With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional⎯including confocal⎯fluorescence microscopy. Nonlinear optical microscopy, in particular two photon–excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.

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Deep tissue two-photon microscopy

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

Recent startling interest for lanthanide luminescence is stimulated by the continuously expanding need for luminescent materials meeting the stringent requirements of telecommunication, lighting, electroluminescent devices, (bio-)analytical sensors and bio-imaging set-ups. This critical review describes the latest developments in (i) the sensitization of near-infrared luminescence, (ii) “soft” luminescent materials (liquid crystals, ionic liquids, ionogels), (iii) electroluminescent materials for organic light emitting diodes, with emphasis on white light generation, and (iv) applications in luminescent bio-sensing and bio-imaging based on time-resolved detection and multiphoton excitation (500 references).

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Lanthanide luminescence for functional materials and bio-sciences

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

Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.

https://materias.df.uba.ar/TemasNano2013/files/2013/07/1990_Denk_Science_Two-photon-fluo-microscopy.pdf

Two-Photon Laser Scanning Fluorescence Microscopy

What is to our knowledge the first high-density (>1012 bits/cm3) optical recording of digital information in a multilayered, three-dimensional format is reported. Information is written as submicrometer volume elements of increased refractive index in a photopolymer by two-photon excitation of a photoinitiator at the waist of a highly focused beam from a colliding-pulse mode-locked laser. Quadratic dependence of two-photon excitation on intensity confines polymerization to the focal volume. Information is read with sufficient axial resolution by differential interference contrast microscopy. This write-once, read-many technique should increase the capacity of the spinning disk format by 100-fold.

Three-dimensional optical data storage in refractive media by two-photon point excitation.

Two-photon excitation in laser scanning photolithography allows exposure of patterns not possible with conventional one-photon direct writing. In our experiments two red photons from a highly focused subpicosecond colliding pulse mode-locked dye laser are simultaneously absorbed by initiator molecules to affect a photochemical reaction that is normally driven by single-photon absorption using ultraviolet light. The quadratic dependence of the two-photon absorption rate on the incident intensity confines excitation to a submicron focal volume. By scanning this volume in a 3-d pattern through a thick layer of photoresist it is possible to expose arbitrary three dimensionally defined regions. Preliminary results showing half micron wide trenches of very high aspect ratio, and resist structures with undercutting edges, all produced with only a single development step, demonstrate. the potential utility of two-photon excitation in microfabrication.

Two-photon lithography for microelectronic application

Simultaneous absorption of two red photons from a strongly focused subpicosecond colliding pulse mode4ocked dye laser stimulates visible fluorescence emission from fluorophores having their normal absorption in the ultraviolet1. The quadratic increase of the two-photon excitation rate with excitation intensity restricts fluorescence emission to the focal volume thus providing the same depth resolution as does confocal microscopy. Image degradation due to out of focus backround is thus avoided. Photobleaching and most cellular photodamage are similarly confined to the focus thereby minimizing sample degredation during acquisition of the multiple sections required for 3-d image reconstruction. Fluorescence images of living cells and other thick photolabile fluorescence labled assemblies illustrate the depth discrimination of both two-photon fluorescence excitation and photobleaching. The quadratic intensity dependence of two-photon excitation allows 3-d spatially resolved photochemistry in particular the photolytic release of caged compounds such as neurotransmitters nucleotides fluorescent dyes and second messengers such as 1P3 and Ca. The two-photon release of cased ATP has been measured and release of a caged fluorescent dye has been shown. Point photobleaching and a 3-d " write once read many" optical memory have been demonstrated. Two-photon excitation of photo-initiated polymerization with a sharply focused single beam allows microfabrication of complex structures of arbitrary form. By scanning the focused beam through a liquid polymer with a UV excited initiator it is possible to harden the polymer only at the focus thereby creating© (1991) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

James H. Strickler

Papers

Two-Photon Laser Scanning Fluorescence Microscopy

Three-dimensional optical data storage in refractive media by two-photon point excitation.

Two-photon lithography for microelectronic application

Two-photon excitation in laser scanning fluorescence microscopy

3‐D optical data storage by two‐photon excitation