Two-photon fluorescence excitation in continuous-wave infrared optical tweezers

Proceedings of …, 1995

We describe fluorescence spectroscopy and imaging studies of optically trapped single Chinese hamster ovary (CHO) and motile human sperm cells. The NIR trapping beam was provided by a tunable, multimode continuous wave Ti:Sapphire laser. The beam was introduced into ...

Laser-Tissue Interaction VI, 1995

We describe fluorescence spectroscopy and imaging studies of optically trapped single Chinese hamster ovary (CHO) and motile human sperm cells. The NIR trapping beam was provided by a tunable, multimode continuous wave Ti:Sapphire laser. The beam was introduced into an inverted confocal laser scanning microscope. Fluorescence of cells in the single-beam gradient force optical trap was excited with a 488 nm microbeam (laser scanning microscopy) or with 365 nm radiation from a high-pressure mercury lamp. Modifications to NADH-attributed autofluorescence and Rhodamine-and Propidium Iodide-attributed xenofluorescence indicate a significant cell-damaging effect of 760 nm trapping beams. 760 nm effects produce a biological response comparable to UVA-induced oxidative stress and appear to be a consequence of two-photon absorption.

SPIE Proceedings, 1995

Photoinduced modifications of NAD(P)H attributed autofluorescence of CHO cells in a single-beam gradient force optical trap (optical tweezers) were studied. Fluorescence spectra of single cells in the optical trap were measured using a modified microscope with an JR microbeam at 1064 and 760 nm for trapping, UVA radiation at 365 nm for fluorescence excitation, and an optical multichannel analyzer for spectral recording. No strong effect of the 1064 nm trapping beam on fluorescence intensity and spectral characteristics was found, even for power densities up to 70 MW/cm2. In contrast, 760 nm microirradiation resulted in a significant fluorescence increase, probably indicating cell damage due to absorption by heme-containing molecules. UVA exposure (1 W/cm2) of the trapped cells generated within seconds an initial fluorescence decrease, followed by a significant increase up to 5X of the value prior to irradiation. The UVA-induced modifications reflect NAD(P)H autooxidation and irreversible cell damage due to oxidative stress.

Optical Diagnostics of Living Cells and Biofluids, 1996

We demonstrate multi-photon excitation in optically-trapped living cells. Intracellular non-resonant twophoton excitation of endogenous and exogenous chromophores was induced by CW near infrared (NW) trapping beams of 105 mW power. In the case of fluorescent chromophores, detection of NIR-excited visible fluorescence was achieved by imaging and spectroscopy methods. Trap-induced, two-photon excited fluorescence was employed as a novel diagnostic method to monitor intracellular redox state and cell vitality of single motile spermatozoa and Chinese hamster ovary cells. We found, that nonlinear absorption of NIR photons <800 nm may lead to oxidative stress and severe cell damage. Biological response was amplified in multimode CW lasers due to longitudinal mode-beating and partial modelocking. As a result, we recommend the use of longwavelength-NIR, single-frequency traps ("optical tweezers") for micromanipulation of vital cells.

Multiphoton Microscopy and Fluorescence Lifetime Imaging, 2018

Laser tweezers or optical traps are established laser tools for optical noncontact manipulation of micron/submicron sized objects in liquids such as nonadherent biological cells in medium. Typical laser traps are based on optical gradient forces generated with high numerical aperture near-infrared (NIR) continuous wave (cw) laser microscopes. The laser-cell interaction is determined by a change of the momentum due to the beam direction being altered by refraction. In order to avoid laser absorption, NIR cw lasers such as the Nd:YAG laser at 1064 nm, the frequency doubled erbium:YAG fiber laser at 760 nm, the tunable cw Ti:sapphire ring laser, and laser diodes at wavelengths < 800 nm are employed. They are considered to be safe tweezer sources. However, two-photon absorption effects may occur due to the generation of high MW/cm 2 laser intensities when using tightly focused cw laser beams at a power of 100 mW or more. These nonlinear effects can be used for two-photon excited fluorescence spectroscopy of trapped objects. However, when using low-wavelength NIR (< 800 nm) traps, potential "UV-like" photodamaging effects have to be considered during cell manipulation. The use of the Nd:YAG laser at 1064 nm is recommended when using laser tweezers for optical sperm transport for laser-assisted in vitro fertilization (IVF).

Frontiers in Physics, 2022

Rapid advances in Biophotonics are revolutionizing the illumination of several diseases and, among them, the monitoring of cancer pathogenesis and therapy. Today, several efforts aim to miniaturize the Biophotonics tools, leading to the namely Nanobiophotonics. This scientific field refer to the development of novel technologies, biosensors, and drug delivery systems for prevention, diagnosis, and treatment of diseases at the nanoscale, in sub-cellular and molecular level. Modern non-invasive laser-based techniques are applied in different domains, from practical, clinical applications to molecular and cellular biology fundamental research. Among the plethora of photon-based techniques, optical trapping is a very promising tool for improving the understanding of cancer at cellular level. Recently, optical tweezers are revived as a potential technique for cell characterization, tracking cells behavior and probing interactions forces between cells, cells-biomolecules, and cells-nanopa...

Biophysical Journal, 1999

Multiphoton excitation (MPE) of fluorescent probes has become an attractive alternative in biological applications of laser scanning microscopy because many problems encountered in spectroscopic measurements of living tissue such as light scattering, autofluorescence, and photodamage can be reduced. The present study investigates the characteristics of two-photon excitation (2PE) in comparison with confocal one-photon excitation (1PE) for intracellular applications of fluorescence correlation spectroscopy (FCS). FCS is an attractive method of measuring molecular concentrations, mobility parameters, chemical kinetics, and fluorescence photophysics. Several FCS applications in mammalian and plant cells are outlined, to illustrate the capabilities of both 1PE and 2PE. Photophysical properties of fluorophores required for quantitative FCS in tissues are analyzed. Measurements in live cells and on cell membranes are feasible with reasonable signal-to-noise ratios, even with fluorophore concentrations as low as the single-molecule level in the sampling volume. Molecular mobilities can be measured over a wide range of characteristic time constants from ϳ10 Ϫ3 to 10 3 ms. While both excitation alternatives work well for intracellular FCS in thin preparations, 2PE can substantially improve signal quality in turbid preparations like plant cells and deep cell layers in tissue. At comparable signal levels, 2PE minimizes photobleaching in spatially restrictive cellular compartments, thereby preserving long-term signal acquisition.

Optical Diagnostics of Living Cells III, 2000

Two-photon fluorescence spectroscopy has been performed on rat skeletal muscles to investigate the effect of fixation processes on the micro-environments of the endogenous fluorophors in rat skeletal muscles. The twophoton fluorescence spectra measured for different fixation periods show a differential among those samples that were fixed in water, formalin and methanol, respectively. The results imply that two-photon fluorescence spectroscopy can be a potential technique for identification ofhealthy and malignant biological tissues.

Applied Optics, 2004

We demonstrate how optical tweezers can be incorporated into a multiphoton microscope to achieve three-dimensional imaging of trapped cells. The optical tweezers, formed by a cw 1064 nm Nd:YVO 4 laser, were used to trap live yeast cells in suspension while the 4Ј,6-diamidino-2-phenylindole-stained nucleus was imaged in three dimensions by use of a pulsed femtosecond laser. The trapped cell was moved in the axial direction by changing the position of an external lens, which was used to control the divergence of the trapping laser beam. This gives us a simple method to use optical tweezers in the laser scanning of confocal and multiphoton microscopes. It is further shown that the same femtosecond laser as used for the multiphoton imaging could also be used as laser scissors, allowing us to drill holes in the membrane of trapped spermatozoa.

Optics Letters, 1998

We report on scanning far-and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited f luorescence images are obtained for 50 -200 mW of average power at the sample. A nearly quadratic dependence of f luorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning farfield microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.