Highly nonlinear photodamage in two-photon fluorescence microscopy

被引:354
作者
Hopt, A [1 ]
Neher, E [1 ]
机构
[1] Max Planck Inst Biophys Chem, Abt Membranbiophys, D-37077 Gottingen, Germany
关键词
D O I
10.1016/S0006-3495(01)76173-5
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
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 [Ca2+] 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 approximate to 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 approximate to 2.5) than that of the two-photon signal (mu approximate to 2).
引用
收藏
页码:2029 / 2036
页数:8
相关论文
共 28 条
  • [1] Bewersdorf J, 1998, J MICROSC-OXFORD, V191, P28
  • [2] Booth MJ, 1998, J MICROSC-OXFORD, V190, P298, DOI 10.1046/j.1365-2818.1998.00375.x
  • [3] FEMTOSECOND PULSE-WIDTH CONTROL IN MICROSCOPY BY 2-PHOTON ABSORPTION AUTOCORRELATION
    BRAKENHOFF, GJ
    MULLER, M
    SQUIER, J
    [J]. JOURNAL OF MICROSCOPY-OXFORD, 1995, 179 (03): : 253 - 260
  • [4] CLAYTON RK, 1977, LIGHT LIVING MATTER, V1
  • [5] 2-PHOTON LASER SCANNING FLUORESCENCE MICROSCOPY
    DENK, W
    STRICKLER, JH
    WEBB, WW
    [J]. SCIENCE, 1990, 248 (4951) : 73 - 76
  • [6] Photon upmanship: Why multiphoton imaging is more than a gimmick
    Denk, W
    Svoboda, K
    [J]. NEURON, 1997, 18 (03) : 351 - 357
  • [7] Diels J., 1996, ULTRASHORT LASER PUL
  • [8] HAUGLAND RP, 1996, HDB FLUORESCENT PROB, P549
  • [9] Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation
    Hell, SW
    Booth, M
    Wilms, S
    Schnetter, CM
    Kirsch, AK
    Arndt-Jovin, DJ
    Jovin, TM
    [J]. OPTICS LETTERS, 1998, 23 (15) : 1238 - 1240
  • [10] Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation:: Signal and photodamage
    Koester, HJ
    Baur, D
    Uhl, R
    Hell, SW
    [J]. BIOPHYSICAL JOURNAL, 1999, 77 (04) : 2226 - 2236