We report the observation of two-photon fluorescence excitation and cell confinement, simultaneously, in a continuous-wave (cw) single-beam gradient force optical trap, and demonstrate its use as an in-situ probe to study the physiological state of an optically confined cell sample. At the wavelength of 1064 nm, a single focused gaussian laser beam is used to simultaneously confine, and excite visible fluorescence from, a human sperm cell that has been tagged with propidium iodide, a exogenous fluorescent dye that functions as a viability assay of cellular physiological state. The intensity at the dye peak emission wavelength of 620 nm exhibits a near-square-law dependence on incident trapping beam photon laser power, a behavior consistent with a two-photon absorption process. In addition, for a sperm cell held stationary in the optical tweezers for a period of several minutes at a constant trapping power, red fluorescence emission was observed to increase the time, indicating that the cell has gradually transitioned between a live and dead state. Two-photon excited fluorescence was also observed in chinese hamster ovary cells that were confined by cw laser tweezers and stained with either propidium iodide or Snarf, a pH-sensitive dye probe. These results suggest that, for samples suitably tagged with fluorescent probes and vital stains, optical tweezers can be used to generate their own in-situ diagnostic optical probes of cellular viability or induced photodamage, via two-photon processes.

T-cell contact with antigen-presenting B cells initiates an activation cascade which includes an increase in T-cell [Ca 2+] and leads to T-cell differentiation and proliferation. We evaluated cell-cell contact requirements for T-cell activation by using an optical trap to control the orientation of T-cell/B-cell pairs and fluorescence microscopy to measure subsequent T-cell [Ca 2+] responses. B cells were trapped with a titanium-sapphire laser tuned to 760 nm and placed at various locations along the T cells, which had a polarized appearance defined by shape and the direction of crawling. T-cell intracellular [Ca 2+] was detected as an emission shift from the combination of fura-red and fluo-3, two cytoplasmic [Ca 2+] indicators. T cells which were presented antigen at the leading edge had a higher probability of responding (84% vs. 31%) and a shorter latency of response (42 s vs. 143 s) than those contacting B cells with their trailing end. Similar results were obtained using beads coated with antibodies to the T-cell receptor. These findings demonstrate a role for initial T-cell/B-cell contact geometry in T-cell activation by showing that the T cell is a polarized antigen sensor.

The stimulated emission process has been rarely exploited in spectroscopy and microscopy. Instead, a spectroscopy method based on the pump-probe principle has been frequently used to observe picosecond and femtosecond processes. This common approach has not been applied to microscopy due to the relatively slow acquisition time and the lack of 3D information. We have exploited an idea originally proposed by F. Lytle group in which two pulsed lasers are simultaneously focused on the sample. One laser is used to excite a population of molecules and the second laser to induce stimulated emission. The stimulated radiation is carried away in the same direction of the stimulating laser beam. By collecting the fluorescence emission in other directions, we observe a modulation of the fluorescence signal as a function of the delay between the two laser pulses. The repetition rate of the two lasers is slightly different producing a frequency beating at the laser overlapping volume. We have extended this method to achieve very high spatial and temporal resolution in the microscope environment. By recording only the beating frequency, we obtain a 3D sectional effect similar to two-photon excitation. The harmonic content of the beating signal is limited by the laser pulse width and by the sample frequency response. Information of picosecond processes are extracted by standard frequency-domain methods. Using this principle, we built a stimulated emission microscope that has 3D and fluorescence lifetime capabilities.

We have designed and constructed a near-infrared spectrometer for the non-invasive optical study of biological tissue. This instrument works in the frequency-domain and employs multiple source-detector distances to recover the absorption coefficient (μa) and the reduced scattering coefficient (ixj) of tissue. The light sources are eight light emitting diodes (LEDs) whose intensities are modulated at a frequency of 120 MHz. Four LEDs emit light at a peak wavelength of 715 nm (λ1), while the other four LEDs emit at a peak wavelength of 850 nm (X2). From the frequency-domain raw data of phase, DC intensity, and AC amplitude obtained from each one of the eight light sources, which are located at different distances from the detector fiber, we calculate μa and μs at the two wavelengths λ1 and λ2. The concentrations of oxy- and deoxy-hemoglobin, and hence hemoglobin saturation, are then derived from the known extinction coefficients of oxy- and deoxy-hemoglobin at λ1 and λ2. The statistical error in the measurement of the optical coefficients due to instrument noise is about 1-2%. The accuracy in the determination of the absolute value of the optical coefficients is within 10-20%. Preliminary results obtained in vivo on the forearm of a volunteer during an ischemia measurement protocol are presented.