Two-Photon Microscopy of Single Molecules

2000 ◽  
Vol 6 (S2) ◽  
pp. 804-805
Author(s):  
R. Yuste ◽  
A. Majewska ◽  
K. Holthoff ◽  
K. Holthoff
Keyword(s):  
Dendritic Spines ◽  
Pyramidal Neurons ◽  
Calcium Influx ◽  
Pyramidal Cells ◽  
Scattering Media ◽  
Decay Kinetics ◽  
Two Photon ◽  
Photon Excitation ◽  
Cortical Pyramidal Neurons ◽  
Two Photon Excitation

Two-photon excitation has enabled investigators to image living cells in highly scattering media like the central nervous system (1). We have used a custom-built two-photon microscope to image dendritic spines from living cortical pyramidal neurons. Pyramidal cells form the majority of the neuron in the mammalian cortex and they receive practically all their synaptic contacts through dendritic spines. Dendritic spines are small (<1 μm diameter) appendages that have been practically inaccessible to physiological measurements until the application of two-photon excitation to their study (2). We have concentrated in two questions:A- Calcium compartmentalization of spines: Mechanisms of calcium decay kinetics.Dendritic spines can compartmentalize calcium (2). Although the mechanisms of calcium influx into spines have been explored (3), it is unknown what determines the calcium decay kinetics in spines. We investigate calcium dynamics in spines from rat CA1 pyramidal neurons in slices.

scholarly journals Evidence of calcium-permeable AMPA receptors in dendritic spines of CA1 pyramidal neurons

2014 ◽  
Vol 112 (2) ◽  
pp. 263-275 ◽  
Author(s):  
Hayley A. Mattison ◽  
Ashish A. Bagal ◽  
Michael Mohammadi ◽  
Nisha S. Pulimood ◽  
Christian G. Reich ◽  
...  
Keyword(s):  
Dendritic Spines ◽  
Ampa Receptors ◽  
Pyramidal Neurons ◽  
Calcium Influx ◽  
Hippocampal Slices ◽  
Pyramidal Cells ◽  
Stratum Radiatum ◽  
Acute Hippocampal Slices ◽  
Cultured Slices ◽  
Apical Dendrites

GluA2-lacking, calcium-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) have unique properties, but their presence at excitatory synapses in pyramidal cells is controversial. We have tested certain predictions of the model that such receptors are present in CA1 cells and show here that the polyamine spermine, but not philanthotoxin, causes use-dependent inhibition of synaptically evoked excitatory responses in stratum radiatum, but not s. oriens, in cultured and acute hippocampal slices. Stimulation of single dendritic spines by photolytic release of caged glutamate induced an N-methyl-d-aspartate receptor-independent, use- and spermine-sensitive calcium influx only at apical spines in cultured slices. Bath application of glutamate also triggered a spermine-sensitive influx of cobalt into CA1 cell dendrites in s. radiatum. Responses of single apical, but not basal, spines to photostimulation displayed prominent paired-pulse facilitation (PPF) consistent with use-dependent relief of cytoplasmic polyamine block. Responses at apical dendrites were diminished, and PPF was increased, by spermine. Intracellular application of pep2m, which inhibits recycling of GluA2-containing AMPARs, reduced apical spine responses and increased PPF. We conclude that some calcium-permeable, polyamine-sensitive AMPARs, perhaps lacking GluA2 subunits, are present at synapses on apical dendrites of CA1 pyramidal cells, which may allow distinct forms of synaptic plasticity and computation at different sets of excitatory inputs.

Spatially localized ballistic two-photon excitation in scattering media

1998 ◽  
Vol 4 (5) ◽  
pp. 303-310 ◽  
Author(s):  
Henryk Szmacinski ◽  
Ignacy Gryczynski ◽  
Joseph R. Lakowicz
Keyword(s):  
Scattering Media ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

scholarly journals Two-photon excitation in scattering media by spatiotemporally shaped beams and their application in optogenetic stimulation

2013 ◽  
Vol 4 (12) ◽  
pp. 2869 ◽  
Author(s):  
Aurélien Bègue ◽  
Eirini Papagiakoumou ◽  
Ben Leshem ◽  
Rossella Conti ◽  
Leona Enke ◽  
...  
Keyword(s):  
Scattering Media ◽  
Two Photon ◽  
Optogenetic Stimulation ◽  
Photon Excitation ◽  
Two Photon Excitation

scholarly journals Human cortical pyramidal neurons: From spines to spikes via models

2018 ◽  
Author(s):  
Guy Eyal ◽  
Matthias B. Verhoog ◽  
Guilherme Testa-Silva ◽  
Yair Deitcher ◽  
Ruth Benavides-Piccione ◽  
...  
Keyword(s):  
Dendritic Spines ◽  
Cortical Neurons ◽  
Pyramidal Neurons ◽  
Temporal Cortex ◽  
Pyramidal Cells ◽  
Physiological Data ◽  
Excitatory Synapses ◽  
Basal Dendrites ◽  
Cortical Pyramidal Neurons ◽  
Dendritic Branches

AbstractWe present the first-ever detailed models of pyramidal cells from human neocortex, including models on their excitatory synapses, dendritic spines, dendritic NMDA- and somatic/axonal- Na+ spikes that provided new insights into signal processing and computational capabilities of these principal cells. Six human layer 2 and layer 3 pyramidal cells (HL2/L3 PCs) were modeled, integrating detailed anatomical and physiological data from both fresh and post mortem tissues from human temporal cortex. The models predicted particularly large AMPA- and NMDA- conductances per synaptic contact (0.88 nS and 1.31nS, respectively) and a steep dependence of the NMDA-conductance on voltage. These estimates were based on intracellular recordings from synaptically-connected HL2/L3 pairs, combined with extra-cellular current injections and use of synaptic blockers. A large dataset of high-resolution reconstructed HL2/L3 dendritic spines provided estimates for the EPSPs at the spine head (12.7 ± 4.6 mV), spine base (9.7 ± 5.0 mV) and soma (0.3 ± 0.1 mV), and for the spine neck resistance (50 – 80 MΩ). Matching the shape and firing pattern of experimental somatic Na+-spikes provided estimates for the density of the somatic/axonal excitable membrane ion channels, predicting that 134 ± 28 simultaneously activated HL2/L3- HL2/L3 synapses are required for generating (with 50% probability) a somatic Na+ spike. Dendritic NMDA spikes were triggered in the model when 20 ± 10 excitatory spinous synapses were simultaneously activated on individual dendritic branches. The particularly large number of basal dendrites in HL2/L3 PCs and the distinctive cable elongation of their terminals imply that ~25 NMDA- spikes could be generated independently and simultaneously in these cells, as compared to ~14 in L2/3 PCs from the rat temporal cortex. These multi-sites nonlinear signals, together with the large (~30,000) excitatory synapses/cell, equip human L2/L3 PCs with enhanced computational capabilities. Our study provides the most comprehensive model of any human neuron to-date demonstrating the biophysical and computational distinctiveness of human cortical neurons.

Two‐photon excitation spectroscopy and excited state decay kinetics of isolated diphenylbutadiene

1985 ◽  
Vol 83 (5) ◽  
pp. 2186-2190 ◽  
Author(s):  
James S. Horwitz ◽  
Bryan E. Kohler ◽  
Thomas A. Spiglanin
Keyword(s):  
Excited State ◽  
Decay Kinetics ◽  
Two Photon ◽  
State Decay ◽  
Photon Excitation ◽  
Kinetics Of ◽  
Two Photon Excitation

Two-photon excitation microscopy in cellular biophysics

1995 ◽  
Vol 53 ◽  
pp. 62-63
Author(s):  
David W. Piston ◽  
Brian D. Bennett ◽  
Robert G. Summers
Keyword(s):  
Confocal Microscopy ◽  
Laser Beam ◽  
Duty Cycle ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10-5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

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