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

Cell monolayers have serious limitations for cell biological investigations and for cell-based assays in drug screening and toxicity studies. However, the establishment of three-dimensional cultures as a mainstream approach requires the development of reliable protocols, new cell lines and suitable imaging techniques.

The third dimension bridges the gap between cell culture and live tissue

Large, living biological specimens present challenges to existing optical imaging techniques because of their absorptive and scattering properties. We developed selective plane illumination microscopy (SPIM) to generate multidimensional images of samples up to a few millimeters in size. The system combines two-dimensional illumination with orthogonal camera-based detection to achieve high-resolution, optically sectioned imaging throughout the sample, with minimal photodamage and at speeds capable of capturing transient biological phenomena. We used SPIM to visualize all muscles in vivo in the transgenic Medaka line Arnie, which expresses green fluorescent protein in muscle tissue. We also demonstrate that SPIM can be applied to visualize the embryogenesis of the relatively opaque Drosophila melanogaster in vivo.

https://science.sciencemag.org/content/sci/305/5686/1007.full.pdf

Optical sectioning deep inside live embryos by selective plane illumination microscopy

A long-standing goal of biology is to map the behavior of all cells during vertebrate embryogenesis. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 hours of development. Multiview in vivo imaging at 1.5 billion voxels per minute provides “digital embryos,” that is, comprehensive databases of cell positions, divisions, and migratory tracks. Our analysis of global cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo's cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.

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Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.

Summary Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three-dimensional fluorescence imaging based on non-resonant two-photon or three-photon fluorophor excitation requires light intensities in the range of MW cm 22 to GW cm 22 , which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi-gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non-invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depthresolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non-invasive fluorophore loading into single living plant cells. Non-destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis-like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two-photon excitation process rather than a onephoton or three-photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two-photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid-induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm 22 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non-invasive NIR laser surgery within a living cell or within an organelle is therefore possible.

Multiphoton microscopy in life sciences

Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy

We show the point-spread function of a confocal microscope with an increased detection aperture. The increase in aperture is accomplished by coherent collection of the light from the specimen with two opposing objective lenses, i.e., type-B 4Pi confocal microscopy. We demonstrate experimentally its feasibility for detecting scattered or fluorescently emitted light. The 4Pi confocal point-spread functions are shown for constructive and destructive interference of the collected wave fronts and are compared with the point-spread functions of comparable confocal microscopes.

Confocal microscopy with an increased detection aperture: type-B 4Pi confocal microscopy.

In a 4Pi‐confocal microscope the specimen is illuminated and observed coherently from above and below such that the numerical aperture is increased [S. W. Hell, European Patent Application 91121368.4 (filed 1990, published 1992), S. W. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992)]. The point spread functions of 4Pi‐confocal and confocal microscopes were measured. Our measurements prove a three‐ to seven‐fold increase of axial resolution, thus opening the prospect for a powerful three‐dimensional imaging technique with an axial resolution down to 75 nm.

Measurement of the 4Pi‐confocal point spread function proves 75 nm axial resolution

Nonlinear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the illumination volume

We demonstrate theoretically and experimentally a fourfold increase in axial point resolution in far-field light microscopy. The resolution enhancement is achieved by coherently illuminating the specimen with two opposing objective lenses (4Pi confocal microscopy) and applying two-photon excitation. The point spread function and the axial resolution of this set-up are calculated in optical units. The axial resolution is measured and compared with predictions, both for the confocal and for the 4Pi confocal set-up, in single as well as in two-photon excitation mode.

Steffen Lindek

Papers

Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy

Confocal microscopy with an increased detection aperture: type-B 4Pi confocal microscopy.

Measurement of the 4Pi‐confocal point spread function proves 75 nm axial resolution

Nonlinear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the illumination volume

Enhancing the Axial Resolution in Far-field Light Microscopy: Two-photon 4Pi Confocal Fluorescence Microscopy