Two-Photon near Infrared Excitation in Living Cells

1997 ◽  
Vol 5 (1) ◽  
pp. 27-34 ◽  
Author(s):  
Karsten König

Non-linear effects due to two-photon near infrared (NIR) excitation of endogenous and exogenous cellular chromophores allow novel techniques in tissue, cell and biomolecule diagnostics, as well as in intracellular micromanipulation (e.g. intracellular photochemistry). Two-photon NIR excitation may also result in cell damage effects. The high photon intensities (1024 photons cm−2 s−1) required for non-resonant two-photon excitation can be achieved by diffraction-limited focusing of continuous wave (cw) laser beams (cw microbeams) with powers in the mW range. For example, NIR traps (“laser tweezers”) used as force transducers and micromanipulation tools in cellular and molecular biology are sources of two-photon excitation. NIR traps can induce two-photon excited visible fluorescence and, in the case of <800 nm-traps, UVA-like cell damage. Multimode cw microbeams may enhance non-linear effects due to longitudinal mode-beating. To perform high scan rate two-photon fluorescence imaging, the application of ultrashort laser pulses of moderate peak power but low average power (pulsed microbeams) is required. In NIR femtosecond microscopes, non-destructive imaging of two-photon excited fluorophores in various human and culture cells was demonstrated for <2 mW average powers, <200 mW peak powers and 400 GW cm−2 intensities (700–800 nm, ∼150 fs, ∼100 MHz). However, higher average power levels may result in failed cell reproduction and cell death due to intracellular optical breakdown. In addition, destructive transient local heating and μN force generation may occur.

2021 ◽  
Author(s):  
WEN-SHUO KUO ◽  
Chia-Yuan Chang ◽  
Ping-Ching Wu ◽  
Jiu-Yao Wang

Abstract BackgroundNitrogen doping and amino-group functionalization, which result in strong electron donation, can be achieved through chemical modification. Large π-conjugated systems of graphene quantum dot (GQD)-based materials acting as electron donors can be chemically manipulated with low two-photon excitation energy in a short photoexcitation time for improving the charge transfer efficiency of sorted nitrogen-doped amino acid–functionalized GQDs (sorted amino-N-GQDs). ResultsIn this study, a self-developed femtosecond Ti-sapphire laser optical system (222.7 nJ pixel−1 with 100-170 scans, approximately 0.65-1.11 s of total effective exposure times; excitation wavelength: 960 nm in the near-infrared II region) was used for chemical modification. The sorted amino-N-GQDs exhibited enhanced two-photon absorption, post-two-photon excitation stability, two-photon excitation cross-section, and two-photon luminescence through the radiative pathway. The lifetime and quantum yield of the sorted amino-N-GQDs decreased and increased, respectively. Furthermore, the sorted amino-N-GQDs exhibited excitation-wavelength-independent photoluminescence in the near-infrared region and generated reactive oxygen species after two-photon excitation. An increase in the size of the sorted amino-N-GQDs boosted photochemical and electrochemical efficacy and resulted in high photoluminescence quantum yield and highly efficient two-photon photodynamic therapy. ConclusionThe sorted dots can be used in two-photon contrast probes for tracking and localizing analytes during two-photon imaging in a biological environment and for conducting two-photon photodynamic therapy for eliminating infectious microbes.


1998 ◽  
Vol 4 (S2) ◽  
pp. 424-425
Author(s):  
G.Y. Fan ◽  
H. Fujisaki ◽  
R.-K. Tsay ◽  
R.Y. Tsien ◽  
Mark H. Ellisman

A video-rate scanning two-photon excitation microscope (TPEM) has been successfully constructed and tested. The TPEM, based on a Nikon RCM-8000, incorporates a femtosecond pulsed laser, a pre-chirper, and a non-confocal detection box for ratio imaging. Fig. 1 shows the schematic layout of the main components of the instrument, each of which is briefly discussed below.Laser System: A Tsunami Ti: Sapphire laser (from Spectra-Physics) is optically pumped by a 5 W green laser (Millennia from Spectra-Physics) and is capable of generating 100 fs pulses at a repetition rate of 82 MHz and an average power of 0.8 W. The output wavelength is tunable from 690 to 1050 nm with three optical sets, each covering part of the spectrum with some overlapping.Pre-chirper: After leaving the Tsunami, the laser beam enters an optic unit known as a pre-chirper which pre-chirps laser pulses to compensate for the group velocity dispersion which will result when the laser beam goes through the microscope optics.


2001 ◽  
Vol 79 (6) ◽  
pp. 830-832 ◽  
Author(s):  
A. M. van Oijen ◽  
R. Verberk ◽  
Y. Durand ◽  
J. Schmidt ◽  
J. N. J. van Lingen ◽  
...  

1993 ◽  
Vol 48 (1) ◽  
pp. 192-219 ◽  
Author(s):  
M. S. Fee ◽  
S. Chu ◽  
A. P. Mills ◽  
R. J. Chichester ◽  
D. M. Zuckerman ◽  
...  

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