Applications of Imaging Interferometry - SPIE Digital Library

Invited Paper

Applications of Imaging Interferometry Jason Reeda, Paul Wilkinsona, Keith O’Dohertya, Joanna Schmitb, Sen Hanb, Josh Trokec, Michael Teitellc , William Klugd, and James Gimzewskia. a Department of Chemistry and Biochemistry; California Nanosystems Institute, UCLA, b

Los Angeles CA 90095;

Veeco Metrology, 2650 E. Elvira Road, Tucson, AZ 85706; UCLA Department of Pathology and Laboratory Medicine; California Nanosystems Institute, UCLA, Los Angeles CA 90095; d Department of Mechanical and Aerospace Engineering, UCLA, Los Angeles CA 90095 c

ABSTRACT Here we report application of imaging interferometry to the study of nanomechanical motion in biosensors and living biological systems. Using strobed interferometric microscopy we are able to probe the dynamic behavior of individual (100 x 500 x 1 micron) cantilevers in an eight cantilever array over frequencies from 0 – 1 MHz. In a related approach, we have developed an interferometric method to measure cell-specific mechanical signals in real time. This yields real-time diagnostic information about cell structure, metabolism and movement, along with response to chemical and physical stimuli. Our new approach makes use of “nanomirrors” fixed to the cell membrane. These mirrors act as nanoscopic displacement probes and can be interrogated, rapidly, by optical profiling metrology. Keywords: Optical profiler, microcantilever, live cell imaging

1. MICRO CANTILEVER DYNAMICS Silicon micro cantilevers are used as transducers for a wide range of physical, chemical and biochemical stimuli, where the exhibit exquisite sensitivity (10-18 g, 10-15 J, 10-9 M, etc) over a wide range of temperatures (100 mK – 1300 K). [1-4] [5] [6, 7] [5, 8] This is accomplished by inducing static bending in the cantilever structure or by changing the cantilever’s resonant behavior, both easily measurable responses. There is increasing interest in using higher-order resonant modes to achieve extra sensitivity; however this raises the question of exactly which modes are excited in the cantilever. Using strobed interferometric microscopy we are able to probe the dynamic behavior of individual (100 x 500 x 1 micron) cantilevers in an eight cantilever array over frequencies from 0 – 1 MHz.

1.1 Microcantilever Arrays Eight silicon cantilevers are arranged in an array on a silicon base, each of which is 500 microns long by 100 microns wide and 0.9 microns thick (Fig. 1). The cantilever pitch is 250 microns, which was originally chosen to make optical communication arrays. These arrays were obtained from IBM Zurich Research Laboratories, and fabricated from a oriented wafer, so any horizontal face on the chip is orientation . The cantilevers are extend in the direction. The cantilever array was fixed to a cylindrical piezo actuator driven in the z-axis by a sinusoidal signal (0 – 50 v; 0-150 nm) at the observation frequencies (0 – 1 MHz). Interferometry XIII: Applications, edited by Erik L. Novak, Wolfgang Osten, Christophe Gorecki, Proc. of SPIE Vol. 6293, 629301, (2006) · 0277-786X/06/$15 · doi: 10.1117/12.683948

Proc. of SPIE Vol. 6293 629301-1

Fig. 1. An eight micro cantilever array shown to scale

1.2 Instrument The measurement of the static and dynamic modes of cantilevers was performed on the Veeco interference microscope DMEMS 1100 with a red LED used for stroboscopic illumination and 5X 0.13NA Michelson objective (Fig. 2). The DMEMS 1100 in principle is an optical microscope with a Michelson interference objective that allows for the observation of not only lateral features with typical optical resolution but also height dimensions below the scale of one nanometer [9]. The interference images are collected by a CCD camera during the scan of the interference objective through focus. Each pixel’s interference intensity from all collected images is used to determine the shape of the object.

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. Fig. 2. Diagram of the experimental set up showing the optical profiler and cantilever array


1. 3 Results The DMEMS system enabled spatial visualization of 16 cantilever modes with nanometer-scale amplitudes; combined with FEA calculations we directly assignd mode indicies (Fig. 3). We observed the reversal of specific modes between experiment and theory. We also observed small non-uniformity of individual cantilevers in the array (Fig. 4), and the clear relationship between boundary conditions and resonant behavior. Our conclusion is that the assignment of a resonant frequency spectrum is fairly complex and doesn’t necessarily follow simple intuition.

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Fig. 4. In-phase freeze frame interferometric images from the cantilever array driven at (A) 161 kHz, (B) 400 kHz and (C) 953 kHz, obtained by the Veeco Metrology DMEMS stroboscopic interference microscope.

2. NANOMIRRORS Atomic force microscopy studies by us and others indicates that cell-specific mechanical signals yield realtime diagnostic information about cell structure, metabolism and movement, along with response to chemical and physical stimuli.[10, 11] Using imaging interferometry, we have developed a higherthroughput experimental approach. Our new approach makes use of magnetic “nanomirrors” fixed to the cell membrane, in place of the AFM tip. These mirrors act as nanoscopic displacement probes and can be interrogated, rapidly, by optical profiling metrology. This approach enables parallel, simultaneous nanomechanical measurements of hundreds-to- thousands of individual cells.


2.1 Optical Profiler Modifications Imaging live cells required that the optical profiler be modified to accommodate the dispersive effects of the liquid cell media in the optical path. Schematically, this required replacement of the high magnification Mirau objective (20x) in the DMEMS 1100 optical profiler with a Michelson objective module and a separate illumination module (Fig. 5). The system is designed to optimize a conventional profiler’s performance when used to test a sample covered by a dispersive medium in the optical path of the test beam T. This required that a compensating, dispersive cell be placed in the reference path. Fig. 6 shows a full schematic of the modified system.

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Fig. 5. Modified Michelson objective shown with illumination source and dispersive compensating cell.

Fig. 6. Schematic representation of a Michelson type interferometric objective.

2.2 Nanomirror Fabrication White light Vertical Scanning Interferometry (VSI) has accuracy better than 1 nm in the z-axis [12]. We constructed nanomirrors to act as tiny retroreflectors on the cell membrane, which we would tracked with in real time with VSI. Fig. 7 shows an SEM image of a 10 micron diameter, 2 micron thick nanomirror.


Nanomirrors of this design were fabricated by e-beam vacuum depositing gold and nickel through a 20 micron polycarbonate shadow mask with mono-disperse, track-etched 10 micron holes.

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Fig. 7. SEM image of a 10 micron nickel-gold magnetic nanomirror

2.3 Tracking Nanomirror Motion We accurately measured the nanometer-scale motion of a prototype nanomirror using a soft gel substrate to simulate the cell surface (Fig. 8). We recorded nanomirror indentation into the gel of up to 40 nm, which varied linearly with magnetic field strength. The gel substrate has a Young’s modulus of approximately 1 MPa, therefore we estimate that we were able to generate approximately 1 nN of force on the nanomiror.

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Fig. 8. Testing force generation by nanomirrors on a gel substrate


2.4 Imaging Interferometry of Live Cells In addition to nanomechanical measurements with nanomirrors, we directly observed mammalian cells with the optical profiler, using no staining or other labeling technique. Imaging live cells required that we construct a special reference cell that matched exactly the dispersion of the liquid cell media, as discussed above. We also constructed a live cell perfusion chamber with a fixed viewing window so that the dimensions of the test path and the compensating cell were identical. This perfusion chamber functions much like a conventional Petri dish to grow and sustain the cell during observation. Fig. 9 shows a phase scanning interferometric image of live NIH 3T3 mouse fibroblast cells taken with the apparatus. The image shows considerable detail of the cell membrane, including intra-cellular organelles.

Fig. 9. Live NIH 3T3 mouse fibroblast cells observed by the optical profiler.

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