Moderne Methoden Optischer Mikroskopie und … · Themen 0. Grundlagen optischer Spektroskopie 1....

Moderne Methoden Optischer Mikroskopie und Spektroskopie

Themen

0. Grundlagen optischer Spektroskopie

1. Laser

2. Konventionelle Mikroskopie

3. Konfokale Mikroskopie, Zweiphotonen-, 4π-, STED- Mikroskopie

4. Nahfeldmikroskopie

5. Tieftemperatur SMS, Lochbrennen und Photonecho

6. Raumtemperatur SMD

7. Fluoreszenzkorrelationsspektroskopie

8. SERS/CARS Spektroskopie/Mikroskopie

Literatur

• Molekülphysik und Quantenchemie, Haken, Wolf, Springer, 1998

• Experimentalphysik III, Demtröder, Springer, 1996• Optik, Hecht, Oldenbourg 2001• Laser, Kneubühl, Sigrist, Teubner 1995• Confocal laser scanning microscopy, C. J. R. Sheppard;

D. M. Shotton, Oxford : Bios Scientific Publ., 1997• Near-field microscopy and near-field optics, Daniel

Courjon, London : Imperial College Press, 2003• Principles of Fluorescence Spectroscopy, Lakowicz,

Kluwer, 1999• Single Molecule Spectroscopy, Rigler, Orrit, Basché (eds), Springer 2001

Publikationen

101

102

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104

# Pu

blic

atio

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2000199019801970Year

confocal SNOM SMS fluorescence

Das elektromagnetische Spektrum

Amplitude + PhaseAmplitude

Licht-Materie-Wechselwirkung

Wechselwirkung Licht-Materie → Übergänge zwischen definierten Energieniveaus

Strahlungstyp Wellenlängenbereich Übergänge

Gammastrahlung <1 pm Kernübergänge

Röntgen 1 nm-1 pm Rumpfelektronen

UV 400 nm-1 nm Valenzelektronen

Vis 750 nm-400 nm Valenzelektronen

NIR 2.5 µm-750 nm Valenzelektronen/Molekülschwingungen

IR 25 µm-2.5 µm Molekülschwingungen

Mikrowellen 1 mm-25 µm Molekülrotationen, Elektronenspinübergänge

Radiowellen >1 mm Kernspinübergänge

kBT (300K)=25meV ≙ 200 cm-1

Licht-Materie-Wechselwirkung

Zweiatomiges Molekül

1eV

Elektronische Niveaus

Schwingungsniveaus

Kernverschiebung

0

0.2

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1.4

200 250 300 350 400 450 500

700650600550500450400350

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20x10-3

15

10

5

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ission (b.E.)

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orpt

ion

N

O

O

N

O

O

Fluorescein

YOYO-1

Ethidiumbromid

Beispiele für Fluorophore

Fluoreszenz - Anregungs- und Emissionsspektrum

Wellenlänge in nm

340 360 380 400 420 440 460 480 500 520 540 560 580 600

rel. Fluoreszenz

0.0

2.0e+6

4.0e+6

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1.0e+7

1.2e+7

1.4e+7

1.6e+7

1.8e+7AnregungsspektrumEmissionsspektrum

www.atto-tec.de

Alexa®488, www.probes.com

Texas Red® 1,2-dihexadecanoyl-sn-glycero-3- phosphoethanolamine, triethylammonium salt (Texas Red® DHPE)

www.probes.com

10000

8000

6000

4000

2000

0

140120100806040200x103

∑sin (x+n*∂x)

Biospektroskopie 5

GrößenordnungenGrößenordnungen biologischer Objektebiologischer Objekte

1.0

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3000200010000-1000-2000-3000

Bild der Lichtquelle Bild des Spiegels

f1 f1 f2 f2 f3 f3

www.olympus.comTransmitted and reflected light

UIS Objectives for Transmitted Light and Reflected Light

Type ArticleCode

Objective Name N.A. W.D.(mm)

F.N. C.C.(mm)

imm. Remarks Trans.DIC

Reflect.DIC

Phasering

Note

PlanApochromat

3758137582375833758437585376513765237656376533765437657376553766537666

PLAPO 1,25xPLAPO 2xPLAPO 40xPLAPO 60x OPLAPO 100x OMPLAPO 1,25xMPLAPO 2,5xMPLAPO 20xMPLAPO 50xMPLAPO 60xMPLAPO 100xMPLAPO 100x OLMPLAPO 150xLMPLAPO 250x

0.040.080.951.401.400.040.080.600.950.900.951.400.900.90

5.16.20.130.100.103.610.70.900.300.400.350.101.00.80

26.526.526.526.526.52226.526.526.526.526.526.526.526.5

--

0.11-0.230.170.17

--0000000

oiloil oil

Sp, CcSpSpDepolaryzerDepolaryzerSpSpSpSpSpSpSp

A, B

A BB

UniversalPlanApochromat

37561375623759637563375643756537566375683759937567

UPLAPO 4xUPLAPO 10xUPLAPO 10x WUPLAPO 20xUPLAPO 20x OUPLAPO 40xUPLAPO 40x OIUPLAPO 60xUPLAPO 60x WUPLAPO 100x OI

0.160.400.400.700.800.85

0.50-1.000.901.20

0.50-1.35

13.03.10.500.650.190.200.120.200.250.10

26.526.526.526.526.526.526.526.526.526.5

-0.170.170.17

-0.11-0.23

-0.11-0.230.15-0.20

0.17

water oil oil wateroil

SpSpSpSp, CcSp, IrisSp, CcSp, CcSp, Iris

A, CA, CA, BA, BA, CA, B

B, C, DA, B, C

UniversalApochromat

3757737597375783759837579

UAPO 20x /340UAPO 20x W/340UAPO 40x /340UAPO 40x W/340UAPO 40x OI/340

0.750.700.901.15

0.65-1.35

0.550.400.200.260.10

2222222222

0.170.17

0.11-0.230.13-0.25

0.17

water

water

oil

Sp, CcSp, CcSp, Iris

A, CA, BA, B

Good transmission at 340nm, (for Fura-2)Good transmission at 340nm, (for Fura-2)Good transmission at 340nm, (for Fura-2)Good transmission at 340nm, (for Fura-2)Good transmission at 340nm, (for Fura-2)

Universal PlanFluorite

375413754237543375443754537547375483744137442374433744437446376213762237623376243762537626375873758837589375913759037592

UPLFL 4xUPLFL 10xUPLFL 20xUPLFL 40xUPLFL 60x OIUPLFL 100x OUPLFL 100x OIUPLFL 4x PUPLFL 10x PUPLFL 20x PUPLFL 40x PUPLFL 100x OPUMPLFL 5xUMPLFL 10xUMPLFL 20xUMPLFL 40xUMPLFL 50xUMPLFL 100xUMPLFL 10x WUMPLFL 20x WLUMPLFL 40x WLUMPLFL 40x W/IRLUMPLFL 60x WLUMPLFL 60x W/IR

0.130.300.500.75

0.65-1.251.30

0.60-1.300.130.300.500.751.300.150.300.460.750.800.950.300.500.800.800.900.90

17.010.01.60.510.100.100.1013.03.11.60.510.1020.010.13.10.630.660.313.33.33.33.32.02.0

26.526.526.526.526.526.526.526.526.526.526.526.526.526.526.526.526.526.526.526.526.526.526.526.5

--

0.170.170.170.170.17

--

0.170.170.17

--0000--0000

oiloiloil

oil

waterwaterwaterwaterwaterwater

SpSpSp, IrisSpSp, Iris SpSpSp SpSpSp

A, CA, BA, B

B, C, DA, B, CA, B, C

A, CA, BA, B

A, B, C

A, B DDDD

B, DB, D

AAAAAA

Strain-freeStrain-freeStrain-freeStrain-freeStrain-free For IR-DIC (775nm) For IR-DIC (775nm)

PlanFluorite

37529374613746237463374653765937660376613766337664

PLFL 100xLCPLFL 20xLCPLFL 40xLCPLFL 60xSLCPLFL 40xLMPLFL 5xLMPLFL 10xLMPLFL 20xLMPLFL 50xLMPLFL 100x

0.950.400.600.700.550.130.250.400.500.80

0.206.9 *2.6 *1.7*7.7*22.521.012.010.63.4

26.522222222

26.526.526.526.526.5

0.14-0.200-2.50-2.50-2.50-2.6

--000

Sp, CcCap-G1,2Cc,Cap-G1,2Cc,Cap-G1,2Cc

C

A, CC

A, C

BBBBB

*:in case CG=1mm*:in case CG=1mm*:in case CG=1mm*:in case CG=1mm

PlanAchromat

3752037521375223752737523375243752537526374313760137602376033760537606

PL 2xPL 4xPL 10xPL 10x CYPL 20xPL 40xPL 50x OIPL 100x OPL 4x PMPL 5xMPL 10xMPL 20xMPL 50xMPL 100x

0.050.100.250.250.400.65

0.50-0.901.250.100.100.250.400.750.90

5.022.010.510.51.20.560.200.1522.019.610.61.30.380.21

2222222222222222222222222222

----

0.170.17

-----000

oiloil

ND filterSpSpSp, IrisSp SpSpSp

Marker pen (U-MARKER) attachable Strain-free

Achromat

37501375023750337504375053750637432374333743437435

ACH 10xACH 20xACH 40xACH 60xACH 100x OACH 100x OIACH 10x PACH 20x PACH 40x PACH 100x OP

0.250.400.650.801.25

0.60-1.250.250.400.651.25

6.13.00.450.150.130.136.13.00.450.13

22222222222222222222

-0.170.170.17

---

0.170.17

-

oiloil

oil

SpSpSpSp, Iris SpSp

Strain-freeStrain-freeStrain-freeStrain-free

TransmittedDIC -

A: U-UCDB/AX-UCDM condenser (NA: 0.9, WD: 1.5 mm)B: U-UCDB/AX-UCDM condenser (NA: 1.4, WD: 0.6 mm)C: IX-LWUCD condenser (NA: 0.55, WD: 23.3 mm)D: U-LWUCD condenser (NA: 0.8, WD: 5.7 mm)

Reflected DIC-

Sp: SpringCc: Correction collarA: U-DICR prism lever -- push posit ionB: U-DICR prism lever -- pull posit ion

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(a.u

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700650600550500Wavelength (nm)

100

80

60

40

20

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Emission2 Emission1Absorption1

Strahlteiler Laser Strahlteiler Detektor 1/2

Human Basal Cell Carcinoma

Alfalfa Stem

www.microscopyu.com/galleries/confocal/

Dinoflagellates

http://www.mpibpc.gwdg.de/abteilungen/200/

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4155Cell biology beyond the diffraction limit

exploits the bending of a cantilever attached to the probe as adirect measure of the probe-sample interaction force, in NSOMan indirect method, based on shear-force damping, iscommonly used. For this, the NSOM probe, or a piezoelectrictuning fork attached to it, is oscillated at its resonancefrequency in a lateral vibrational mode (with a <1 nmamplitude); when in proximity to the sample, shear-forcesdampen this motion and induce measurable changes in theoscillation amplitude and phase. An electronic feedbacksystem, controlling the probe-sample distance directly throughthe piezoelectric scan stage, is subsequently used to maintaina constant oscillation amplitude/phase during scanning. In thisway, a constant probe-sample distance of <10 nm is realized(Fig. 2). The feedback signal itself, as in AFM, is used togenerate a topographic map of the sample surface (Fig. 1). Ofcourse, unique to NSOM is the fact that a correspondingfluorescence map is simultaneously generated.

The optical detection sensitivity of NSOM depends largelyon the extremely small excitation/detection volume set by theaperture dimensions as well as the depth of penetration of thenear-field into the specimen. Together, these propertieseffectively reduce background fluorescence and therebyenhance detection sensitivity. Betzig and Chichester exploitedthis eight years ago, providing the first observation of single-molecule fluorescence under ambient conditions (Betzig and

Optical fiber

Sample

Distance

detectionFeedback

x

y

z

Scanners

Polarizers

Laser

Detector 1

Long-pass filter

Objective

De

tecto

r 2

objective

Fig. 1. The principle of scanning probe microscopy. In AFM andNSOM, a sharp probe is used to map the topographic features on thesample surface accurately. This is done by physically scanning theprobe over the surface while maintaining a constant probe-sampledistance by force feedback.

Fig. 2. Schematic lay out of a near-field scanning optical microscope.The NSOM probe is a tapered optical fiber (Fig. 3A). Laser light iscoupled into the fiber and is used to excite fluorophores as the probescans the sample surface. The probe-sample distance is maintainedconstant at <10 nm during scanning by shear-force-based distancedetection in combination with an electronic feedback systemcontrolling the piezoelectric scan stage. Fluorescence is collected bya conventional inverted microscope. Dual-channel optical detectionallows wavelength and/or polarization discrimination.

Fig. 3. The near-field optical probe. (A) An optical fiber is pulled to afinal diameter of 20-120 nm and subsequently coated withaluminum. This coating serves to confine the light to the tip region.A subsequent etching step results in a flat and circular endpoint andaperture. The aperture functions as a miniature light source, and itsdiameter primarily determines the optical resolution of themicroscope. (B) The principle of surface-specific excitation. Theoptical near-field generated at the aperture has significant intensityonly in a layer of <100 nm from the aperture; lower lyingfluorophores are therefore not excited. Hence, backgroundfluorescence is effectively suppressed. This forms the basis for thehigh optical detection sensitivity of this technique.

4155Cell biology beyond the diffraction limit

exploits the bending of a cantilever attached to the probe as adirect measure of the probe-sample interaction force, in NSOMan indirect method, based on shear-force damping, iscommonly used. For this, the NSOM probe, or a piezoelectrictuning fork attached to it, is oscillated at its resonancefrequency in a lateral vibrational mode (with a <1 nmamplitude); when in proximity to the sample, shear-forcesdampen this motion and induce measurable changes in theoscillation amplitude and phase. An electronic feedbacksystem, controlling the probe-sample distance directly throughthe piezoelectric scan stage, is subsequently used to maintaina constant oscillation amplitude/phase during scanning. In thisway, a constant probe-sample distance of <10 nm is realized(Fig. 2). The feedback signal itself, as in AFM, is used togenerate a topographic map of the sample surface (Fig. 1). Ofcourse, unique to NSOM is the fact that a correspondingfluorescence map is simultaneously generated.

The optical detection sensitivity of NSOM depends largelyon the extremely small excitation/detection volume set by theaperture dimensions as well as the depth of penetration of thenear-field into the specimen. Together, these propertieseffectively reduce background fluorescence and therebyenhance detection sensitivity. Betzig and Chichester exploitedthis eight years ago, providing the first observation of single-molecule fluorescence under ambient conditions (Betzig and

Optical fiber

Sample

Distance

detectionFeedback

x

y

z

Scanners

Polarizers

Laser

Detector 1

Long-pass filter

Objective

Dete

cto

r 2

objective

Fig. 1. The principle of scanning probe microscopy. In AFM andNSOM, a sharp probe is used to map the topographic features on thesample surface accurately. This is done by physically scanning theprobe over the surface while maintaining a constant probe-sampledistance by force feedback.

Fig. 2. Schematic lay out of a near-field scanning optical microscope.The NSOM probe is a tapered optical fiber (Fig. 3A). Laser light iscoupled into the fiber and is used to excite fluorophores as the probescans the sample surface. The probe-sample distance is maintainedconstant at <10 nm during scanning by shear-force-based distancedetection in combination with an electronic feedback systemcontrolling the piezoelectric scan stage. Fluorescence is collected bya conventional inverted microscope. Dual-channel optical detectionallows wavelength and/or polarization discrimination.

Fig. 3. The near-field optical probe. (A) An optical fiber is pulled to afinal diameter of 20-120 nm and subsequently coated withaluminum. This coating serves to confine the light to the tip region.A subsequent etching step results in a flat and circular endpoint andaperture. The aperture functions as a miniature light source, and itsdiameter primarily determines the optical resolution of themicroscope. (B) The principle of surface-specific excitation. Theoptical near-field generated at the aperture has significant intensityonly in a layer of <100 nm from the aperture; lower lyingfluorophores are therefore not excited. Hence, backgroundfluorescence is effectively suppressed. This forms the basis for thehigh optical detection sensitivity of this technique.

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4158

interactions or intramolecular dynamics at the single-moleculelevel on cells. The first such examples have already beenreported (Kirsch et al., 1999); however, although clearlyshowing resolution below the diffraction limit, single-moleculedetection sensitivity has not yet been reached. Importantly,

NSOM bridges the gap between the diffraction-limitedresponse of normal light microscopy and the 5-10 nm distancesensitivity inherent in FRET and, as such, already providesimportant additional but otherwise unobtainable information.

In the past decade, especially the past two years, severalother approaches to break the diffraction limit have beendeveloped. These include interferometric microscopy methodssuch as 4Pi confocal microscopy (Hell and Stelzer, 1992),I5M (Gustafsson et al., 1999), standing-wave-total-internal-reflection fluorescence microscopy (Cragg and So, 2000) andharmonic excitation light microscopy (Frohn et al., 2000).These methods, however, do not have single-moleculedetection sensitivity, and all require extended electronic post-processing of the images. Recently, a novel techniqueinvolving ‘point spread function (PSF) engineering’, whichexploits stimulated emission depletion (STED), has beendescribed (Klar et al., 2000). This method involves theinduced quenching of fluorescence by stimulated emission atthe rim of the diffraction-limited focal spot, thereby squeezingit to an almost spherical shape of ~100 nm in diameter. Thusfar, this is the only technique that seriously rivals the smallexcitation/detection volume and therefore sensitivity ofNSOM. The maximal resolution obtained with aperture-typeNSOM relates to the limited energy throughput of the near-field probe. This limits the minimum size of the aperture to beused and hence the resolution of the microscope to ~20 nm.A possible way around this involves exploiting single-molecule emitters, attached to a scanning probe, which act aslight source to excite molecules in the sample (Michaelis etal., 2000).

The most important technical challenge that remains is theconstruction of an NSOM instrument that operates underphysiologically relevant conditions and allows the study ofsoft, rough and motile surfaces, such as the plasma membraneof living cells. When combined with single-molecule detectionsensitivity and an optical resolution that is comparable totransmission electron microscopy, this will prove to be aninvaluable tool in cell biology. These are truly exciting times.

We thank Jeroen Korterik, Frans Segerink, Marjolein Koopman andBen Joosten for expert technical assistance. Frank de Lange issupported by grant FB-N/T-1a from the Netherlands Foundation forFundamental Research of Matter (FOM). Bärbel de Bakker issupported by grant TTN4812 from the Dutch Technology Foundation(STW). The research by Maria Garcia-Parajo was made possible bya fellowship of the Royal Netherlands Academy of Arts and Sciences(KNAW).

References

Allen, T. D., Cronshaw, J. M., Bagley, S., Kiseleva, E. and Goldberg, M.W. (2000). The nuclear pore complex. J. Cell Sci. 113, 3885-3886.

Ambrose, W. P., Goodwin, P. M., Martin, J. C. and Keller, R. A. (1994).Alterations of single molecule fluorescence lifetimes in near-field opticalmicroscopy. Science 265, 364-367.

Balaban, N. Q., Schwarz, U. S., Riveline, D., Goichberg, P., Tzur, G.,Sabanay, I., Mahalu, D., Safran, S., Bershadsky, A., Addadi, L. andGeiger B. (2001). Force and focal adhesion assembly: a close relationshipstudied using elastic micropatterned substrates. Nat. Cell Biol. 3, 466-472.

Betzig, E. and Chichester, R. J. (1993). Single molecules observed by near-field scanning optical microscopy. Science 262, 1422-1425.

Betzig, E., Chichester, R. J., Lanni, F. and Taylor, D. L. (1993). Near-fieldfluorescence imaging of cytoskeletal actin. Bioimaging 1, 129-133.

JOURNAL OF CELL SCIENCE 114 (23)

Fig. 4. (A) Single molecule detection on cells by NSOM. This figureshows a 40 nm optical resolution near-field ‘zoom-in’ on the indicatedarea (3.2 ! 3.2 µm2) in the bright-field image of a fibroblastexpressing LFA-1-GFP. GFP excitation is accomplished using 488 nmlight (Ar-Kr laser line) linearly polarized along 90°. Fluorescence iscollected by a 1.3 numerical aperture oil-immersion objective incombination with standard optical filters. A polarizing beam-splittercube (Newport, Fountain Valley, CA) is used to split the fluorescencesignal into two perpendicular polarized components (compare withFig. 2). Both signals are then detected by photon-counting avalanchephotodiode detectors (APD, SPCM-100, EG&G Electro optics,Quebec). The red/green color-coding of the signals reflects theorientation of the GFP molecules in the plane of the sample.Examples of clustered molecules (arrows) as well as examplesshowing clear single-molecule detection sensitivity are indicated(circles and squares). The squares show the fast-blinking behaviortypical of single molecule GFP fluorescence. The circles presentdemonstrations of discrete photodissociation phenomena.(B) Estimation of the resolution in the near-field image. This figureshows a line trace through the feature marked with the hexagon in thenear-field image. The full width at half maximum (FWHM; arrows) ofsuch traces can be used to obtain an estimate for the maximalresolution (half the FWHM) in the near-field image. On this basis, weestimate the resolution in the near-field image to be ~40 nm.

4158

interactions or intramolecular dynamics at the single-moleculelevel on cells. The first such examples have already beenreported (Kirsch et al., 1999); however, although clearlyshowing resolution below the diffraction limit, single-moleculedetection sensitivity has not yet been reached. Importantly,

NSOM bridges the gap between the diffraction-limitedresponse of normal light microscopy and the 5-10 nm distancesensitivity inherent in FRET and, as such, already providesimportant additional but otherwise unobtainable information.

In the past decade, especially the past two years, severalother approaches to break the diffraction limit have beendeveloped. These include interferometric microscopy methodssuch as 4Pi confocal microscopy (Hell and Stelzer, 1992),I5M (Gustafsson et al., 1999), standing-wave-total-internal-reflection fluorescence microscopy (Cragg and So, 2000) andharmonic excitation light microscopy (Frohn et al., 2000).These methods, however, do not have single-moleculedetection sensitivity, and all require extended electronic post-processing of the images. Recently, a novel techniqueinvolving ‘point spread function (PSF) engineering’, whichexploits stimulated emission depletion (STED), has beendescribed (Klar et al., 2000). This method involves theinduced quenching of fluorescence by stimulated emission atthe rim of the diffraction-limited focal spot, thereby squeezingit to an almost spherical shape of ~100 nm in diameter. Thusfar, this is the only technique that seriously rivals the smallexcitation/detection volume and therefore sensitivity ofNSOM. The maximal resolution obtained with aperture-typeNSOM relates to the limited energy throughput of the near-field probe. This limits the minimum size of the aperture to beused and hence the resolution of the microscope to ~20 nm.A possible way around this involves exploiting single-molecule emitters, attached to a scanning probe, which act aslight source to excite molecules in the sample (Michaelis etal., 2000).

The most important technical challenge that remains is theconstruction of an NSOM instrument that operates underphysiologically relevant conditions and allows the study ofsoft, rough and motile surfaces, such as the plasma membraneof living cells. When combined with single-molecule detectionsensitivity and an optical resolution that is comparable totransmission electron microscopy, this will prove to be aninvaluable tool in cell biology. These are truly exciting times.

We thank Jeroen Korterik, Frans Segerink, Marjolein Koopman andBen Joosten for expert technical assistance. Frank de Lange issupported by grant FB-N/T-1a from the Netherlands Foundation forFundamental Research of Matter (FOM). Bärbel de Bakker issupported by grant TTN4812 from the Dutch Technology Foundation(STW). The research by Maria Garcia-Parajo was made possible bya fellowship of the Royal Netherlands Academy of Arts and Sciences(KNAW).

References

Allen, T. D., Cronshaw, J. M., Bagley, S., Kiseleva, E. and Goldberg, M.W. (2000). The nuclear pore complex. J. Cell Sci. 113, 3885-3886.

Ambrose, W. P., Goodwin, P. M., Martin, J. C. and Keller, R. A. (1994).Alterations of single molecule fluorescence lifetimes in near-field opticalmicroscopy. Science 265, 364-367.

Balaban, N. Q., Schwarz, U. S., Riveline, D., Goichberg, P., Tzur, G.,Sabanay, I., Mahalu, D., Safran, S., Bershadsky, A., Addadi, L. andGeiger B. (2001). Force and focal adhesion assembly: a close relationshipstudied using elastic micropatterned substrates. Nat. Cell Biol. 3, 466-472.

Betzig, E. and Chichester, R. J. (1993). Single molecules observed by near-field scanning optical microscopy. Science 262, 1422-1425.

Betzig, E., Chichester, R. J., Lanni, F. and Taylor, D. L. (1993). Near-fieldfluorescence imaging of cytoskeletal actin. Bioimaging 1, 129-133.

JOURNAL OF CELL SCIENCE 114 (23)

Fig. 4. (A) Single molecule detection on cells by NSOM. This figureshows a 40 nm optical resolution near-field ‘zoom-in’ on the indicatedarea (3.2 ! 3.2 µm2) in the bright-field image of a fibroblastexpressing LFA-1-GFP. GFP excitation is accomplished using 488 nmlight (Ar-Kr laser line) linearly polarized along 90°. Fluorescence iscollected by a 1.3 numerical aperture oil-immersion objective incombination with standard optical filters. A polarizing beam-splittercube (Newport, Fountain Valley, CA) is used to split the fluorescencesignal into two perpendicular polarized components (compare withFig. 2). Both signals are then detected by photon-counting avalanchephotodiode detectors (APD, SPCM-100, EG&G Electro optics,Quebec). The red/green color-coding of the signals reflects theorientation of the GFP molecules in the plane of the sample.Examples of clustered molecules (arrows) as well as examplesshowing clear single-molecule detection sensitivity are indicated(circles and squares). The squares show the fast-blinking behaviortypical of single molecule GFP fluorescence. The circles presentdemonstrations of discrete photodissociation phenomena.(B) Estimation of the resolution in the near-field image. This figureshows a line trace through the feature marked with the hexagon in thenear-field image. The full width at half maximum (FWHM; arrows) ofsuch traces can be used to obtain an estimate for the maximalresolution (half the FWHM) in the near-field image. On this basis, weestimate the resolution in the near-field image to be ~40 nm.

Text

fibroblast expressing LFA-1(integrin)-GFP

Alexa 488

Multiple-photon excitation

fluorescence microscopy

Multiple-photon excitation fluorescence

microscopy is a technique that uses non-linear

optical effects to achieve optical sectioning.

How it works: The sample is illuminated with a wavelength

around twice the wavelength of the absorption peak of thefluorophore being used. For example, in the case offluorescein which has an absorption peak around 500nm,1000 nm excitation could be used. Essentially no excitationof the fluorophore will occur at this wavelength. However, ifa high peak-power, pulsed laser is used (so that the meanpower levels are moderate and do not damage the specimen),two-photon events will occur at the point of focus. At thispoint the photon density is sufficiently high that two photonscan be absorbed by the fluorophore essentiallysimultaneously. This is equivalent to a single photon with anenergy equal to the sum of the two that are absorbed. In thisway, fluorophore excitation will only occur at the point offocus (where it is needed) thereby eliminating excitation ofout-of-focus fluorophore and achieving optical sectioning.

A cuvette of fluorescent dye excited by

single photon excitation (right line) and

multiphoton excitation (localized spot of

fluorescence at left) illustrating that two

photon excitation is confined to the focus of

the excitation beam (courtesy of Brad Amos

CBIT Home

The Center for Biomedical Imaging Technology has engineered atwo-photon laser scanning microscope, which is available for use. Dr.Paul Campagnola has adapted the center's BioRad MRC600 fortwo-photon excitation of fluorescence while still maintaining theMRC600's one-photon CLSM.

Two-photon excitation of fluorescence is based on the principle thattwo photons of longer wavelength light are simultaneously absorbedby a fluorochrome which would normally be excited by a singlephoton, with a shorter wavelength. The nonlinear optical absorptionproperty, of two-photon excitation, limits the fluorochrome excitationto the point of focus. This is graphically demonstrated in the imageabove. The images below, of pollen granules which were obtained onour system, clearly show the difference between one and two-photonexcitation. The two-photon image was generated using 704nmexcitation light while the one-photon image was generated using488nm.

Limiting the excitation light to the point of focus rather than exposing the entire sample greatlyreduces total photobleaching and photodamage. This is one major advantage to using two-photonexcitation.

Two-photon microscopy has several other advantages over confocal microscopy; optical sections canbe obtained deeper within a specimen, a detection pinhole is no longer necessary, thus not limitingthe number of photons being detected. (However, a pinhole may still be used to slightly improve theresolution of two-photon excitation.) In addition, the use of UV fluorophores is no longer limited toUV corrected objectives. Two-photon excitation utilizes visible wavelengths to excite UV fluorescentstains and indicators, therefore the objectives do not have to pass UV light.

Optical sections may be obtained from deeper within a

tissue that can be achieved by confocal or wide-fieldimaging. There are three main reasons for this:

the excitation source is not attenuated by absorption byfluorophore above the plane of focuslonger excitation wavelengths suffer less scatteringfluorescence signal is not degraded by scattering fromwithin the sample as it is not imaged

When images of optical sections that are deep within alight-scattering sample are obtained using confocalmicroscopy, the fluorescence signal is attenuated by lightscatter. Furthermore, some fluorescence originating fromregions away from the point being instantaneouslyilluminated will be scattered such that this fluorescence willpass through the confocal pinhole thereby increasingbackground. Confocal imaging therefore suffers adeterioration in signal-to-background when obtaining imagesfrom deep within a sample. Multiphoton imaging is largelyimmune from these effects as little fluorescence is generatedaway from the point of illumination and all detectedfluorescence photons may be used for imaging regardless ofwhether they have been scattered or not.

Images of acid fucsin stained monkey kidney taken at a

depth of 60 µm by confocal (left) and multiphoton

microscopy (right). Laser intensities were adjusted to

produce the same mean photons per pixel. The confocal

image shows a significant increase in local background

resulting in a lower contrast image. However, the

multiphoton image maintains contrast even at significant

depths within a light scattering sample.(Reproduced from

Figure 7 of Centonze,V.E and J.G.White. (1998)

Biophysical J. 75:2015-2024)

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Bestandteil des aktiven Zentrum der Oxidase ist ein Flavin-Adenin Dinukleotid (FAD)

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und kann dann wieder durch O2 oxidiert werden (wieder fluoreszierend).

Zeitabhängige Beobachtung

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FluoreszenzspektroskopieAnwendung: Enzymologie

Anwendung: Enzymologie


Einzelmolekül Enzymologie Flavoenzym Cholesterol Oxidase (COx)

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Lu et al. Science 1998

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C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

ADP

ADPADP

ADP

NaNa

ATP

ADP

Cover-Slip

Dye

{{

ATP Hydrolysis

No Na

low ATP (~0.5µM)

low ATP (~0.5µM)

+

High Na (2mM) +

P = III II I+

III II I-Polarization:

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OH O

HN2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OH O

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

HN2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

+

P = III II I+


H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OH O

HN2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OH O

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

HN2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

HN2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

HN2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

+

P = III II I+


H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OH O

HN2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OH O

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

HN2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

HN2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

HN2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

H N2

CH2

CH2

CH2

CH2

CH

CH2

N

C

OH

O

C

OH

O

C

OHO

-1.0

-0.5

0.0

0.5

1.0

0.50.40.30.20.10.0Time (s)

Po

lariza

tio

n

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.80.60.40.20.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2P

ola

riza

tio

n

4.54.03.53.02.52.01.51.00.50.0

Time (s)

Immobilized F0F1 ATPase + Vesicle

Cover slip

Drop of buffer

F F0 1

Vesicle

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KK

F F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KK

F F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KK

F F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KK

F F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KK

F F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KK

F F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KKKK

KK

KKKK

KK

KKKK

KKKK

KK

KK

KK

KK

KK

KK

KK

Na+

K+

ATP

ADP

NaNa

ATP

ADP

His-tagsF F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KKKK

KK

KKKK

KK

KKKK

KKKK

KK

KK

KK

KK

KK

KK

KK

+

+

NaNa

ATP

ADP

F F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KKKK

KK

KKKK

KK

KKKK

KKKK

KK

KK

KK

KK

KK

KK

KK

+

+

NaNa

ATP

ADP

F F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KKKK

KK

KKKK

KK

KKKK

KKKK

KK

KK

KK

KK

KK

KK

KK

+

+

NaNa

ATP

ADP

F F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KKKK

KK

KKKK

KK

KKKK

KKKK

KK

KK

KK

KK

KK

KK

KK

+

+

NaNa

ATP

ADP

F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KKKK

KK

KKKK

KK

KKKK

KKKK

KK

KK

KK

KK

KK

KK

KK

+

+

NaNa

ATP

ADP

F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KKKK

KK

KKKK

KK

KKKK

KKKK

KK

KK

KK

KK

KK

KK

KK

+

+

NaNa

ATP

ADP

F0 1

Na

Na

Na

Na

Na

Na

NaNa

Na

Na

Na

Na

Na

Na

Na

Na

ATP

ATP

ATP

ATP

OH

OH

ADPADP

ADP

ADPADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

KKKK

KK

KKKK

KK

KKKK

KKKK

KK

KK

KK

KK

KK

KK

KK

+

+

NaNa

ATP

ADP

ATP Synthesis

-1.0

-0.5

0.0

0.5

1.0 1.00.80.60.40.20.0

-1.0

-0.5

0.0

0.5

1.0P

ola

riza

tio

n

Po

lariza

tio

nP

ola

riza

tio

n

Norm

aliz

ed A

CF

1.00.80.60.40.20.0

-0.2

-1.0

-0.5

0.0

0.5

1.0 1.00.80.60.40.20.0

-0.2

Time (s)

0.40.30.20.10.0

Time lag (ms)

100806040200

-1.0

-0.5

0.0

0.5

1.0

Time (s)

Time (s)

-1.0

-0.5

0.40.30.20.10.0

0.40.30.20.10.0

0.0

0.5

1.0

Membrane potential = 0ADP in saturationmembrane potential >0 ATP synthesis

ADP in saturationMembrane potential >0With inhibitor DCCD

FRET


DonorFluorescein

AkzeptorRhodamin

Am Einzelmolekül:

single pair FRET (spFRET)

Förster Fluoreszenz-Resonanz-Energietransfer (FRET)

Energieüberlapp

Hovius et al., TIBS, 2000

Fluoreszenzspektroskopie: Anwendungen auf Einzelmoleküle

FRET


Prinzip des Förster-Prozesses

Förster Radius R0

Radius beim Energietransfer E = 0.5

Beispiel hier (fluorescein-rhodamin): R0 = 5.6 nm

Für die meisten D-A Paare:• R0 = 1.5 - 8.0 nm• Sensitiver Bereich: 0.5 R0 - 1.5 R0

Strahlungslose Energieübertragung: Dipol-Dipol Wechselwirkung mit Abstandsabhängikeit ~ r6

R0 = (8.79 ·10-5 n-4 !D J "2)1/6 Å

n Brechungsindex des Mediums (z.B. Wasser n = 1.33)

!D Quantenausbeute des Donors (0.2 -1)J Austauschenergie (Energieüberlapp)

[nm4 M-1 cm-1]" Orientierungsfaktor

(mit 2/3 für nicht orientierte Dipole)

D A

r µA

µD


FRET - Anwendung


spFRET Anwendungsmöglichkeiten

• Nanometer Abstandsmessung

• Micro-Millisekunden Zeitauflösung

Konformationsänderungen

Protein-Protein/Substrat Wechselwirkung

Proteinfaltung


FRET - Proteinfaltung


Proteinfaltung beobachtet mit spFRET

Schuler et al. Nature 2002

Schematische Strukturen vom cold-shock protein

(Csp) und von der Modelstruktur polyprolin

a: gefaltetes Csp

b: entfaltetes Csp

c: (Pro)6

d: (Pro)20


Donor: Alexa Fluor 488

Akzeptor: Alexa Fluor 594

R0 = 5.4 nm


FRET - Proteinfaltung


Biospektroskopie 223Schuler et al. Nature 2002

spFRET mit csp: (a,c) Pro6 (b,d); Pro20; (e) Csp


(a) Pro20 (b); Csp; (c) Csp+Pro20

Fluoreszenzkorrelationsspektros-kopie


Fluoreszenz-Korrelations-Spektroskopie (FCS):

Studium der Moleküle in Lösung

Die Anzahl von Molekülen in dem fokalen Volumen kann zu jeder Zeit mit einer Poisson-Verteilung beschrieben werden. Für die Fluktuation der Partikelanzahl gilt:

Unter der Annahme, dass die Anregungsintensität konstant ist, kann die Fluktuation desFluoreszenzsignals als Funktion der Zeit wie folgt geschrieben werden:

)()()( tFtFtF !=!

Für die praktische Beobachtbarkeit von zeitlichen Änderungen im Fluoreszenzsignal sind die folgenden Randbedingungen wichtig:! Möglichst wenig Moleküle im Volumen

führen zu großen Schwankung von F(t)!Genügend viele Moleküle im Volumen sind

wichtig für ausreichendes gutes S/N


( ) ( )

NN

NN

N

N 122

=!

=!

"=

T

dttFT

tF0

)(1

)(mit

Fluoreszenzkorrelationsspektros-kopie


Bei dieser Fluktuation der Fluoreszenzintensität gehen verschiedene Faktoren ein:

und der Korrelationszeit tc . Es gilt G(t=! )=0

2

)()()(

F

tFttFtG

c

c

+=


[ ]!=V

dVtrCrWtF ),()()( "##W(r): räumlich Ausdehnung des

Belichtungsvolumen (Ellipsoid)

" = I0·#$%$qq: Quantenausbeute

I0: max. Amplitude des Laserstrahls

#: Detektionseffizienz

%: molekularer Absorptionsquerschnitt

C: Konzentration der Fluorophore im

Volumen V (z.B 1 femtoliter).

mit der zugehörigen

Autokorrelationsfunktion

Das Signal wird mit Bezug auf seine Selbstähnlichkeit nach einer Korrelationszeit tc

analysiert. Die Autokorrelation G(t=0) ist deshalb die normalisierte Varianz des

fluktuierenden Signals und gibt damit Auskunft über die absolute Konzentration im

Anregungsvolumen.

=> Bestimmung von N und damit von C aus der Autokorrelationskurve !

FCS - Anwendung


FCS: Anwendung Proteinfaltung

(P. Schwille Göttingen).

Der hydrodynamische Radius RG

beeinflusst den Diffusionskoeffizienten Dund zeigt sich somit in eine veränderte Autokorrelation im FCS-Experiment.

Stokes-Einstein Relation


GR

TkD

!"6

!=

unfolded: RG ~ 58Åfolded: RG ~ 45 Å

FCS - Anwendung


Anwendung der Fluoreszenz-Korrelations-Spektroskopie:

Wieviele Liganden kann ein Rezeptor binden ?

Großes Potential der FCS

beim Einsatz in der Analytik

(Medizin, Pharmazie, Biotechnologie):

• schnelle Messungen

• eindeutige Ergebnisse

• sehr geringe Probemengen

Hovius et al., TIBS, 2000

Fluoreszenzmarkierte Liganden (Kugeln)Zu c:

Anzahl der Liganden (im Volumenelement)

als Funktion der Rezeptorkonzentration.

• : die Zahl der Liganden bleibt auch mit steigenden

Zahl der Rezeptoren konstant

(=> ein Ligand pro Rezeptor)

O: Kontrolle mit bekanntem Bindungspaar

Streptavidin-Biotin mit (4:1 Stöchiometrie)


FCS - AnwendungZwei-Farben FCS / Kreuzkorrelationen


(P. Schwille Göttingen).

Vergleich von Einzel –und

Zwei Farben FCS

Schematischer Aufbau für

Zwei-Farben FSC

!Anregung zweier Farbstoffe im gleichen

konfokalen Volumen

!Berechnung der Kreuzkorrelationen

=> Beobachtung eines starken Signals

(Kreuzkorrelation) bei Assoziation

von zwei unterschiedlich markierten

Molekülen.

Single-Molecule SERS

Cars - Prinzip

CARS Anwendung

Moderne Methoden Optischer Mikroskopie und … · Themen 0. Grundlagen optischer Spektroskopie 1....

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