Laser TIRF 3 - Core Facility for Integrated...

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Microskopy from Carl Zeiss Laser TIRF 3 Simply Excite More The New Standard: Six Laser Lines for the Investigation of Processes Near the Cell Membrane

Transcript of Laser TIRF 3 - Core Facility for Integrated...

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M i c r o s k o p y f r o m C a r l Z e i s s

Laser TIRF 3Simply Excite More

The New Standard: Six Laser Lines for the

Investigation of Processes Near the Cell Membrane

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1. Epi-fluorescence green (excitation with 458 nm), αPlan-Fluar 100x/1.45 Oil Oberbanscheidt, van den Boom, Bähler, Institute of General Zoology and Genetics, university of Münster, Germany

2. under TIrF illumination you only excite structures within the evanescent field. You can see structures that you cannot see in Epi-fluorescence

TIRF Microscopy from Carl Zeiss.

TIRF microscopy (Total Internal Reflection Fluorescence), has firmly established itself in

life science research as a standard imaging technique for the investigation of processes

near the cell membrane. Now with Laser TIRF 3, Carl Zeiss brings a new instrument

generation onto the market, which allows even more advanced applications: e.g. syn-

chronous observation of differently labeled molecules at high temporal resolution.

More flexible, more economical, safer: Laser TIRF 3 from Carl ZeissAs a method for the investigation of molecular mecha-nisms in the vicinity of the cell membrane, TIRF micros-copy has proven itself in cell biology and membrane biophysics for years. With the new product generation Laser TIRF 3 Carl Zeiss elevates this technique to a new performance level.

• Newmotorizedsliderforreproduciblesettingoftheillumination angle

• Newmanualslider as an economic entry version • New,flexibleandupgradeablelasermodulewithup

to 4 solid state lasers• Rapidswitchingbetweentheexcitationwavelengths

and intensities via AOTF• Maximumprotectionbymeansofintelligentlasersafety• Excellentopticalperformance:bestcontrastand

highestresolutioninx,y,andz BasedonthemodularresearchplatformofAxioObserver, the Imaging System Laser TIRF 3 can be adapted to changing requirements at any time. Handling and oper-ation is simple and comfortable. TIRF sliders are merely inserted into the luminous field diaphragm plane without complexadd-ons.Inaddition,differentincubatorsfromCarl Zeiss for long term observation under physiological conditions are available.

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laser TIrF 3 Imaging System with incubation

The sum of the advantages – Laser TIRF 3 in overview

Compact complete system • Themotorized/manualTIRFsliderinsertsinthefield

diaphragm, small footprint, all operating elements are easily accessible

• Nocomplexexternaladd-ons• Availableascompletesystemorforretrofittingon

AxioObserver

Multi-color laser module• Newlasermodulehousesuptofoursolidstatelasers• Laserlineswithdifferentwavelengthandoutput

power: 405 nm (50 mW), 488 nm (100 mW and 20 mW), 532nm(75mW/20mW)or561nm(40mW/20mW)and635nm(30mW),respectively

• Foroptimalexcitationofmorefluorophorescombi-nations

• Alllaserlinesareguidedthroughonebroadbandfiber into the microscope

Excellent optics• EnhancedbrightnesswiththeαPlan-FLuAR100x/1.45Oil (TIRF angle up to 72°) andαPlan-ApOchROMAT

100x/1.46Oil(TIRFanglesupto73,2°)

• hightransmission,verygoodcontrastandcolorcor-rection, homogenous illumination

• Apochromaticbeampathresultsinoneoptimalsetting for laser beam divergence for all used wave lengths

Standardized system software • SeamlessintegrationintoAxioVision:fromFastImage

Acquisitionuptocompleximageanalysis

Numerous combination options • TIRF/Epi-fluorescence,TIRF/DIc,TIRF/phasecontrast• TIRF/incubation,TIRFwithTime-lapseImaging

Sophisticated laser safety system• Maximumprotectionoftheuserinallapplications:

invisible, cannot be deactivated or demounted• SystemalsoprovideslasersafetyinTIRF/transmission

andTIRF/incubation

Modular structure•Modulescanbeexpandablefornewapplications

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Features of the New Laser TIRF Generation from Carl Zeiss.

Laser TIRF 3 is oriented toward the high requirements of live cell research in every

detail.Withacompact,easilyoperatedsystem.providingextremelyfinecontroland

automatedreproducibilityoversettingtheilluminationanglewithamotorizedTIRF

slider. With a new laser module, which is controlled by AOTF and which handle up

to6laserlines.Forsignficantlymorequalityinscientificpractice.

• TheTIRFangleisstoredintheAxioVisionsystemsoftware and can be reset again at any time.

• Allwavelengthsavailableinthelasermoduleareguided through the same broad band fiber and via the TIRF slider into the microscope’s beam path.

TheextendedfocusingrangeoftheTIRFsliderallows theuseofalargenumberofstagesaswellaspiezofocusingdevicesorthecombinationwithAFM(AtomicForceMicroscopy).Overall,thishighlyergo-nomicsystemprovidesflexiblehandlingandabroadrange of applications with numerous combination options.

Best optics for an optimum TIRF angle: the objectivesIn order to make the interaction of individual molecules or objects close to the interface from glas to medium

Advanced technology for complex research projects: the complete systemIntegrated in the Axio Observer research platform, Laser TIRF3 combines amaximumof information andsafety with extremely simple operation. The apochro-matically corrected beam path ensures the best possible imagequality.ThesoftwareAxioVisioncanbeoperatedintuitively and easily adapted to your individual require-ments.

More than just an option: the new TIRF sliders Optimized for even more application variety: the mo-torized TIRF slider for reproducible angle settings. The mechanical design of the slider allows a resolution of achievable angle difference of about 5 nm within the center range of TIRF angles

laser TIrF 3 Imaging System with Dual Camera setup

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visible, an objective must have a numerical aperture that achievesaTIRFangleofatleast61°.carlZeisshasdevel-opedoroptimizedtwoobjectivesespeciallyforTIRF:

•αPlan-ApOchROMAT100x/1.46Oilachievesanangle of up to 73,2°

•αPlan-FLuAR100x/1.45Oilachievesanangleof up to 72°.

Asaresult,penetrationdepthsof<100nmarerealizeddepending on the wavelength. Together with filter sets whichareoptimizedforTIRFandthenewlyconceivedbeam path in the TIRF slider, reflections and interferences in the image have been reduced to a minimum.

Sophisticated and well-conceived: laser safetyOften neglected, yet crucial for your safety: protection against laser light. For Laser TIRF Carl Zeiss developed its own laser safety device which allows you to work freely without impediments.

New possibilities: four laser wavelengths for Multi-color TIRFWiththenewlasermoduleinLaserTIRF3,uptosixla-ser wavelengths can be selected. Therewith even more fluorophorescanbeoptimallyexcitedifmorethanonefluorescent marker is used.

Laser lines 405 458 488 514 532 561 635BFP (Blue Fluorescent Protein) X

CFP (Cyan Fluorescent Protein) X X

FITC X

EcFp(EnhancedcyanFluorescentprotein) X

GFp(S65T) X X

wtGFP X X

EGFp(EnhancedGreenFluorescentprotein) X X

YFP X

EYFp(EnhancedYellowFluorescentprotein) X

DsRed X X

mRFP X X

HcRed X X

mRaspberry X

mPlum pH 7 X

mPlum pH 11 X

TexasRed-Xconjugate X

Cy2 X X

Cy3 X X

Cy3.5 X X

Cy5 X

Cy5.5 X

Alexa405 X

Alexa430 X X

Alexa488 X

Alexa532 X

Alexa546 X

Alexa555 X X

Alexa568 X

Alexa594 X X

Alexa633 X X

Alexa647 X

Alexa660 X

DiI X X

DiO X X

TIrF slider with motorized illumination angle adjustment

αPlan-Fluar 100x/1.45 Oil with DIC slider αPlan-aPOChrOMaT 100x/1.46 Oil with DIC slider Setting the TIrF angle with the manual slider

TIrF slider with manual illumination angle adustment

Examples of dyes and markers for the available wavelenghts

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Possible Applications with Laser TIRF.

Exocytosis,endocytosisorthedisplayofindividualmoleculesinthecellmembrane–

anyone who conducts research at the limits of visibility needs technology which provides

thebestpossibleimagequalityandflexibility.LaserTIRF3nowsuppliesevenmore

information: with four laser lines and a wide variety of possible applications.

Multi-color TIRF is used to visualize the distribution ofstress fibers showing which part of the cell is solid and which part is moving. With Laser TIRF 3 this application is substantially simplified: up to three differently labeled proteins canbe excited andhencebeendetected syn-chronously.Duringsequentialexcitationofthedifferentproteins, also the illumination angle and therefore the penetration depth can be kept constant for each wave length.

TIRF/Epi-fluorescenceThe combination of TIRF and Epi-fluorescence is usedwhen it is also of interest where the molecules under ob-servationorginatefrom.WithTIRF/Epi-fluorescence,forexample,thetransportofvesiclesfromtheGolgiappara-tus in the depths of the cell can be followed to their secret locationof themembrane.Whenusing themotorizedTIRF slider, only the illumination angle is changed to switchbetweenTIRFandEpi-fluorescenceillumination.

Modular and economical: the new laser module With the new laser module in Laser TIRF 3 the user has evenmore applications at his disposal.Amaximumoffour solid-state lasers with different performance classes can be installed. An Argon laser (458 nm, 488 nm, 514 nm) can be incorporated in the new laser module instead of the 488 nm line by means of a coupling module. Thus, onehasamaximumof6differentexcitationwavelengthsavailable. Control via AOTF allows rapid switching be-tween the different wavelengths as well as the regulation of the intensity.

Multi-color TIRF with up to three laser lines for synchronous excitationMulti-colorTIRFprovidesinformationontheinteractionsof severalproteins ina specificprocess.Anexample isthe observation of the molecular aspects of cell move-ment. It provides information on how the molecules op-erate, which the cell needs for movement. In this work,

1. B16/F1 melanoma cells (mouse) – TIrF illumination, blue: CFP-actin. Exitation 458 nm, green: dsred-Clathrin light, Chain a. Exitation 541 nm, αPlan-Fluar 100x/1.45 Oil, Oberbanscheidt, van den Boom, Bähler, Institute of General Zoology a. Genetics, university of Münster, Germany 2. Single channel display – CFP-actin. Exitation 458 nm, 3. Single channel display – dsred-Clathrin light Chain a. Single channel display 514 nm

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theexposuretimeofthecameralimittheimageacqui-sition rate. With the integration of Laser TIRF 3 into the conceptofFastImageAcquisitioninAxioVisionitisnowpossible toanalyzenear-membraneprocesseswith thisstandard for the first time.

TIRF and FRETWith new filter sets and the complete integration into theAxioVision system software, Laser TIRF3nowalsoallowsthecombinationofTIRFwithFRET.Forhightem-poralandspatialresolutionthenewDualcamerasetupis recommended. Two cameras simultaneously detect the differentfluorescencesignalswhichareexcitedby twodifferent laser wavelengths simultaneously in TIRF with full resolution. The subsequent image analysis can be done very conveniently using the physiology module of theAxioVisionsoftware.

TIRF/Transmitted-light contrasting methodsWithTIRFonlythestructuresinclosestproximitytothecoverslip are visible, never the entire cell nor the entire supporting surface of the cell. Frequently, however, it is critical to compare the fluorescence signals from Epi-fluorescence and TIRF with the overall morphologyto get a more comprehensive view of the cell. Carl Zeiss Laser TIRF is the only TIRF system which can com-bine TIRF with brightfield contrast techniques such as DifferentialInterferencecontrastorphasecontrastwhile simultaneously maintaining laser safety.

TIRF and High Speed ImagingHigh time resolution is required where processes occur at high speed or when several different markers are to be detected as quickly as possible. The answer to the question of colocalisation of events is only possible when neither the switching of the excitation wave-length nor the adjustment of the illumination angle nor

The Sacharomyces cerevisae plus-end binding protein Bim1p (green – Excitation 488 nm) decorates the ends of rhodamine-labelled microtubules (red – Excitation 561nm). TIrF microscopy allows to visualize the interactions of microtubule-associated proteins with dynamic microtubules in highly purified in-vitro assays.αPlan-aPOChrOMaT 100x/1.46 OilTomasz Zimniak and Stefan Westermann, IMP Vienna, austria

hela Cells – Cytoplasmic vesicles in red (Excitation with 561 nm), focal adhesions in Green (Excitation with 488 nm),αPlan-aPOChrOMaT 100x / 1.46 OilNatalia andreyeva and Vic Small, IMBa Vienna, austria

hela Cells – Cytoplasmic vesicles in red (excitation with 561 nm), focal adhesions in green (excitation with 488 nm),αPlan-aPOChrOMaT 100x/1.46 OilNatalia andreyeva and Vic Small, IMBa Vienna, austria

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Laser TIRF in System.

Carl Zeiss has developed components especially for Laser TIRF 3, which are at

the cutting-edge of high performance technology. All of them are thoughtfully

matched to one another. In the Cell Observer® system they provide an unrivaled

performancespectrumandanexceptionalvarietyofapplicationsrangingfrom

simpleobservationuptolong-termexperimentswithincubation.

Perfect conditions and maximum protection for the user: incubation with laser safetyFor long-term observation under physiological conditions, several incubators are available for different applications: Incubator pM (laser safe), Incubator XL (multi-compat-ible:inredforallwavelengthsupto561nmandblackforallwavelengthsupto650nm)aswellasIncubatorSTIRF.The control of the incubation equipment is completely integrated into AxioVision. The ingenious concept for laser safety assures laser safety when working with all laser lines and energies. At the push of a button it is possible to switch between laser operation and normal widefield illumination for examination of the samplethroughtheeyepieces.Extremelyconvenient:thisfunc-tion can even be assigned to the control buttons on the microscope.

Cutting-edge microscopy: Axio Observer from Carl ZeissAs a research platform with the acknowledged best fluo-rescence technology, unmatched stability and excellentoptics,AxioObserverhasestablisheditselfinresearchasthe standard to be met. In combination with Laser TIRF 3, thesemi-motorizedvariantAxioObserver.D1orthefullymotorized Axio Observer.Z1 stand is extremely appro- priate for state-of-the art research. It provides outstand-ing performance in all important aspects. Perfectly matched to the apochromatic reflected-light beam path of Axio Observer, the optics of the Laser TIRF 3 sliderprovides unprecedented image quality. The motorized z-Focusallowsextremelyfinefocusingin10nmintervals.

TIrF sample cover – laser spot can be seen through the opaque mate-rial. The laser beam can be adjusted either to the front or to the back

Incubator S Dark for TIrF

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Simply intelligent: control and calibration with AxioVisionAxioVisionprovidesavarietyofmoduleswhichcanbecombinedwithLaserTIRF3.MultichannelImageAcqui-sition, Fast Image Acquisition, Mark&Find, physiology,Colocalisation, etc.

• high safety: all calibration andadjustmentprocessesare performed under laser safety conditions.

• Simple calibration: when using the motorized TIRFslider, the angle adjustment is calibrated with regard to the actual sample via a calibration routine in the software. The routine automatically determines at the sample location the critical angle for total internal reflection in two different directions. A possible offset is then calculated in the future angle adjustment. The actualTIRFangleandtherefractionindexofthesam-ple or the penetration depth of the evanescent field, respectively, result from the measurement of the criti-cal angle.

Ultrafast and high resolution: cameras for high image acquisition ratesFor the highest speeds, Carl Zeiss offers a range of highlysensitivedetectors,includingtheEMccDcamera QuantEMfromRoperScientific.IntheFastAcquisitionmodetheyarecompletelyintegratedinAxioVision.Thiscamerawith13µmpixel sizeanda512x512chip isparticularly appropriate for low-light applications and for single molecule detection. For all other TIRF applica-tions Axiocam MR monochrome from carl Zeiss with 12 bit dynamic range and high resolution is the camera of choice.

Interference-free: the special filter sets for TIRFSpecially developed for TIRFmicroscopy andoptimizedfor the most common fluorescent proteins and dyes. Doubleandtripleband-passfilters,whichallowtheex-citation waves to pass synchronously or sequentially. The new filters are coated on a special glass substrate which minimizesinterferencephenomena.

axioCam Mrm

axioVision

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640 nm 488 nm 532 nm 561 nm 405 nm

The Functional Principle of TIRF Microscopy

The analysis of images which have been acquired with conventional widefield

fluorescenceexcitationnearthecellmembraneisdifficultduetothebackground

fluorescence from other planes which are not in focus. Only under TIRF conditions

aresolutionof<200nminzisachievedbyselectiveillumination.

The penetration depth of the evanescent field is defined as the distance d from the interface in which the energy of light has fallen to 37 % of its original value. This de-pends on the angle of incidence and the wavelength of the light used (3).

(3) d = (With λ= 488 nm, n1 = 1.518, n1 = 1.33 αg=61.2°)

Because of the energy distribution, fluorophores which arefurtherawayfromtheboundaryarenotexcited.Thus,athinopticalsectionwithathicknessofapproximately100to200nmandwithaveryhighsignal/backgroundratio is formed.The imagecontrastandresolution inzare significantly improved.

The principle of total reflectionTotal reflection occurs at the interface between an op-tically denser medium and an optically less dense one when a light beam is incident at an angle which, accord-ing to Snell’s law of refraction (1), is larger than the so-called critical angle αg (i.e. α2=90°).

(1) n1* sin(α1 ) = n2* sin(α2 )

So the following applies: n1* sin(α1 ) = n2* 1

For the transition from coverslip (n1 = 1.518) to water (n2=1.33)acriticalangleof61°results.Underconditionsof total reflection a standing evanescent (= ephemeral) wavewhoseintensitydecaysexponentiallywithrespectto the distance from the interface is formed (2).

(2) I(z) = I(0)•e

theo. max. TIrF area with αPlan-Fluar

TIr

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Fluorophores

Excited fluorophores

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Cover glass: n1 = 1.518

Immersion oil

Total reflected lightExcitation light

Emittierted fluorescent light

Objective

λ0

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-zd

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• thelaserlightcomingfromthemonomodefiberisagainselectedforthepolarizationdirectionand

• themodulecansimultaneouslybecombinedwithconventional HBO or other white light.

The technology of the TIRF sliderThe decisive performance characteristic of the TIRF slider: linearlypolarizedlaserlightisintroducedintothebeampath via the TIRF slider, which is inserted into the lumi-nous field diaphragm plane of the reflected-light beam path. A double polarization-maintaining prism in theslider ensures that

1. TIrF slider2. Polarization-maintaining double prism3. reflector module4. Objective5. Sample6. Cone of diffuse light produced by the conven-

tional Epi-fluorescence illumination. This cone is not seen in TIrF-mode

a No deflection of the laser beam. The sample is excited as in conventional Epi-fluorescence

b The incident angle of the laser beam into the sample can be manipulated via the angle adjust-ment screw on the slider until TIrF conditions are reached

c under TIrF conditions, excitation light is not transmitted into the sample but reflected back into the objective lens and efficiently blocked by the reflector module

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Carl Zeiss MicroImaging GmbH07740 Jena, Germany

BioSciences | Göttingen LocationPhone: +49 551 5060 660Telefax: +49 551 5060 464E-Mail: [email protected]

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