UConn nmLabs: Coupled AFM and Optics

The NanoMeasurement Labs now features a combined AFM/3d-Optics system. An Asylum Research MFP-3d AFM, with a variable temperature fluid cell, allows systems to be probed from above with nanoscale precision and all of the functionality expected of an atomic force microscope. But, this system is integrated on top of an inverted optical microscope (Nikon TE-2000), providing simultaneous AFM and bright field, dark field, phase, or fluorescence imaging. In addition to standard 10x, 40x, and 100x air objectives (0.95 NA), the scope is configured with a 60x oil objective (1.45 NA) for ultimate resolution studies. A broadband (UV-vis) illumination source with rapid switching between 4 independent filters allows simple multi-color excitation. A high speed micro-shutter allows photobleaching studies. And, a dual-view detection module allows simultaneous multi-color detection, ideal for FRET measurements. Images are acquired by a low light, high speed, on chip amplification (EMCCD) Hamamatsu camera. Finally, 3d imaging is achievable with a PI piezodriven objective mount for rapid and precise z-focus adjustments. Acquisition of individual images, in addition to 3-d volumes as a function of time, wavelength, or z-stacks (or combinations for 4d or 5d imaging), are controlled, analyzed, visualized, and quantified by Improvision and Autoquant software. The entire system is run by a high speed, high RAM, dual core-duo Pentium based PC computer with a top of the line graphics card and a direct-to-hard-drive Raid0 configuration for continuous high speed image acquisition and analysis.

Multi-color imaging

The following figure displays four consecutive images of microspheres with varying fluorescent profiles, excited and detected with distinct filter-sets as follows:

Top left: Transmission (the triangular dark shape at left is the shadow from the AFM probe).

Top right: Excitation 528-552 nm, emission 578-632 nm

Base left: Excitation 465-495, emission 515-555

Base right: Excitation 465-495, emission 500

High speed imaging

A movie of 30 ms/frame images (100x air) displays an agglomeration of Iron particles coated with a fluorescent dye (4 um), floating in water. Over time, they can be seen to spin and dance due to manipulation by an external magnetic field (waving a nearby magnet by hand).

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Z-stacks and Deconvolution

A series of 134 z-slices, spaced 200 nm apart over a range of 26 um, reveals fluorescent F-actin filaments (100x air). 2 data sets are included: first, the raw data; second, the z-stack after a deconvolution routine. Significant improvement of the resolution is clear.

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3-d Optical Reconstruction

The same F-actin filaments are displayed in a 3-d format.

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AFM and Optics

AFM images of isolated filaments and bundles (right and left, respectively) reveal filament diameters on the order of 20 nm.

3-d living mammalian cells

A HaCaT cell (courtesy B. Aneskievich) reconstructed from a deconvolved z-series.

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Current Projects

Leveraging this unique combination of optical and AFM equipment, the mechanical properties of living cells are being measured by nanoindentation in the AFM and simultaneously their optically resolved shape and response is being monitored. This includes efforts to characterize cell responses (mechanical and optical) to specific forces as well as to solution and direct tip-delivery of various biochemicals (i.e. drugs).