Leica AM TIRF MCVisualize Life’s Secrets with True MultiColor TIRF

FlexibilityReproducibilityDynamicsIntegrationTIRF (Total Internal Reflection Fluorescence) microscopy is theideal technique for the study of cellular membranes and theirenvironment. TIRF uses the evanescent field generated by totalreflection to excite fluorophores. Instead of illuminating an entirespecimen with excitation light, as in widefield fluorescencemicroscopy, the evanescent field only penetrates the specimen toa depth of 70–300 nm. Fluorochromes at deeper levels of thespecimen are not excited. This method enables a substantiallyimproved signal-to-noise ratio, providing the highest qualityresults. The integration of four AOTF-controlled laser lines into theLeica TIRF system allows fast wavelength switching and a highimage recording rate with multiple fluorophores.

Multiple contrast techniquesNo more arduous searching for TIRF angles. The Leica AM TIRFMC system features an integrated sensor that does the searchingfor you and enables the evanescent field to be reproducibly intro-duced at a defined depth of the specimen. TIRF, widefield fluores-cence, and transmitted light contrast methods can be usedsequentially – simply by the push of a button.

True MultiColor Laser TIRFThe new MultiColor TIRF from Leica Microsystems is an all-in-onesystem offering four integrated solid state lasers for excitation offluorophores in all important wavelengths. The extremely shortswitching times, the automatically constant TIRF penetrationdepth when switching from one wavelength to another and theextremely high and synchronized image recording rate open upcompletely new horizons for researching dynamic processes inliving cells.

The elegant solution for fluorescence microscopyLeica’s TIRF system features a dynamic scanner that can be usedto precisely position the laser beam and determine the exact pen-etration depth of the evanescent field. The powerful Leica AF6000fluorescence software offers full control of the TIRF system. Allmicroscope functions and many analysis tools are included.

Superior imaging with superb opticsThe Leica TIRF system combines superior imaging performance, highsensitivity and an optimum signal-to-noise ratio to ensure accurateresults for all examinations of close-to-membrane structures.

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The ideal configuration for your applicationTIRF microscopy is an excellent method for examining processesin live cells. Leica offers the best configuration for specific appli-cations. Whether for vesicle transport, interactions betweenmolecules, FRET (Fluorescence Resonance Energy Transfer), mem-brane research or single molecule examination, Leica has the rightsolution for every application – tailored precisely to your needs.

The Leica TIRF module consists of a Leica HCX Plan Apo objectivewith a 1.46 numerical aperture, a high-performance 3D scanner,and a laser unit with four maintenance-free diode lasers. Thismodule easily adapts to Leica’s inverted microscopes and systemsto suit user requirements and can be customized through a widerange of accessories including stages and objectives. For epifluo-rescence excitation, Leica offers the powerful EL6000 externallight source.

Superlative performance for dynamic living cell researchProviding the wavelengths 405 nm, 488 nm, 561 nm and 635 nm formulticolor applications, the Leica AM TIRF MC offers maximumflexibility for TIRF applications with multiple fluorophores. Theindividual laser lines are coupled and controlled quickly and pre-cisely via AOTF (Acousto-Optical Tunable Filters), achieving aswitching time of only 1 ms. Also, the position of the laser is auto-matically adjusted when the wavelength is changed to ensurethat the selected TIRF penetration depth remains constant. A

Your Personal TIRF SystemFluorescence microscopy of the future

Tubulin YFP Widefield

Tubulin YFP TIRF 120 nm

Tubulin YFP TIRF 90 nm

Tubulin YFP TIRF 70 nm

Courtesy of: Prof. Dr. R. Jacob,University Marburg, Department of ClinicalCytobiology and Cytopathology,Marburg, Germany

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newly designed SyncBoard controls fast image recording of up to30 frames per second. This high temporal resolution for excitingseveral fluorophores combined with the spatial TIRF resolutionare key prerequisites for visualizing transport processes in cellmembranes or interactions of proteins in real time.

Leica’s new fluorescence axis and TIRF objectiveTo meet the exacting requirements of TIRF users, Leica has furtherimproved the fluorescence axis of the microscope, while the highnumerical aperture of 1.46 enables a specimen penetration depthof 70 to 300 nm to be achieved. Like the fluorescence axis, Leica’sTIRF objective features excellent apochromatic correction andcombines enhanced imaging performance with high sensitivity.

Reproducibility and improved technologyThrough the coupling of the laser unit and TIRF module the light isprecisely directed onto a scanning mirror. The positioning of thismirror, through the easy-to-use operating software, determines thepenetration depth and direction of the evanescent field within thespecimen. This software control ensures that these parameterscan be precisely reproduced at any time.

Leica has not only eliminated the well-known TIRF operating diffi-culties, but has also made fundamental improvements to TIRFfluorescence technology. The fluorescence axis implementsspecifically corrected wavefronts for the TIRF method. And, forthe first time, Leica offers a system without color aberration.

With TIRF microscopy techniques, cells are not directly irradiatedwith laser light. The method of exciting fluorochromes via anevanescent field is particularly gentle and allows examination oflive cell functions to be conducted over longer periods of time.

“Leica Microsystems offers superioroptical quality with user-friendlycontrol of practical techniques inquantitative biology and biomedi-cine. The revolutionary total in-ternal reflection objective designgives fully automated TIRF con-trol, precise penetration depth,and excitation light correctionswithout ambiguous optical han-dling.”

Prof. Gregory HarmsUniversity Würzburg, Rudolf-Virchow-Zentrum,DFG Research Center for ExperimentalBiomedicine, Würzburg, Germany

TIRF scanner and MultiColor laser controlThe unique scanner-sensor technology in the Leica AM TIRF MCallows automatic setting of the TIRF angle and the adjustment ofthe required TIRF penetration depth to the various wavelengths.Thanks to the automatic AOTF control, it is possible to switchquickly between the laser lines at a constant penetration depth.This explains the superior performance, reproducibility and ultrahigh speed of the Leica AM TIRF MC system, which has the addedbenefit of being easy to use.

Research of molecular interactions at cell membranes, of pro-teins and receptors involved in transport mechanisms, requiresfast switching of the excitation wavelength. With Leica Multi-Color TIRF, wavelengths are switched in a few ms without alter-ation of the penetration depth. The system’s fast control and highimage recording rate of up to 30 frames per second is also anadvantage for FRET analysis. Using the new FRET wizard, experi-ments can be conducted with ease and fully evaluated automati-cally. If alternating wavelengths are required in the widefield lightpath of the TIRF module, Leica’s fast filter wheels can be used.Switching times of approximately 30–50 ms are easily achievable.

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TIRF Membrane ResearchView dynamic structures and events

Tubulin CFP Widefield

Tubulin CFP TIRF

Gal3 YFP Widefield

Gal3 YFP TIRF

Courtesy of: Prof. Dr. R. Jacob,University Marburg, Department of ClinicalCytobiology and Cytopathology,Marburg, Germany

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With the Leica TIRF system it is easy to image single moleculesclose to the cell membrane. Typically, a powerful objective’sresolving power in the z axis is physically limited to approximately400–500 nm by the wavelength of the light used. This physicalrestriction is eliminated by the high accuracy of the TIRF excitationof ~ 100 nm in the z axis. Even interactions of single molecules, thekinetics or co-localization of molecules can be visualized andmeasured with Leica’s highly sensitive TIRF system. The constantpenetration depth when switching the laser lines is a key prereq-uisite for using multiple fluorophores. Due to the excellent signal-to-noise ratio, the entire dynamic range of this high-performancecamera can be exploited. Even with weak fluorescent signals, theEMCCD camera provides highest resolution images while ensur-ing the uncompromising sensitivity essential for the gentle treat-ment of light-sensitive specimens.

Due to strong background fluorescence, vesicle transport studiesrequire an optimum signal-to-noise ratio. At the same time, stabil-ity for experiments lasting several hours or days is a must. Vesicletransport experiments are made simple with the Leica AM TIRFMC. With the addition of Leica’s special climate chamber to theTIRF system, temperature and CO2 levels remain constant. TheLeica AF6000 multidimensional fluorescence workstation withintelligent application software and hardware provides accurateresults. For brilliant photographs of vesicle transport investiga-tions, a high-sensitive and high-speed EMCCD camera can beused.

TIRF Vesicle TransportHigh resolution and minimum light stress

TIRF Single MoleculeExplore the world of single molecules

“Timing is particularly importantfor the understanding of cellulartransport processes. Cells workquickly and precisely – just likeour Leica AM TIRF MC system.”

Prof. Dr. R. JacobUniversity Marburg, Department of ClinicalCytobiology and Cytopathology,Marburg, Germany

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Light path of the Leica TIRF module. The laser beam is posi-tioned in the objective with the aid of a scanner. The return-ing beam of the total reflection is measured on a special sensor.This feedback allows precise, fully automatic, and repro-ducible setting of the penetration depth of the generatedevanescent field.

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Automatic correlation determinationLeica’s auto-alignment function automatically finds the correla-tion between the penetration depths of the evanescent field andthe TIRF angles (to be set). An integrated TIRF sensor detects totalreflection and enables the user to easily travel to specific pene-tration depths. The penetration depth of the evanescent field alsodepends on the wavelength of the laser, in addition to the refrac-tive indices of the coverslip and specimen. The integrated refrac-tometer function automatically determines the refractive indicesof the specimens being examined. Intelligent mathematic algo-rithms precisely calculate the penetration depth of the TIRF fieldfrom the wavelength, the refractive index, and the incident angleof the laser beam. The selected contrast method is always TIRFand not a mixture of laser widefield excitation and TIRF. The usercan start examining a specimen at the smallest penetration depthof the evanescent field and go continuously deeper. Penetrationdepth can be selected according to the specimen structure underexamination and the orientation of the specimen. In this way thevisualized structures or processes are assigned to an exact posi-tion close to the cell membrane. TIRF gives many structural andfunctional insights into a cell.

All contrast methods and colors are a click awayLeica’s laser coupling technology allows both simultaneous andsequential use of TIRF, widefield fluorescence, and transmittedlight contrast methods: the user can activate epifluorescencewith a quick shutter mechanism to observe structures deeperthan the evanescent field. All contrast methods are readily avai-lable and can be combined as required (for example transmittedlight and fluorescence, or DIC and TIRF). If you use multiple fluo-rophores in your TIRF experiments, you can switch easily andquickly between the four laser lines, knowing that the penetrationdepth will automatically remain constant.

TIRF Auto-alignmentReliable contrast methods and MultiColor TIRF

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Leica AM TIRF MC at a glance:

• Integration of four wavelengths405 nm, 488 nm, 561 nm and 635 nmwith fast AOTF control

• Correction of the penetration depthwhen switching wavelength

• Combination of TIRF and fast FRETanalysis

• Highest transmission of all opticalTIRF components for maximum TIRFimaging speed and brilliance

• Fast image recording of up to30 fps with simultaneous changeof wavelength

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Control all functionsLeica’s powerful Leica AF6000 fluorescence software guides theoperation of the TIRF system. TIRF and microscope functions arecontrolled via the clearly designed interface. Leica AF6000 alsooffers a wide range of analysis tools and software modules forFRET, time lapse, multi-dimensional imaging, and deconvolution,making light work of complex applications.

Make it personalThere are a number of options that tailor the Leica TIRF system toindividual user needs.

• The TIRF module adapts to the Leica DMI 6000 B inverted,fully automated research microscope to deliver extremelydetailed and reproducible results. This advanced solution isideally suited to complex tasks in live cell microscopy.

• For multidimensional TIRF applications, the module can befully integrated into the high-performance Leica AF6000 LXfluorescence platform.

• For Multicolor TIRF applications the module can be equippedwith a four solid state laserbox (405, 488, 561, 635 nm)and AOTF control.

• Leica’s made-to-measure TIRF solution for micromanipulation,the TIRF climate box, is available with micromanipulators.

TIRF ScannerThe elegant solution for fluorescence microscopy

Top: Widefield fluorescence image of breastcarcinoma tumor cells expressing a GFP taggedcell adhesion Molecule CD44 that is expressedon the cell membrane.

Bottom: The same cells imaged in TIRF.

Courtesy of: Dr. Maria C. Montoya, CNIO,Spanish National Cancer Center, Madrid, Spain

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TIRF ObjectiveHigh performance imaging through superb optics

TIRF MultiColorFluorescence analysis in the entire color spectrum

Reliable, exact resultsLeica’s TIRF objective offers maximum apochromatic correction,an optimum signal-to-noise ratio and high performance imaging.The objective’s correction ring compensates for spherical aberra-tions caused by temperature from 23–37°C, as well as coverslipthicknesses of 0.12–0.22 mm. Leica’s TIRF objective meets all therequirements for consistent, high-quality imaging performanceand exact analysis in TIRF microscopy.

All colors automatically in focusAs a MultiColor TIRF system, the Leica AM TIRF MC offers wave-lengths in the entire color spectrum from ultraviolet to red. Fourlonglife and maintenance-free diode lasers in the wavelengths405 nm, 488 nm, 561 nm and 635 nm are integrated into the system.The automatic coupling and control via AOTF ensures extremelyshort switching times of 1 ms and adjusts the laser beam so thatthe penetration depths are not changed when the wavelength isswitched. Key practical advantages are the compact design ofthe overall system with its small footprint and the optimized safetyclass I laser unit with quiet air cooling. All four wavelengths areguided via one single multi-mode fiber.

Focus stability is the key to obtaining superior images with TIRF.Image quality is ensured when the distance between the objec-tive interface and the coverslip remains constant. To achieve this,Leica has developed a powerful Adaptive Focus Control and fullyintegrated it into the Leica AM TIRF MC system. The Leica Adap-tive Focus Control actively holds the focus over time – even underdemanding environmental conditions. The principle is based onthe surface reflection of the glass bottom of the Petri dish. Effec-tive for all contrasting methods, it can easily be combined with adigital autofocus. This combination enables you to keep cells infocus, even if they change their shape over time.

Easily accessible specimen and high laser safety

Leica AM TIRF MC with AdaptiveFocus Control:

• Reliable: holds focus positionactively over time

• Fast: corrects for focus shiftsin the blink of an eye

• Flexible: in combination with digitalautofocus, corrects the focus tocompensate for changes in cellularmorphology

TIRF AFCLong-term stability in TIRF with the Leica Adaptive Focus Control

“With the user, for the user”Leica Microsystems

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