Near-Field Scanning Optical Microscopy
We have recently reported a new NSOM/AFM tip design that has the potential to circumvent many of the problems that currently plague the conventional fiber optic NSOM probes. The idea revolves around the use of focused ion beam (FIB) instrumentation to sculpt a small glass sphere that has been attached to the end of a conventional AFM cantilever. Once coated with aluminum, an aperture is cut into the end of the tip using an FIB to provide a well-defined aperture for light delivery. These tips offer several advantages over the conventional fiber optic approach that include: (1) a taper angle that can be precisely controlled thus maximizing the amount of light delivered, (2) greater flexibility in using high index materials, thus reducing the ultimate resolution possible, (3) a tip-sample feedback control that is identical to the well understood and developed AFM techniques, and (4) a much reduced spring constant for the tip which opens the possibility of imaging viable living cells.
Figure 1 illustrates the progression in AFM tip modifications necessary to implement the NSOM tip design. The milling and high resolution imaging shown in Fig. 1 was carried out with the use of a Micrion 9000 FIB. Figure 1(a) shows a typical silicon nitride AFM tip with a spring constant of approximately 0.12 N/m. Figure 1(b) shows the same AFM cantilever where the pyramid normally used as the stylus in AFM measurements has been removed with the FIB and a 5 mm hole has been cut through the end of the cantilever. Next, the hole is painted lightly with epoxy and a 7 mm, high index glass sphere (n = 1.9, MO-SCI Corporation) is placed in the hole using standard micromanipulation techniques. This is shown in Figs. 1(c) and 1(d).
As shown in Fig. 1(c), the glass sphere projects from both sides of the AFM cantilever which allows FIB modification of both the light input and output sides of the sphere. The glass sphere is cut with the FIB to form a pyramid structure similar in dimensions to the pyramid stylus utilized in AFM measurements. This is illustrated in Figs. 1(e) and 1(f). It is important to stress that the use of the FIB for tip fabrication allows complete control over both the shape of the tip (pyramid, cone, etc.) and the aspect ratio, which is important for efficient light delivery. Finally, the tips are evaporatively coated with approximately 100 nm of aluminum and an aperture is cut into the very end of the tip using the FIB. The top of the sphere (not shown) is also milled flat with the FIB to (1) increase the coupling efficiency for light into the tip, (2) remove the aluminum coating, and (3) increase the feedback signal.
Once fabricated, the tip is mounted into a modified Dimension AFM head (Digital Instruments). The normal diode laser used for tip feedback in the AFM head has been removed and replaced with an optical fiber (Newport) that delivers the desired excitation line from an argon ion laser (Liconix, 5000 series). The light coupled into the AFM head serves the dual purpose of excitation source for the NSOM tip and signal for the tip feedback loop. The dual use for the excitation light avoids many of the problems associated with eliminating stray feedback light in NSOM emission measurements.
Below shows near-field fluorescence and force measurements taken with the nanofabricated tip shown above. The test sample consisted of a thin film of 50 nm fluorescent latex spheres (Duke Scientific Corporation) embedded in a host acetate matrix. Small fluorescent features are clearly visible in Fig. 3(a) which map the locations of the fluorescent spheres embedded in the film. These are not correlated with structural features in the force image. The full-width-half-maximum of the smallest fluorescent features is approximately 130 nm which represents the convolution between the actual tip aperture size and the size of the fluorescent spheres (50 nm). This resolution is comparable to that reported using conventional fiber optic NSOM probes and demonstrates the feasibility of using these tips for high resolution fluorescence measurements. Currently, the resolution is limited only by our ability to fabricate the small aperture in the end of the tip with the FIB, which we are currently working to improve.
Kapkiai, L. K., Moore-Nichols, D. Carnell, J., and Dunn, R. C. Hybrid near-field scanning optical microscopy tips for live cell measurements. Appl. Phys. Lett. 84, 3750 (2004).
Krogmeier, J.R. and Dunn, R. C., "Focused Ion Beam Modification of Atomic Force Microscopy Tips for Near-Field Scanning Optical Microscopy", Appl. Phys. Lett., 79, 4494 (2001).
Fluorescence resonance energy transfer (FRET) proves an increasingly important technique for measuring proximity relations in proteins, nucleic acids, and membranes. The method takes advantage of the strong distance dependence of non-radiative energy transfer from an excited donor molecule to an unexcited acceptor molecule. This dipole-dipole interaction, first described in1948, has an inverse sixth power dependence on the intermolecular separation making it sensitive to angstrom scale distance variations.
We reported a new technique that combines FRET measurements with near-field scanning optical microscopy (NSOM). NSOM is an imaging technique that provides subwavelength optical resolution by scanning a small light source within nanometers of a sample surface. The light source is usually fashioned from a single mode optical fiber that is heated and drawn to a fine point and then coated with aluminum around the sides to confine the light. By positioning the probe within nanometers of the sample surface, optical measurements can be made with 50 to 100 nm spatial resolution.
We have demonstrated a variation on the NSOM technique that incorporates the FRET mechanism to increase imaging capabilities. The idea is shown schematically below. In this technique, an acceptor dye is attached to an NSOM probe that has not been coated with a metal. Light exiting the NSOM probe (blue arrow) is resonant with the donor dye in the sample but not the acceptor dye attached to the tip. As the NSOM tip approaches the sample surface, energy transfer can occur between the excited donor dye in the sample and the acceptor dye bound to the tip (dark green arrow). This coupling gives rise to a red-shifted fluorescence (red arrow) which can be selectively monitored using filters to block the donor dye emission (light green arrows).
There are several advantages in using the FRET/NSOM configuration above. The novel coupling of microscopic techniques makes it possible to optically probe only those structures located closest to the NSOM tip. The strong distance dependence of FRET effectively reduces the interaction region between the tip and sample to that of closest approach. Therefore, NSOM probes do not have to be coated with an opaque metal such as aluminum to obtain sub-diffraction limit spatial resolution. This increased z sensitivity may also lead to new ways of non-invasively monitoring small height changes in reduced sample regions. Moreover, because the technique does not require the formation of a small light source, it can be extended to other non-optical probes such as AFM tips. This may result in a new generation of probes capable of much higher spatial resolution in fluorescence imaging than that currently attainable with conventional fiber optic NSOM probes. Finally, since the acceptor dye attached to the tip is non-resonant with the excitation light exiting the tip, photobleaching is dramatically reduced leading to an increased probe lifetime.
To investigate the feasibility of FRET/NSOM, LB coated NSOM probes were used to fluorescently image the multi-layer films of DPPC/fluorescein and arachidic acid. Two experimental conditions are compared to investigate the energy transfer to the NSOM probe and the resulting effect on imaging capabilities. The left image shown below is a 50 µm x 50 µm NSOM fluorescence image at 548 nm of the multi-layer film following excitation at 458 nm. This emission wavelength corresponds to the donor emission and thus this image contains contributions from both the top and bottom layers of the multilayer film. However, when the emission from the tip attached acceptor dye is monitored (right image), some features become much less intense and some become brighter. Both observations support that energy transfer is occurring from the uppermost monolayer of the film to the tip. By comparing the two images taken on the same region of the film, one can assign which layer of the film each bright domain resides. This represents an optical sectioning power on the order of nanometers.
The goals of combining the r-6 distance dependence of FRET with the NSOM technique include the following: (1) a reduction in the probe volume in NSOM for applications on thick samples such as cells, (2) an increase in the z sensitivity for single point dynamic measurements, (3) a simplification of NSOM probe fabrication by eliminating the need for the metal coating, and (4) the introduction of a scheme that can be extended to non-waveguide type probes such as atomic force microscopy (AFM) tips.
Vickery, S. A. and Dunn, R. C., “Combining AFM and FRET for High Resolution Fluorescence Microscopy”, J. Microscopy, 202, 408-412 (2001).
Vickery, S. A. and Dunn, R. C., “Scanning Near-Field Fluorescence Resonance Energy Transfer Microscopy”, Biophys. J., 76, 1812-1818 (1999).