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

Research into terahertz technology is now receiving increasing attention around the world, and devices exploiting this waveband are set to become increasingly important in a very diverse range of applications. Here, an overview of the status of the technology, its uses and its future prospects are presented.

Cutting-edge terahertz technology

BODIPY dyes and their derivatives: syntheses and spectroscopic properties.

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.

/pdf/deep-tissue-two-photon-microscopy-2aqu6d6n65.pdf

Deep tissue two-photon microscopy

I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

This review examines the photophysical properties of an interesting and biologically important class of proteins known as the rhodopsins. The visual rhodopsins are responsible for the conversion of light into nerve impulses in the image resolving eyes of mullusks, arthropods, and vertebrates. Despite independent evolutionary development, these systems have converged on proteinand chromophore-binding site designs that are remarkably similar. The bacterial rhodopsins represent a much broader class of proteins that serve both photosynthetic and phototactic functions. The best known of this latter group is bacteriorhodopsin, which converts light into energy via photon-activated transmembrane proton pumping. Regardless of function, all rhodopsins appear to share common features, which include a retinyl chromophore in a protein-binding site formed within the inner segments of seven transmembrane helices. Upon the absorption of light, the chromophore isomerizes to generate a bathochromically shifted photoproduct that stores a significant fraction of the photon energy in both electrostatic and conformational forms. Subsequent dark reactions carry out the relevant biological function. The nature of the primary photochemical events in the rhodopsins has

Photophysics and Molecular Electronic Applications of the Rhodopsins

Nature of the primary photochemical events in rhodopsin and bacteriorhodopsin

Light-harvesting arrays containing one, two, or eight boron-dipyrrin (BDPY) pigments and one porphyrin (free base or Zn chelate) have been synthesized using a modular building block approach. The reaction of pyrrole and 4-(BDPY)benzaldehyde or 3,5-bis(BDPY)benzaldehyde, prepared by Pd-mediated ethynylation with the corresponding iodo-benzaldehydes, affords the desired BDPY-porphyrin array in yields of 10−58%. The arrays are soluble in organic solvents and have been characterized by static and time-resolved absorption and fluorescence spectroscopy. The blue-green BDPY absorption complements spectral coverage of the porphyrin chromophores and rivals the intensity of the porphyrin Soret band when eight BDPY accessory pigments are present. Efficient energy transfer from the BDPY pigment(s) to the porphyrin (free base or Zn-chelate) is observed in arrays containing one or two (>90%) or eight (>85%) accessory pigments per porphyrin. Biphasic excited-state decay behavior is exhibited by the BDPY pigments in isol...

http://chembio.uconn.edu/Group_pubs/1995-1999/li_1998_jacs.pdf

Design, Synthesis, and Photodynamics of Light-Harvesting Arrays Comprised of a Porphyrin and One, Two, or Eight Boron-Dipyrrin Accessory Pigments

Boron−dipyrrin chromophores containing a 5-aryl group with or without internal steric hindrance toward aryl rotation have been synthesized and then characterized via X-ray diffraction, static and time-resolved optical spectroscopy, and theory. Compounds with a 5-phenyl or 5-(4-tert-butylphenyl) group show low fluorescence yields (∼0.06) and short excited-singlet-state lifetimes (∼500 ps), and decay primarily (>90%) by nonradiative internal conversion to the ground state. In contrast, sterically hindered analogues having an o-tolyl or mesityl group at the 5-position exhibit high fluorescence yields (∼0.9) and long excited-state lifetimes (∼6 ns). The X-ray structures indicate that the phenyl or 4-tert-butylphenyl ring lies at an angle of ∼60° with respect to the dipyrrin framework whereas the angle is ∼80° for mesityl or o-tolyl groups. The calculated potential energy surface for the phenyl-substituted complex indicates that the excited state has a second, lower energy minimum in which the nonhindered aryl...

/pdf/structural-control-of-the-photodynamics-of-boron-dipyrrin-1ct59k7v3m.pdf

Structural Control of the Photodynamics of Boron-Dipyrrin Complexes

Optical control of the primary step of photoisomerization of the retinal molecule in bacteriorhodopsin from the all-trans to the 13-cis state was demonstrated under weak field conditions (where only 1 of 300 retinal molecules absorbs a photon during the excitation cycle) that are relevant to understanding biological processes. By modulating the phases and amplitudes of the spectral components in the photoexcitation pulse, we showed that the absolute quantity of 13-cis retinal formed upon excitation can be enhanced or suppressed by ±20% of the yield observed using a short transform-limited pulse having the same actinic energy. The shaped pulses were shown to be phase-sensitive at intensities too low to access different higher electronic states, and so these pulses apparently steer the isomerization through constructive and destructive interference effects, a mechanism supported by observed signatures of vibrational coherence. These results show that the wave properties of matter can be observed and even manipulated in a system as large and complex as a protein.

/pdf/coherent-control-of-retinal-isomerization-in-41my3uv7lt.pdf

Robert R. Birge

Papers

Photophysics and Molecular Electronic Applications of the Rhodopsins

Nature of the primary photochemical events in rhodopsin and bacteriorhodopsin

Design, Synthesis, and Photodynamics of Light-Harvesting Arrays Comprised of a Porphyrin and One, Two, or Eight Boron-Dipyrrin Accessory Pigments

Structural Control of the Photodynamics of Boron-Dipyrrin Complexes

Coherent control of retinal isomerization in bacteriorhodopsin.