
It can be operated in three different ways: In a 4Pi microscope of type A, the coherent superposition of excitation light is used to generate the increased resolution. The emission light is either detected from one side only or in an incoherent superposition from both sides. In a 4Pi microscope of type B, only the emission light is interfering. When operated in the type C mode, both excitation and emission light are allowed to interfere leading to the highest possible resolution increase (~7 fold along the optic axis as compared to confocal microscopy).
In a real 4Pi microscope light is not applied from all sides, leading to so-called side lobes in the point spread function. Typically (but not always) two-photon excitation microscopy is used in a 4Pi microscope in combination with an emission pinhole to lower these side lobes to a tolerable level.
History
In 1971, Christoph Cremer and Thomas Cremer proposed the creation of a perfect hologram, i.e. one that carries the whole field information of the emission of a point source in all directions, a so-called hologram.[1][2]
The first description of a practicable system of 4Pi microscopy, i.e. the setup with two opposing, interfering lenses, was invented by Stefan Hell in 1991.[3] He could demonstrate it experimentally also in 1994.[4]
In the following years the number of applications for this microscope have grown. Parallel excitation and detection with 64 spots in the sample simultaneously combined with the improved spatial resolution resulted in the successful recording of the dynamics of mitochondria in yeast cells with a 4Pi microscope in 2002.[5]
A commercial version was launched by microscope manufacturer Leica Microsystems in 2004.[6]
Up to now, the best quality in a 4Pi microscope was reached in conjunction with the STED principle.[7] Using a 4Pi microscope with appropriate excitation and de-excitation beams it was possible to create a uniformly 50 nm sized spot which corresponds to a decreased focal volume compared to confocal microscopy by a factor of 150–200.
See also
References
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- ↑ Cremer C, Cremer T (1971) Punkthologramme: Physikalische Grundlagen und mögliche Anwendungen. Enclosure to Patent application DE 2116521 „Verfahren zur Darstellung bzw. Modifikation von Objekt-Details, deren Abmessungen außerhalb der sichtbaren Wellenlängen liegen" (Procedure for the imaging and modification of object details with dimensions beyond the visible wavelengths). Filed April 5, 1971; publication date October 12, 1972.Deutsches Patentamt, Berlin. http://depatisnet.dpma.de/DepatisNet/depatisnet?action=pdf&docid=DE000002116521A
- ↑ Considerations on a laser-scanning-microscope with high resolution and depth of field: C. Cremer and T. Cremer in M1CROSCOPICA ACTA VOL. 81 NUMBER 1 September, pp. 31—44 (1978)
Basic design of a confocal laser scanning fluorescence microscope & principle of a confocal laser scanning 4Pi fluorescence microscope, 1978 - ↑ European patent EP 0491289
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A vendor's stamp duty has been launched on industrial property for the primary time, at rates ranging from 5 per cent to 15 per cent. The Authorities might be trying to reassure the market that they aren't in opposition to foreigners and PRs investing in Singapore's property market. They imposed these measures because of extenuating components available in the market." The sale of new dual-key EC models will even be restricted to multi-generational households only. The models have two separate entrances, permitting grandparents, for example, to dwell separately. The vendor's stamp obligation takes effect right this moment and applies to industrial property and plots which might be offered inside three years of the date of buy. JLL named Best Performing Property Brand for second year running
The data offered is for normal info purposes only and isn't supposed to be personalised investment or monetary advice. Motley Fool Singapore contributor Stanley Lim would not personal shares in any corporations talked about. Singapore private home costs increased by 1.eight% within the fourth quarter of 2012, up from 0.6% within the earlier quarter. Resale prices of government-built HDB residences which are usually bought by Singaporeans, elevated by 2.5%, quarter on quarter, the quickest acquire in five quarters. And industrial property, prices are actually double the levels of three years ago. No withholding tax in the event you sell your property. All your local information regarding vital HDB policies, condominium launches, land growth, commercial property and more
There are various methods to go about discovering the precise property. Some local newspapers (together with the Straits Instances ) have categorised property sections and many local property brokers have websites. Now there are some specifics to consider when buying a 'new launch' rental. Intended use of the unit Every sale begins with 10 p.c low cost for finish of season sale; changes to 20 % discount storewide; follows by additional reduction of fiftyand ends with last discount of 70 % or extra. Typically there is even a warehouse sale or transferring out sale with huge mark-down of costs for stock clearance. Deborah Regulation from Expat Realtor shares her property market update, plus prime rental residences and houses at the moment available to lease Esparina EC @ Sengkang - ↑ One of the biggest reasons investing in a Singapore new launch is an effective things is as a result of it is doable to be lent massive quantities of money at very low interest rates that you should utilize to purchase it. Then, if property values continue to go up, then you'll get a really high return on funding (ROI). Simply make sure you purchase one of the higher properties, reminiscent of the ones at Fernvale the Riverbank or any Singapore landed property Get Earnings by means of Renting
In its statement, the singapore property listing - website link, government claimed that the majority citizens buying their first residence won't be hurt by the new measures. Some concessions can even be prolonged to chose teams of consumers, similar to married couples with a minimum of one Singaporean partner who are purchasing their second property so long as they intend to promote their first residential property. Lower the LTV limit on housing loans granted by monetary establishments regulated by MAS from 70% to 60% for property purchasers who are individuals with a number of outstanding housing loans on the time of the brand new housing purchase. Singapore Property Measures - 30 August 2010 The most popular seek for the number of bedrooms in Singapore is 4, followed by 2 and three. Lush Acres EC @ Sengkang
Discover out more about real estate funding in the area, together with info on international funding incentives and property possession. Many Singaporeans have been investing in property across the causeway in recent years, attracted by comparatively low prices. However, those who need to exit their investments quickly are likely to face significant challenges when trying to sell their property – and could finally be stuck with a property they can't sell. Career improvement programmes, in-house valuation, auctions and administrative help, venture advertising and marketing, skilled talks and traisning are continuously planned for the sales associates to help them obtain better outcomes for his or her shoppers while at Knight Frank Singapore. No change Present Rules
Extending the tax exemption would help. The exemption, which may be as a lot as $2 million per family, covers individuals who negotiate a principal reduction on their existing mortgage, sell their house short (i.e., for lower than the excellent loans), or take part in a foreclosure course of. An extension of theexemption would seem like a common-sense means to assist stabilize the housing market, but the political turmoil around the fiscal-cliff negotiations means widespread sense could not win out. Home Minority Chief Nancy Pelosi (D-Calif.) believes that the mortgage relief provision will be on the table during the grand-cut price talks, in response to communications director Nadeam Elshami. Buying or promoting of blue mild bulbs is unlawful.
A vendor's stamp duty has been launched on industrial property for the primary time, at rates ranging from 5 per cent to 15 per cent. The Authorities might be trying to reassure the market that they aren't in opposition to foreigners and PRs investing in Singapore's property market. They imposed these measures because of extenuating components available in the market." The sale of new dual-key EC models will even be restricted to multi-generational households only. The models have two separate entrances, permitting grandparents, for example, to dwell separately. The vendor's stamp obligation takes effect right this moment and applies to industrial property and plots which might be offered inside three years of the date of buy. JLL named Best Performing Property Brand for second year running
The data offered is for normal info purposes only and isn't supposed to be personalised investment or monetary advice. Motley Fool Singapore contributor Stanley Lim would not personal shares in any corporations talked about. Singapore private home costs increased by 1.eight% within the fourth quarter of 2012, up from 0.6% within the earlier quarter. Resale prices of government-built HDB residences which are usually bought by Singaporeans, elevated by 2.5%, quarter on quarter, the quickest acquire in five quarters. And industrial property, prices are actually double the levels of three years ago. No withholding tax in the event you sell your property. All your local information regarding vital HDB policies, condominium launches, land growth, commercial property and more
There are various methods to go about discovering the precise property. Some local newspapers (together with the Straits Instances ) have categorised property sections and many local property brokers have websites. Now there are some specifics to consider when buying a 'new launch' rental. Intended use of the unit Every sale begins with 10 p.c low cost for finish of season sale; changes to 20 % discount storewide; follows by additional reduction of fiftyand ends with last discount of 70 % or extra. Typically there is even a warehouse sale or transferring out sale with huge mark-down of costs for stock clearance. Deborah Regulation from Expat Realtor shares her property market update, plus prime rental residences and houses at the moment available to lease Esparina EC @ Sengkang - ↑ Review article 4Pi microscopy
- ↑ One of the biggest reasons investing in a Singapore new launch is an effective things is as a result of it is doable to be lent massive quantities of money at very low interest rates that you should utilize to purchase it. Then, if property values continue to go up, then you'll get a really high return on funding (ROI). Simply make sure you purchase one of the higher properties, reminiscent of the ones at Fernvale the Riverbank or any Singapore landed property Get Earnings by means of Renting
In its statement, the singapore property listing - website link, government claimed that the majority citizens buying their first residence won't be hurt by the new measures. Some concessions can even be prolonged to chose teams of consumers, similar to married couples with a minimum of one Singaporean partner who are purchasing their second property so long as they intend to promote their first residential property. Lower the LTV limit on housing loans granted by monetary establishments regulated by MAS from 70% to 60% for property purchasers who are individuals with a number of outstanding housing loans on the time of the brand new housing purchase. Singapore Property Measures - 30 August 2010 The most popular seek for the number of bedrooms in Singapore is 4, followed by 2 and three. Lush Acres EC @ Sengkang
Discover out more about real estate funding in the area, together with info on international funding incentives and property possession. Many Singaporeans have been investing in property across the causeway in recent years, attracted by comparatively low prices. However, those who need to exit their investments quickly are likely to face significant challenges when trying to sell their property – and could finally be stuck with a property they can't sell. Career improvement programmes, in-house valuation, auctions and administrative help, venture advertising and marketing, skilled talks and traisning are continuously planned for the sales associates to help them obtain better outcomes for his or her shoppers while at Knight Frank Singapore. No change Present Rules
Extending the tax exemption would help. The exemption, which may be as a lot as $2 million per family, covers individuals who negotiate a principal reduction on their existing mortgage, sell their house short (i.e., for lower than the excellent loans), or take part in a foreclosure course of. An extension of theexemption would seem like a common-sense means to assist stabilize the housing market, but the political turmoil around the fiscal-cliff negotiations means widespread sense could not win out. Home Minority Chief Nancy Pelosi (D-Calif.) believes that the mortgage relief provision will be on the table during the grand-cut price talks, in response to communications director Nadeam Elshami. Buying or promoting of blue mild bulbs is unlawful.
A vendor's stamp duty has been launched on industrial property for the primary time, at rates ranging from 5 per cent to 15 per cent. The Authorities might be trying to reassure the market that they aren't in opposition to foreigners and PRs investing in Singapore's property market. They imposed these measures because of extenuating components available in the market." The sale of new dual-key EC models will even be restricted to multi-generational households only. The models have two separate entrances, permitting grandparents, for example, to dwell separately. The vendor's stamp obligation takes effect right this moment and applies to industrial property and plots which might be offered inside three years of the date of buy. JLL named Best Performing Property Brand for second year running
The data offered is for normal info purposes only and isn't supposed to be personalised investment or monetary advice. Motley Fool Singapore contributor Stanley Lim would not personal shares in any corporations talked about. Singapore private home costs increased by 1.eight% within the fourth quarter of 2012, up from 0.6% within the earlier quarter. Resale prices of government-built HDB residences which are usually bought by Singaporeans, elevated by 2.5%, quarter on quarter, the quickest acquire in five quarters. And industrial property, prices are actually double the levels of three years ago. No withholding tax in the event you sell your property. All your local information regarding vital HDB policies, condominium launches, land growth, commercial property and more
There are various methods to go about discovering the precise property. Some local newspapers (together with the Straits Instances ) have categorised property sections and many local property brokers have websites. Now there are some specifics to consider when buying a 'new launch' rental. Intended use of the unit Every sale begins with 10 p.c low cost for finish of season sale; changes to 20 % discount storewide; follows by additional reduction of fiftyand ends with last discount of 70 % or extra. Typically there is even a warehouse sale or transferring out sale with huge mark-down of costs for stock clearance. Deborah Regulation from Expat Realtor shares her property market update, plus prime rental residences and houses at the moment available to lease Esparina EC @ Sengkang