We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

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Imaging intracellular fluorescent proteins at nanometer resolution.

1. Introduction 2. Theoretical foundations 3. Propagation and focusing of optical fields 4. Spatial resolution and position accuracy 5. Nanoscale optical microscopy 6. Near-field optical probes 7. Probe-sample distance control 8. Light emission and optical interaction in nanoscale environments 9. Quantum emitters 10. Dipole emission near planar interfaces 11. Photonic crystals and resonators 12. Surface plasmons 13. Forces in confined fields 14. Fluctuation-induced phenomena 15. Theoretical methods in nano-optics Appendices Index.

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Principles of nano-optics

Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.

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Nonlinear magic: multiphoton microscopy in the biosciences

In 1873, Ernst Abbe discovered what was to become a well-known paradigm: the inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. However, for its most popular imaging mode, fluorescence microscopy, the diffraction barrier is crumbling. Here, I discuss the physical concepts that have pushed fluorescence microscopy to the nanoscale, once the prerogative of electron and scanning probe microscopes. Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.

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Far-Field Optical Nanoscopy

A multiphoton microscopy based on coherent anti-Stokes Raman scattering is accomplished with near-infrared ultrashort laser pulses. We demonstrate vibrational imaging of chemical and biological samples with high sensitivity, high spatial resolution, noninvasiveness, and three-dimensional sectioning capability. {copyright} {ital 1999} {ital The American Physical Society}

Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering

It is demonstrated that third-harmonic generation (THG) near interfaces in the refractive index or the third-order nonlinear susceptibility (chi(3)) permits three-dimensional imaging of transparent objects. The nonlinear dependence of THG on the excitation power provides inherent optical sectioning. At the same time, the nonresonant nature of THG, in combination with the near-IR excitation wavelengths used (1-2 µm), render this technique potentially (biologically) nondamaging and nonbleaching. A specific property of THG imaging is its sensitivity to - and potential use for imaging of - the relative orientation of interfaces with respect to the axis of propagation of the excitation radiation.

3d microscopy of transparent objects using third-harmonic generation

The excitation efficiency in two-photon absorption (TPA) microscopy depends strongly — owing to the square dependence of the TPA fluorescence on the excitation intensity — on the temporal width of the excitation pulse. Because of their inherently large frequency bandwidth, ultrashort optical pulses tend to broaden substantially because of dispersion from propagation through the dispersive elements in the microscope. In this paper, the dispersion characteristics of a wide range of microscope objectives are investigated. It is shown that the induced dispersion can be pre-compensated in all cases for pulses as short as 15 fs. Because of the excellent agreement between the results from theoretical modelling and the experimental data, predictions of the possibility of dispersion control for microscope objectives in general, as well as for even shorter pulses, can be inferred. Since for TPA imaging the background due to single photon absorption processes and scattering is independent of the pulse width, proper dispersion pre-compensation — which minimizes the pulse duration at the focal point and hence maximizes the excitation efficiency — provides optimal image contrast in TPA microscopy.

Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives

We demonstrate a method for the measurement of femtosecond optical pulses in the focal point of a high-NA lens, using interferometric autocorrelation through two-photon absorption. A chirp-free input pulse of 47 fs is found to broaden by ≈50% after focusing by a well-compensated objective. With proper prechirp compensation, the actual pulse width in the focus of such a lens system can be restored to (almost) its initial value. The unique value of the presented two-photon autocorrelation technique is its capability of measuring the actual pulse width at the focal point of a high-NA lens, an aspect that is of direct importance to two-photon imaging approaches, for example.

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Measurement of femtosecond pulses in the focal point of a high-numerical-aperture lens by two-photon absorption

Far-field fluorescence microscopy beyond the diffraction limit

The fundamental features of third-harmonic generation microscopy are examined both theoretically and experimentally, and the technique is applied to the characterization of layered structures. Measurements and model calculations have been performed of the third-harmonic yield generated from homogeneous layers. Model calculations based on the paraxial approximation show good agreement with the experimental results, despite the conditions of high numerical aperture. The method proposed here allows for the determination of (i) the layer’s third-order susceptibility relative to that of the substrate, (ii) its index of refraction at the third-harmonic frequency relative to that at the fundamental frequency, and (iii) its thickness and for the identification of a gradient region between two adjacent layers.

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G. J. Brakenhoff

Papers

3d microscopy of transparent objects using third-harmonic generation

Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives

Measurement of femtosecond pulses in the focal point of a high-numerical-aperture lens by two-photon absorption

Far-field fluorescence microscopy beyond the diffraction limit

Determination of material properties by use of third-harmonic generation microscopy.