Hybrid near-field scanning optical microscopy tips for live cell measurements

Abstract

We report a near-field scanning optical microscopy ͑NSOM͒ probe that enables high-resolution imaging of living cells under physiological buffered conditions. The hybrid design combines a conventional fiber optic near-field probe with a standard atomic force microscopy cantilever. Imaging of fluorescent latex spheres suspended in an acetate matrix demonstrates the subdiffraction limited fluorescence and topography capabilities of the tips. The reduced spring constant of the hybrid tip is also shown to be amenable to measurements on living cells. Near-field scanning optical microscopy ͑NSOM͒ is a scanning probe technique that utilizes specially fabricated probes to deliver light down to the nanometric dimension.1,2 NSOM provides opportunities to simultaneously measure both optical and topographic features of samples with subdiffraction limited spatial resolution. These capabilities have been utilized to probe thin films, solid-state devices, and due to the unique electromagnetic fields emerging from the nearfield aperture, single molecule orientations.2 The potential impact of NSOM is arguably the greatest in the biological sciences, where there is a well-developed history of using fluorescence probes to tag specific proteins or structures. This field seems particularly well suited to take advantage of the single molecule fluorescence sensitivity, high spatial resolution, and simultaneous force information that NSOM offers. However, current applications of NSOM to biological samples are largely limited to isolated protein samples, model membranes, or chemically fixed biological cells. 2-5The extension to viable, unfixed biological tissues has previously proven problematic.The difficulty in conducting NSOM measurements on viable biological tissues arises from the forces generated in maintaining the NSOM tip close to the specimen. Highresolution NSOM measurements require that the tip be held within nanometers of the sample while scanning. This necessitates the implementation of a feedback system for sensing the sample surface and maintaining the tip-sample gap. [6][7][8] Traditionally, a force feedback approach is implemented in which the tip is either dithered laterally or vertically to the sample surface, depending on probe geometry. The dampening in the amplitude of the oscillating NSOM tip as the tip interacts with the sample surface is monitored and used to hold the tip-sample gap constant during scanning. While straightforward and highly successful for most applications, the large forces generated using conventional fiber optic NSOM probes often damage fragile biological samples such as living cells.A number of approaches have been reported in attempts to circumvent the large forces generated under normal NSOM feedback operation and thus open applications in the biological sciences. These have largely involved either changing the feedback mechanism utilized to hold the tip close to the sample or modifying the probes themselves to lower the spring constant and thus the forces generated in force feedback. 2,5,9 ...

NSOM/QD-based nanoscale immunofluorescence imaging of antigen-specific T-cell receptor responses during an in vivo clonal Vγ2Vδ2 T-cell expansion

“…8,9,[12][13][14][15]35,36 However, it should be pointed out that, although another type of NSOM, apertureless NSOM (ANSOM) or NSOM with pointed probes, 10,11 has overcome the limitation of aperture probes of aperture NSOM and reached a great spatial resolution, 37,38 ANSOM has not been successful for such nanoscale resolution imaging of cell membrane proteins because of AN-SOM's high-background signal and photobleaching of dyes caused by the metal tip of ANSOM. 11,38 Because all commercially available NSOM instruments use the aperture NSOM technique, the combined aperture NSOM and QD may provide a powerful tool for high-resolution imaging of TCR and other membrane proteins.…”

“…[8][9][10][11] Complicated natures of cell membranes or biologic molecules make it difficult for NSOM to generate high spatial resolution images. Whereas home-made NSOM operating in liquid can yield images of biologic molecules, the current commercial NSOM instruments are all designed for in-air imaging, [12][13][14][15] posing a challenge for nanoscale imaging of cell membrane proteins. Although NSOM combined with some common fluorescent materials were used for imaging, 16 the absence of highly photostable fluorophores for use in NSOM is perhaps one of the major reasons why NSOM has not been reproducibly used for nanoscale imaging of functional cellular molecules.…”

Tip‐enhanced optical spectroscopy for surface analysis in biosciences

“…Recent developments such as novel hybrid probes that combine commercial cantilevers and optical fibers and novel modes for tuning-fork operation significantly improve the applicability of aperture-based SNOM. 5,47 For tip-enhanced spectroscopy, the same problems arise in principle. However, since solid metal tips or particles can be far smaller, highly sensitive feedback schemes are more readily developed.…”

Near-Field Scanning Optical Microscopy: A New Tool for Exploring Structure and Function in Biology

“…A similar probe was fabricated using fiber-optic NSOM probes incorporated into silicon nitride AFM cantilevers [65]. Again with the use of FIB technology, a small hole is drilled into the end of an AFM probe.…”