Multiphoton fluorescence excitation in continuous-wave infrared optical traps.

The multiphoton fluorescence excitation observed in acontinuous-wave (cw) single-beam gradient force optical trap(optical tweezers) is reported for latex beads labeled with ayellow-green fluorescent dye (BODIPY). The fluorescenceemission spectra of the yellow-green beads trapped and excited by thesame 1064-nm laser light are identical to the spectra excited by the365-nm UV light. The influence of the numerical aperture of theobjective on the slope of the log-log power-dependence has beendemonstrated for BODIPY-Oil solution under cw and pulsed-laserconditions.

Detection of respiratory enzyme activity in Giardia cysts and Cryptosporidium oocysts using redox dyes and immunofluorescence techniques.

The fluorescent redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), combined with fluorescein-labeled antibodies, was tested for the simultaneous detection of the respiratory electron transport system (ETS) activity and enumeration of Giardia cysts and Cryptosporidium oocysts by spectral microfluorometry and epifluorescence microscopy. The reduction of CTC and p-iodonitrotetrazolium violet (INT), a non-fluorescent redox dye, was compared with propidium iodide (PI) and fluorescein diacetate (FDA) for the measurements of Giardia cyst viability over time. According to the PI and FDA staining techniques, nearly 60% of the cysts tested viable at the beginning of the observations; after 21 days their viability decreased to 5%.

Effect of pentoxifylline on the intrinsic swimming forces of human sperm assessed by optical tweezers.

It is still controversial whether in vitro exposure of sperm to pentoxifylline increases sperm motility and force, which is defined as the product of velocity by beat frequency of the tail. Laser optical tweezers have been successfully used in the past to evaluate sperm force in basal conditions. The aim of this prospective study was to determine whether exposure of human sperm to pentoxifylline has any effect on sperm intrinsic forces.

Cell Viability and DNA Denaturation Measurements by Two-Photon Fluorescence Excitation in CW Al:GaAs Diode Laser Optical Traps.

Cell viability and DNA denaturation are measured through two-photon fluorescence excitation using a single diode laser beam as the trapping and exciting source simultaneously. Two-photon fluorescence emission spectra are presented for CHO cells and T lymphocytes loaded with various fluorescent probes. This single beam method is demonstrated to be a safe tool to monitor the viability of optically trapped cells, even under intense 809 nm diode laser illumination (∼106 W/cm2).

Physiological monitoring of optically trapped cells: assessing the effects of confinement by 1064-nm laser tweezers using microfluorometry.

We report the results of microfluorometric measurements of physiological changes in optically trapped immotile Chinese hamster ovary cells (CHOs) and motile human sperm cells under continuous-wave (CW) and pulsed-mode trapping conditions at 1064 nm. The fluorescence spectra derived from the exogenous fluorescent probes laurdan, acridine orange, propidium iodide, and Snarf are used to assess the effects of optical confinement with respect to temperature, DNA structure, cell viability, and intracellular pH, respectively. In the latter three cases, fluorescence is excited via a two-photon process, using a CW laser trap as the fluorescence excitation source.