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Mach-ZehnderInterference Microscopy
Dual Beam Interference Microscopy
The measurement of the refractive index of microscopic objects conveys a multitude of important material-specific and structural information. In the context of material science, for example, the evaluation of boththe refractive index and the dispersion relation enable the qualification ofmicro and crystal optical components.
A core issue regarding the observationof thin, transparent objects through aconventional light microscope is posedby their low image contrast. An inter-ference microscope applied to live cellimaging resolves this issue without anyreactive agents or dyes: This principleconverts a cell’s refractive index intocontrast, yielding a simple method toquantify cell properties by optical meansusing visible light. Such measurementsallow, for example, the evaluation of a cell’s dry mass. In summary, theapproach opens up many novel appli-cations in biology as well as medicaldiagnostics.
To obtain the interference contrast of a phase object, light passes throughthe object. The contrast originatesfrom the extent to which the objectshifts the phase of the transmittedlight. The superposition of the resultingobject wave with a coherent referencewave visualizes this phase contrast.This highly sensitive process demands a complete separation of the objectand reference wave’s beams as well as an exact wave optical tuning of the optical components. With perfectbeam alignment, white light illuminationcreates an interferogram, resulting incharacteristic interference colors whichdirectly correspond to the object’s re-fractive index.
Mach-Zehnder Interferometry
A key design concept for the dualbeam interference microscope stemsfrom the Mach Zehnder optical setupof mirrors and beam splitter. This setupfor the observation and measurementof transmitted light places the desiredobject in one of the interferometer’sarms. A challenge is posed by the align-ment of the two independent light beams as compared the Michelson in-terferometer setup, which can functionsolely with a single set of high-techoptical components. The dual natureof the Mach Zehnder interferometersetup demands the highest precision in both the selection of the twin opticalcomponents and the tuning of thebeam paths. Modern optical construc-tion and automation concepts inheritedfrom state-of-the-art laser technologysatisfy these higher demands at relativelylow cost and thus guarantee both highprecision and simple calibration.
Measurement of the Refractive Index
All phase objects from biological sampleslike cells to transparent materials likeglass or plastic create contrast followthe same principle: the material’s thick-ness and its refractive index correspondto an optical »thickness«. Dependingon this thickness, the object wave isshifted with respect to the referencewave. The interference of these twolight waves in the microscope amplifiesthis shift. An object’s local difference inrefractive index and/or thickness leadsto a distortion in light pattern of theinterferogram, which appears as a colorshift as seen, for example, on an opticalfiber in the image on the left, below.Note, that the color as well as its shiftdo not result from image processing,but are a direct output of the interfero-meter. Here, a phase shifting algorithmcan represent, for example, the spatialdistribution of the refractive index acrossthe picture from a series of images.
Mach-Zehnder Interference Microscopy
Above: Dual Beam Interferencemicroscope manufactured by Leitz.Middle: Diatom viewed under interference contrast. In the homo-geneous field the object’s refractiveindex creates a characteristic inter-ference color pattern. Below: The refractive index profile of optic fibers can be evaluated withhighest precision.
Stain-free Life Cell Imaging
The advancement of modern biotech-nology, genetics and pharmaceuticalresearch largely depends on the micros-copic investigation of living tissue. Toobtain the necessary contrast in lightmicroscopy nowadays, a diverse andcomplex toolbox of chemical methodsexists to stain specific cell components.These cell compartments are selectivelylinked to dye molecules for the obser-vation with fluorescence excitation.However, all staining procedures moreor less interfere with the cell’s meta-bolism: These invasive processes canlead to unwanted effects ranging fromdiffering gene expression patterns overchanges in protein kinetics to phototoxicity: the light induced disruption ofcell metabolism. Interference contrastmicroscopy, on the other hand, doesnot need to add any compounds to visualize cells, thus allowing for undis-turbed long-term observations of cellphysiology.
The refractive index can measure thedensity distribution within the cell andbetween cell populations. Like this, theexpression of membrane proteins andchanges in membrane potential can beobserved without the application of dyes.
Material Science and Analysis
Interference microscopy also offers thepossibility to measure the refractive in-dex distribution in thin and transparentmaterials like glass, plastics and liquids.The refractive index and the dispersionrelation depend on material specificproperties such as, for example: • Mass density• Concentration of heavy ions• Size and orientation of molecules • Coordination of chemical bonds
In the context of quality management,high-throughput assays could scan forinhomogeneities in glass or for the ag-gregation of crystallites in transparentplastic films in real time. A potentialapplication could also be the charac-terization and measurement of opticalaberrations in microoptics.
Developmental Goals
A cooperation with the department for Technical Optical Systems TOS of theRWTH Aachen University aims at theconstruction of a Mach Zehnder inter-ference microscope, which automati-cally aligns to a specific measurementconfiguration, thus enabling an auto-mation of the measurement process itself. For applications in the context ofcell biology, this system will be coupledwith an upright fluorescence illumina-tion. For special applications in materialscience the system could be enhancedby the ability to measure polarizationcontrast.
Contact
Prof. Dr. P. LoosenPhone +49 241 [email protected]
Dipl.-Phys. D. M. MahlmannPhone +49 241 [email protected]
Applications of Interference Microscopy
Above: Mach-Zehnder inter-ferometer on an optical bench.Middle: Barium bromatecrystals during the growth in a interference contrast. Below: Comparison of abright-field with an interfe-rence contrast image of aneuron. Note, that only theinterference image highlightsthe thinnest membrane seg-ments in the periphery.Very below: A phase shiftingalgorithm can register chan-ges in the density of the cellmembrane of e.g. HEK 293cells with high spatial resolu-tion.
50 µm
Bright Field Interference Contrast
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Subject to alterations in specifications and other technical information.04/2008.
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