Cited by 442Advanced Fluorescence Microscopy TechniquesNonlinear Optical Materials StudiesLaser-Matter Interactions and Applications

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Highly Nonlinear Photodamage in Two-Photon Fluorescence Microscopy

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Hopt, Alexander, Neher, Erwin·2001·Biophysical Journal

DOI 10.1016/s0006-3495(01)76173-5PMC PMC1301392

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Abstract

Two-photon fluorescence excitation is being increasingly used in laser scan microscopy due to very low photodamage induced by this technique under normal operation. However, excitation intensity has to be kept low, because nonlinear photodamage sets in when laser power is increased above a certain threshold. We studied this kind of damage in bovine adrenal chromaffin cells, using two different indicators of damage: changes in resting [Ca(2+)] level and the degranulation reaction. In agreement with previous studies, we found that, for both criteria, damage is proportional to the integral (over space and time) of light intensity raised to a power approximately 2.5. Thus, widening the laser pulse shape at constant average intensity both in time and in focal volume is beneficial for avoiding this kind of damage. Both measures, of course, reduce the two-photon fluorescence excitation. However, loss of signal can be compensated by increasing excitation power, such that, at constant damaging potential, signals may be even larger with long pulses and large focal volumes, because the exponent of the power law of damage is higher (mu approximately 2.5) than that of the two-photon signal (mu approximately 2).

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Authors: Hopt, A., & Neher, E.
Affiliations: Max Planck Institute for Biophysical Chemistry (DE)
Max Planck Society (DE)
Corresponding author: Neher, Erwin
ORCID: Neher, Erwin
Venue: Biophysical Journal
Publisher: Elsevier BV
Published: 2001-04
Volume: 80
Issue: 4
Pages: 2029-2036
ISSN: 0006-3495 (ISSN-L)
DOI: 10.1016/s0006-3495(01)76173-5
PMC: PMC1301392
OpenAlex: W2089975180
Cited by: 442
References: 27
Citations by year: 2026: 15 · 2025: 15 · 2024: 15 · 2023: 16 · 2022: 23 · 2021: 21 · 2020: 16 · 2019: 14 · 2018: 14 · 2017: 18 · 2016: 16 · 2015: 18 · 2014: 24 · 2013: 18 · 2012: 13
FWCI: 9.13
Type: journal article
Language: en
Open access: bronze
doi.org
Licence: www.elsevier.com/tdm/userlicense/1.0/ (tdm)
www.elsevier.com/open-access/userlicense/1.0/ (vor, 4489 d)
Locations: Biophysical Journal (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Max Planck Institute for Plasma Physics (submittedVersion)
MPG.PuRe (Max Planck Society) (submittedVersion)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
Physical Sciences › Materials Science › Electronic, Optical and Magnetic Materials › Nonlinear Optical Materials Studies
Physical Sciences › Physics and Astronomy › Atomic and Molecular Physics, and Optics › Laser-Matter Interactions and Applications
Keywords: two-photon fluorescence microscopy, photodamage, laser pulse shape, focal volume, excitation intensity, adrenal chromaffin cells, calcium imaging, degranulation
MeSH terms: Adrenal Glands, Animals, Calcium, Cattle, Lasers, Light, Microscopy, Fluorescence, Models, Theoretical, Time Factors, Models, Statistical, Photons, Chromaffin Cells

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Highly Nonlinear Photodamage in Two-Photon Fluorescence Microscopy

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