Combined influences of chromatic aberration and scattering in depth-resolved two-photon fluorescence endospectroscopy.
The influence of chromatic aberration of an objective lens in two-photon fluorescence (TPF) endospectroscopy of scattering media has been systematically investigated through both experiments and numerical simulations. Experiments were carried out on a uniform 3D scattering gelatin phantom embedded with TiO(2) granules (to mimic tissue scattering) and fluorescein-tagged polystyrene beads. It was found that fluorescence spectral intensity and lineshape varied as a function of depth when measured with a gradient-index (GRIN) lens which has severe chromatic aberration. The spectral distortion caused by the chromatic aberration became diminishing as the imaging depth increased. Ray tracing analysis and Monte Carlo simulations were carried out to study the interplay of chromatic aberration and scattering in the depth-resolved TPF spectra. The simulation results suggest that the collected fluorescence signals from deeper layers included more out-of-focus photons that experienced a few or multiple scatterings, which diminish the influence of chromatic aberration on the measured TPF spectra. The simulated collection efficiencies of TPF at different wavelengths and depths can be used to properly recover the true depth-resolved TPF spectra of a relatively uniform scattering medium.
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205.
Yicong
Y
Wu
Xingde
X
Li
Appl Opt. 1994 Nov 1;33(31):7397-405
20941301
Opt Express. 2009 May 11;17(10):7907-15
19434122
Microsc Res Tech. 2007 May;70(5):398-402
17393493
Science. 1990 Apr 6;248(4951):73-6
2321027
J Biomed Opt. 2001 Oct;6(4):385-96
11728196
Opt Lett. 2009 Apr 1;34(7):953-5
19340182
J Biomed Opt. 2004 May-Jun;9(3):511-22
15189089
Appl Opt. 2000 Mar 1;39(7):1194-201
18338003
Appl Opt. 1996 Jul 1;35(19):3441-6
21102733
Opt Lett. 2006 Apr 15;31(8):1076-8
16625908
J Neurophysiol. 2004 Apr;91(4):1908-12
14668300
J Opt Soc Am A Opt Image Sci Vis. 1996 May;13(5):952-61
8622177
Opt Lett. 2005 Nov 15;30(22):3048-50
16315717
Opt Lett. 1997 Sep 1;22(17):1323-5
18188227
J Neurophysiol. 2004 Nov;92(5):3121-33
15128753
Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7075-80
12756303
Opt Express. 2006 Feb 6;14(3):1027-32
19503423
Opt Lett. 2004 Aug 1;29(15):1763-5
15352362
Opt Lett. 2005 Sep 1;30(17):2272-4
16190441