Achim K. Kirsch

Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation

We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50-200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.

PICOSECOND MULTIPHOTON SCANNING NEAR-FIELD OPTICAL MICROSCOPY

We have implemented simultaneous picosecond pulsed two- and three-photon excitation of near-UV and visible absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 1064-nm emission from a pulsed Nd:YVO4 laser was used to excite the visible mitochondrial specific dye MitoTracker Orange CM-H2TMRos or a Cy3-labeled antibody by two-photon excitation, and the UV absorbing DNA dyes DAPI and the bisbenzimidazole BBI-342 by three-photon excitation, in a shared aperture SNOM using uncoated fiber tips. Both organelles in human breast adenocarcinoma cells (MCF 7) and specific protein bands on polytene chromosomes of Drosophila melanogaster doubly labeled with a UV and visible dye were readily imaged without photodamage to the specimens. The fluorescence intensities showed the expected nonlinear dependence on the excitation power over the range of 5-40 mW. An analysis of the dependence of fluorescence intensity on the tip-sample displacement normal to the sample surface revealed a higher-order function for the two-photon excitation compared to the one-photon mode. In addition, the sample photobleaching patterns corresponding to one- and two-photon modes revealed a greater lateral confinement of the excitation in the two-photon case. Thus, as in optical microscopy, two-photon excitation in SNOM is confined to a smaller volume.

Continuous Wave Two-Photon Scanning Near-Field Optical Microscopy

We have implemented continuous-wave two-photon excitation of near-UV absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 647-nm emission of an Ar-Kr mixed gas laser was used to excite the UV-absorbing DNA dyes DAPI, the bisbenzimidazole Hoechst 33342, and ethidium bromide in a shared aperture SNOM with uncoated fiber tips. Polytene chromosomes of Drosophila melanogaster and the nuclei of 3T3 Balb/c cells labeled with these dyes were readily imaged. The fluorescence intensity showed the expected nonlinear (second order) dependence on the excitation power in the range of 8-180 mW. We measured the fluorescence intensity as a function of the tip-sample displacement in the direction normal to the sample surface in the single- and two-photon excitation modes (SPE, TPE). The fluorescence intensity decayed faster in TPE than in SPE.

Fluorescence resonance energy transfer detected by scanning near‐field optical microscopy

Fluorescence resonance energy transfer (FRET) between excited fluorescent donor and acceptor molecules occurs via the Förster mechanism over a range of 1–10 nm. Because of the strong (sixth power) distance dependence of the signal, FRET has been used to assess the proximity of molecules in biological systems. We used a scanning near‐field optical microscope (SNOM) operated in the shared‐aperture mode using uncoated glass fibre tips to detect FRET between dye molecules embedded in polyvinyl alcohol films and bound to cell surfaces. FRET was detected by selective photobleaching of donor and acceptor fluorophores. We also present preliminary results on pixel‐by‐pixel energy transfer efficiency measurements using SNOM.

Shear force imaging of DNA in a near-field scanning optical microscope (NSOM)

A near‐field scanning optical module has been constructed as an accessory for a Nanoscope IIIa commercial scanning probe microscope. Distance feedback and topographic registration are accomplished with an uncoated optical fibre scanning tip by implementation of the shear force technique. The tip is driven by a piezoelectric actuator at a resonance frequency of 8–80 kHz. A laser diode beam is scattered by the tip and detected by a split photodiode, with lock‐in detection of the difference signal. The amplitude (r) and phase (τ) responses were characterized as a function of the calibrated tip–sample separation. Using an r cos τ feedback signal, imaging of pUC18 relaxed circular plasmid DNA spread on mica precoated with cetylpyridinium chloride was achieved. The apparent width (28 ± 5 nm) was approximately four times that achieved by scanning force measurements with the same instrument; the apparent height of the DNA (0.6 ± 0.3 nm) was similar with the two techniques. These results demonstrate the applicability of the shear force signal for imaging biological macromolecules according to topography and in conjunction with the optical signals of a near‐field scanning optical microscope (NSOM).

Interferometric measurement of fluorescence excitation spectra

A Michelson interferometer has been adapted as an excitation source for fluorescence spectroscopy. A moving fringe pattern was generated by linear displacement of the movable mirror of the Michelson interferometer coupled to a xenon-arc lamp. This spectrally modulated source was monitored by a reference photomultiplier and used for exciting a Rhodamine B solution. The fluorescence emission at >645 nm was detected by a second photomultiplier. The two interferograms were acquired by a dual-channel digital oscilloscope, and their discrete Fourier transforms and corresponding power spectra were generated in a computer. The power spectrum of the emission signal represented the excitation spectrum, as was confirmed by comparison with the absorption spectrum of Rhodamine B. Thisoptical arrangement is well suited for acquiring fluorescence excitation spectra in the optical microscopy of biological specimens.

Fluorescence SNOM of domain structures of LB films containing electron transfer systems

The morphology of a mixed monolayer consisting of a metal complex and stearic acid on mica was investigated by SNOM. In the topographic shear-force image, round domains of several micrometers diameter protruded several A from the monolayer surface. The fluorescence pattern revealed by SNOM (excitation at 488 nm, emission at 590 nm) matched the monolayer topography, and a higher fluorescence intensity was detected from the interdomain space. The photobleaching kinetics and integrated fluorescence of the different phases of the monolayer film were analyzed, yielding estimates of relative quantum yields and surface densities of the dyad.

Scanning near-field optical microscopy and microspectroscopy of green fluorescent protein in intact Escherichia coli bacteria

Scanning near-field optical microscopy (SNOM) yields high-resolution topographic and optical information and constitutes an important new technique for visualizing biological systems. By coupling a spectrograph to a near-field microscope, we have been able to perform microspectroscopic measurements with a spatial resolution greatly exceeding that of the conventional optical microscope. Here we present SNOM images of Escherichia coli bacteria expressing a mutant green fluorescent protein (GFP), an important reporter molecule in cell, developmental, and molecular biology. Near-field emission spectra confirm that the fluorescence detected by SNOM arises from bacterially expressed GFP molecules.

Integration of Optical Techniques in Scanning Probe Microscopes

During the last 15 years scanning probe microscopes (SPM), in particular the scanning force microscope (SFM) and scanning tunneling microscope (STM), have developed into highly sophisticated instruments for the investigation of surfaces and surface structures under a broad range of environmental conditions. Applications range from imaging single atoms in ultrahigh vacuum 1, to the visualization of strands of DNA on freshly cleaved mica surfaces2, and the investigation of the activation of platelets under physiological conditions 3, 4.