scholarly journals The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse

Development ◽  
1998 ◽  
Vol 125 (23) ◽  
pp. 4627-4635 ◽  
Author(s):  
K.T. Jones ◽  
C. Soeller ◽  
M.B. Cannell
Keyword(s):  
Confocal Microscopy ◽  
Second Messengers ◽  
High Permeability ◽  
Sperm Protein ◽  
Mouse Sperm ◽  
Two Photon ◽  
Local Increase ◽  
Fluorescent Markers ◽  
Observable Event

Mouse sperm-egg fusion was examined using two-photon and confocal microscopy. A delay of several minutes occurred between the first observable event of fusion (which was the diffusion of Ca2+-sensitive dyes from egg into sperm) and any change in egg cytoplasmic Ca2+. When indo-1 dextran was used to obtain ratiometric two-photon images, there was no detectable local increase in egg cytoplasmic Ca2+ near the site of sperm fusion. However, the sperm underwent a Ca2+ transient which appeared to be coincident with the egg cytoplasm Ca2+ transient, which suggested that there was a high permeability pathway for Ca2+ between egg and sperm. To exclude this pathway from providing trigger Ca2+ for the egg transient, we reduced bathing [Ca2+] to approx. 18 microM and 13nM (with EGTA). In these conditions the first egg Ca2+ transient was not prevented, which makes an obligatory role for extracellular Ca2+ in the initiation of the egg Ca2+ transient unlikely. Both FITC-albumin (70 kDa) and 10 kDa dextran-linked Ca2+ indicators were able to diffuse into the sperm from the egg. In addition, phycoerythrin (240 kDa) rapidly diffused into the sperm shortly after fusion (but before any changes in Ca2+ occurred). This suggests that the ‘pore(s)’ that form during sperm-egg fusion must be at least 8 nm in diameter. These data are compatible with the idea that a diffusible sperm protein could trigger the observed changes in intracellular Ca2+ in the egg, but do not exclude the possibility that other second messengers are generated during sperm-egg fusion.

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.

Two-photon excitation fluorescence microscopy in living systems

1993 ◽  
Vol 51 ◽  
pp. 154-155 ◽  
Author(s):  
David W. Piston
Keyword(s):  
Confocal Microscopy ◽  
Fluorescence Microscopy ◽  
Singlet State ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. 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 our fluorescence experiments, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. 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.

Two-Photon Excitation Imaging of Glucose Metabolism in Living Tissue

1997 ◽  
Vol 3 (S2) ◽  
pp. 305-306
Author(s):  
David W. Piston
Keyword(s):  
Confocal Microscopy ◽  
Collection Efficiency ◽  
Three Dimensional ◽  
Spatial Filter ◽  
Input Power ◽  
Photon Absorption ◽  
Focal Volume ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. It provides three-dimensional resolution and eliminates background equivalent to an ideal confocal microscope without requiring a confocal spatial filter, whose absence enhances fluorescence collection efficiency. This results in inherent submicron optical sectioning by excitation alone. In practice, TPEM 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 limits the average input power to less than 10 mW, only slightly greater than the power normally used in confocal microscopy. Because of the intensity-squared dependence of the two-photon absorption, the excitation is limited to the focal volume.

scholarly journals Real‐time two‐photon confocal microscopy using a femtosecond, amplified Ti:sapphire system

1996 ◽  
Vol 181 (3) ◽  
pp. 253-259 ◽  
Author(s):  
G. J. BRAKENHOFF ◽  
J. SQUIER ◽  
T. NORRIS ◽  
A. C. BLITON ◽  
M. H. WADE ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Real Time ◽  
Two Photon

Imaging theory and resolution improvement of two-photon confocal microscopy

2002 ◽  
Vol 45 (11) ◽  
pp. 1468-1478 ◽  
Author(s):  
Tang Zhilie ◽  
Yang Chuping ◽  
Pei Hongjin ◽  
Liang Ruisheng ◽  
Liu Songhao
Keyword(s):  
Confocal Microscopy ◽  
Two Photon ◽  
Resolution Improvement ◽  
Imaging Theory

scholarly journals Luminescence confocal microscopy of 3D components of photonic integrated circuits fabricated by two-photon photopolymerization

2021 ◽  
pp. 100413
Author(s):  
Rilond Pattia Matital ◽  
Danila Anatolievich Kolymagin ◽  
Dmitriy Anatolievich Chubich ◽  
Denis Dmitrievich Merkushev ◽  
Alexei Grigorievich Vitukhnovsky
Keyword(s):  
Confocal Microscopy ◽  
Integrated Circuits ◽  
Photonic Integrated Circuits ◽  
Two Photon

A small-molecule with a large two-photon absorption cross-section serves as a membrane-permeable ribonucleic acid (RNA) probe for live cell imaging

2018 ◽  
Vol 42 (17) ◽  
pp. 14325-14331 ◽  
Author(s):  
Ruiqing Feng ◽  
Longlong Li ◽  
Bing Li ◽  
Jinhui Li ◽  
Dan Peng ◽  
...  
Keyword(s):  
Cross Section ◽  
Cross Sections ◽  
Live Cell Imaging ◽  
Plasma Membranes ◽  
Cell Imaging ◽  
Photon Absorption ◽  
Absorption Cross ◽  
High Permeability ◽  
Two Photon Absorption ◽  
Two Photon

DMI could light up the RNA of the nucleus and the cytoplasm in living systems, which not only exhibits larger two-photon absorption cross-sections (981 GM), but also displays high-permeability to plasma membranes of vigorous cells.

Sign in / Sign up

Export Citation Format

Share Document