scholarly journals Determining composition of micron-scale protein deposits in neurodegenerative disease by spatially targeted optical microproteomics

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Kevin C Hadley ◽  
Rishi Rakhit ◽  
Hongbo Guo ◽  
Yulong Sun ◽  
James EN Jonkman ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Genetic Manipulation ◽  
Ex Vivo ◽  
Confocal Imaging ◽  
Test Case ◽  
Post Mortem ◽  
Two Photon ◽  
Protein Deposits ◽  
Photon Excitation ◽  
Formalin Fixed

Spatially targeted optical microproteomics (STOMP) is a novel proteomics technique for interrogating micron-scale regions of interest (ROIs) in mammalian tissue, with no requirement for genetic manipulation. Methanol or formalin-fixed specimens are stained with fluorescent dyes or antibodies to visualize ROIs, then soaked in solutions containing the photo-tag: 4-benzoylbenzyl-glycyl-hexahistidine. Confocal imaging along with two photon excitation are used to covalently couple photo-tags to all proteins within each ROI, to a resolution of 0.67 µm in the xy-plane and 1.48 µm axially. After tissue solubilization, photo-tagged proteins are isolated and identified by mass spectrometry. As a test case, we examined amyloid plaques in an Alzheimer's disease (AD) mouse model and a post-mortem AD case, confirming known plaque constituents and discovering new ones. STOMP can be applied to various biological samples including cell lines, primary cell cultures, ex vivo specimens, biopsy samples, and fixed post-mortem tissue.

scholarly journals MULTIMODAL NONLINEAR OPTICAL IMAGING OF CELL–MATRIX INTERACTION DURING SPINAL CORD INJURY EX VIVO

2011 ◽  
Vol 23 (03) ◽  
pp. 223-230 ◽  
Author(s):  
Yi-Cheng Huang ◽  
Te-Hsuen Chen ◽  
Wen-Chun Kuo ◽  
Sung-Hao Hsu ◽  
Yi-You Huang ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Ex Vivo ◽  
Second Harmonic ◽  
Two Photon ◽  
Matrix Interaction ◽  
Cell Matrix ◽  
Photon Excitation ◽  
Cord Injury ◽  
Near Future

Neurons within spinal cord injury (SCI) are prevented from regeneration because of scar formation. Chondroitinase ABC (ChABC) was reported to promote functional recovery after spinal cord injury. However, the mechanism and the role of ChABC in the recovery are not clear. In this research, we used second harmonic generation (SHG) and two-photon excitation fluorescence (2PEF) images as probes to observe cell–matrix interaction on fibrosis after SCI followed by ChABC treatment. According to our experimental results, the enzyme ChABC could decrease cystic formation dramatically and consequently allow the spinal cord to regenerate. Using immunohistological analysis, we found that treatment with ChABC at the lesion area resulted in fewer chondroitin sulfate proteoglycans (CSPGs) remaining, longer axonal re-growth, and more new developmental neurons. Furthermore, ChABC 1 U/ml was more effective than 5 U/ml treatment. Using the noninvasive technology, SHG and 2PEF images, we could observe cell–matrix interaction clearly, not only in fixed samples but also in unfixed ex vivo samples. This technology presents a potential for clinical use in the near future.

scholarly journals Exploration of the two-photon excitation spectrum of fluorescent dyes at wavelengths below the range of the Ti:Sapphire laser

2015 ◽  
Vol 259 (3) ◽  
pp. 210-218 ◽  
Author(s):  
J. TRÄGÅRDH ◽  
G. ROBB ◽  
R. AMOR ◽  
W.B. AMOS ◽  
J. DEMPSTER ◽  
...  
Keyword(s):  
Excitation Spectrum ◽  
Fluorescent Dyes ◽  
Ti:Sapphire Laser ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

UV-excited fluorophore images obtained with IR excitation

1996 ◽  
Vol 54 ◽  
pp. 906-907
Author(s):  
D. Wokosin ◽  
V.F. Centonze ◽  
J.G. White
Keyword(s):  
Short Pulse ◽  
Argon Laser ◽  
Confocal Imaging ◽  
Photon Absorption ◽  
Focal Volume ◽  
Two Photon ◽  
Photon Excitation ◽  
Biological Studies ◽  
Short Pulse Lasers ◽  
Two Photon Excitation

The widespread use of two-photon excitation fluorescence imaging has been somewhat inhibited by the necessity to use large, expensive, high-power, short-pulse lasers. These ultra-short pulse lasers are used as an excitation source in a raster scanning configuration to provide sufficient peak power density in a lens focal volume to generate detectable two-photon absorption events for rapid imaging. Biological studies often benefit from multiple fluorescent labels and multi-labelled samples often require different excitation wavelengths for adequate excitation of the various colored fluorophores. This is achieved inexpensively with the three Krypton Argon laser lines in standard confocal imaging systems, but multiple two-photon excitation lasers--if available-- would be a very expensive system. The lasers commonly used for two-photon imaging are tuneable, but this is not a non-trivial and time consuming process. The tuning range on these lasers allows good access to the blue-emitting and green-emitting fluorophores via two-photon excitation; however, we have found that a fixed-wavelength, compact,

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 fluorescence microscopy using a semiconductor laser

Bioimaging ◽  
1995 ◽  
Vol 3 (2) ◽  
pp. 70-75 ◽  
Author(s):  
Pekka E Hänninen ◽  
Martin Schrader ◽  
Erkki Soini ◽  
Stefan W Hell
Keyword(s):  
Fluorescence Microscopy ◽  
Semiconductor Laser ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

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.

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