Cell Imaging and Manipulation by Nonlinear Optical Microscopy

Sacconi, Leonardo; Tolic-Norrelykke, Iva M.; D’Amico, Massimo; Vanzi, Francesco; Olivotto, Massimo; Antolini, R.

doi:10.1385/cbb:45:3:289

Cited by 12 publications

(11 citation statements)

References 75 publications

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“…In particular, the three-dimensional sectioning capability, the penetration depth that can be reached in live tissues and the potentially reduced photo-bleaching and photo-toxicity promote these techniques for an array of important applications in biology and biomedicine. Recently, the use of Second Harmonic Generation (SHG) for imaging biological samples has been explored with regard to both extrinsic and intrinsic SHG (1,2,3,4). The former has mostly been developed with regard to the use of chiral dyes for the measurement of membrane potential first in artificial vesicles (5,6,7), then in live cells (8,9), demonstrating also potential application to tumor cells (10).…”

Section: Introductionmentioning

confidence: 99%

Study of skeletal muscle cross-bridge population dynamics by second harmonic generation

et al. 2007

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The high degree of structural order in skeletal muscle allows imaging of this tissue by Second Harmonic Generation (SHG). Biochemical and colocalization studies have gathered an increasing wealth of clues for the attribution of the molecular origin of the muscle SHG signal to the motor protein myosin. Thus, SHG represents a potentially very powerful tool in the investigation of structural dynamics occurring in muscle during active production of force and/or shortening. A full characterization of the polarization-dependence of the SHG signal represents a very selective information on the orientation of the emitting proteins and their dynamics during contraction, provided that different physiological states of muscle (relaxed, rigor and active) exhibit distinct patterns of SHG polarization dependence. Here polarization data are obtained from single frog muscle fibers at rest and during isometric contraction and interpreted, by means of a model, in terms of an average orientation of the SHG emitters which are structured with a cylindrical symmetry about the fiber axis. The setup is optimized for accurate polarization measurements with SHG, combined with a line scan imaging method allowing acquisition of SHG polarization curves in different physiological states. We demonstrate that muscle fiber displays a measurable variation of the orientation of SHG emitters with the transition from rest to isometric contraction.

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Section: Introductionmentioning

confidence: 99%

Study of skeletal muscle cross-bridge population dynamics by second harmonic generation

et al. 2007

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“…In addition to nanosurgery 9,10,29 a nonlinear microscope could be readily adapted as a tool for other nanomanipulation techniques, such as multiphoton stimulation, 30 multiphoton uncaging of caged neurotransmitters, 31 optical knock out of cell organelles, 32 and intracellular chromosome dissection. 33 The setup we propose provides the possibility of combining all these techniques with real-time confocal or epifluorescene imaging.…”

Section: Discussionmentioning

confidence: 99%

Multimodal optical workstation for simultaneous linear, nonlinear microscopy and nanomanipulation: Upgrading a commercial confocal inverted microscope

Mathew

Santos

Zalvidea

et al. 2009

Review of Scientific Instruments

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In this work we propose and build a multimodal optical workstation that extends a commercially available confocal microscope (Nikon Confocal C1-Si) to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools such as nanosurgery. The setup allows both subsystems (confocal and nonlinear) to work independently and simultaneously. The workstation enables, for instance, nanosurgery along with simultaneous confocal and brightfield imaging. The nonlinear microscopy capabilities are added around the commercial confocal microscope by exploiting all the flexibility offered by this microscope and without need for any mechanical or electronic modification of the confocal microscope systems. As an example, the standard differential interference contrast condenser and diascopic detector in the confocal microscope are readily used as a forward detection mount for second harmonic generation imaging. The various capabilities of this workstation, as applied directly to biology, are demonstrated using the model organism Caenorhabditis elegans.

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“…SHG thus gives a more contrasted image of cell membranes than TPF. 99,100 For this reason, SHG imaging of VSDs has been recently exploited to optically detect action potentials in model membranes, 101 in cultured neurons of Aplysia 102,103 and in mammalian intact neuronal systems. 104,105 In these examples, the optical recording of neuronal APs was performed only in a single position of the neuron using a line scanning procedure.…”

Section: Optical Recording Of Action Potentialsmentioning

confidence: 99%

Advanced Optical Techniques to Explore Brain Structure and Function

Silvestri

Mascaro

Lotti

et al. 2013

J. Innov. Opt. Health Sci.

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Understanding brain structure and function, and the complex relationships between them, is one of the grand challenges of contemporary sciences. Thanks to their°exibility, optical techniques could be the key to explore this complex network. In this manuscript, we brie°y review recent advancements in optical methods applied to three main issues: anatomy, plasticity and functionality. We describe novel implementations of light-sheet microscopy to resolve neuronal anatomy in whole¯xed brains with cellular resolution. Moving to living samples, we show how real-time dynamics of brain rewiring can be visualized through two-photon microscopy with the spatial resolution of single synaptic contacts. The plasticity of the injured brain can also be dissected through cutting-edge optical methods that speci¯cally ablate single neuronal processes. Finally, we report how nonlinear microscopy in combination with novel voltage sensitive dyes allow optical registrations of action potential across a population of neurons opening promising prospective in understanding brain functionality. The knowledge acquired from these complementary optical methods may provide a deeper comprehension of the brain and of its unique features.

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Cell Imaging and Manipulation by Nonlinear Optical Microscopy

Cited by 12 publications

References 75 publications

Study of skeletal muscle cross-bridge population dynamics by second harmonic generation

Study of skeletal muscle cross-bridge population dynamics by second harmonic generation

Multimodal optical workstation for simultaneous linear, nonlinear microscopy and nanomanipulation: Upgrading a commercial confocal inverted microscope

Advanced Optical Techniques to Explore Brain Structure and Function

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