Two-photon excited cellular autofluorescence induced by cw and femtosecond NIR microradiation

Abstract

We report the first two-photon excited autofluorescence measurements in single cells induced by continuous wave NIR laser microbeams. In particular we demonstrate NIR-excited NAD(P)H fluorescence of motile spermatozoa in a single-beam gradient force optical trap ("optical tweezers). In addition, two-photon MR excited autofluorescence imaging and "c-mapping" of CHO cells was performed with laser scanning microradiation of a tunable femtosecond-Ti:sapphire laser. Cellular response on autofluorescence to two-photon absorption of NW radiation was compared with exposure to UVA light (one-photon absorption).Keywords: optical trapping, NIR, microbeam, NADH, laser scanning microscopy, two-photon excited fluorescence, femtosecond laser pulses, lifetime imaging INTRODUCTIONRecently, we reported on nonlinear effects of continuous wave (cw) near infrared (NIR) laser microbeams. In particular, we found that NW trapping beams are able to induce cell damage via a twophoton process1. Microbeams can create an enormous intracellular photon flux. The intensity and photon flux in a diffraction-limited cw 100 mW trapping beam at 760 nm (NA= 1.3) is 4O MW/cm2 and iO27 photons cm2 s-i, respectively. Because typical molecular cross sections are iO48iO5O cm4s (refs.2, 3), the photon flux of the 100 mW microbeam is sufficient to induce two-photon effects. Therefore, optical traps are sources of nonlinear excitation resulting in visible fluorescence and possible cell damage via oxidative stress. However, higher photon fluxes are necessary for two-photon fluorescence imaging with a reasonable frame rate. An increase in cw power would induce thermal damage (mean intracellular temperature increase: 2K/100 mW for 1064 nm microbeams4). In contrast, efficient multiphoton-excitation can be achieved by application of pulsed laser microbeams of moderate peak power. Appropriate laser sources are mode-locked, tunable Ti:sapphire lasers (700 -1000 nm) which provide pulses of about 150 fs with high repetition frequency of 80 MHz. In addition to fluorescence intensity measurements, the application of ultrashort excitation pulses allows fluorescence lifetime measurements. As demonstrated by Piston et al.5 and So et al.6, time-resolved fluorescence imaging ('c-mapping) in the frequency domain can be performed in microscopes using two-photon excitation by the heterodyning technique in combination with microbeam scanning. 12 ISPIE Vol. 2628 O-8194-1992-3/96/$6.OO Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/25/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx

Multi-wavelength fluorescence lifetime spectroscopy: a new approach to the study of endogenous fluorescence in living cells and tissues

“…Proteinbound NADH was described to have longer lifetimes [109,110]. Some have therefore proposed to use the short lifetime (0.4 ns) of endogenous NAD(P)H fluorescence as an indicator of "free" NAD(P)H molecules in cells and tissues, while the longer lifetime (∼ 2 ns) could be attributed to "bound" molecules [57,106,[111][112][113]. In cultivated endothelial cells from the calf aorta, NADH lifetime was tested to separate "free" and "protein-bound" coenzymes, and shown to be sensitive to metabolic modulators [112].…”

Estimating the risk of squamous cell cancer induction in skin following nonlinear optical imaging

“…These lasers can supply high irradiance in the range of GW cm À2 for transient durations as short as 10 À9 to 10 À15 seconds within the focal volume. Even so, during an ultrashort pulse duration, high peak irradiance delivered may still damage the irradiated cells by eliciting a variety of undesired biological responses [4][5][6][7][8][9][10]. However, the mentioned harmful cellular effects are restricted to the irradiated tissue and its immediate surroundings and do not have long term effects.…”

Single Molecule Bioanalysis

“…Cellular autofluorescence has been characterized in terms of spectral properties [76], lifetime [77], and spatial distribution [78]. In the visible regime, flavins [76] and lipofuscin [79] are currently regarded as the major source of endogenous fluorescence.…”