Spectral characteristics of the flavones and anthocyanins present in passionflower (Passiflora incarnata).

Rich in antioxidants with a variety of flavones and anthocyanins, passionflower/fruit has been extensively used in food, beverage, medicinal, and natural dyes industries. The individual components present in passionflower are identified by extracting them in methanol, partitioning them between ethyl acetate and aqueous layers, and recording their ESI mass spectrometric data. The steady-state absorption and fluorescence spectra of the extract in methanol and dimethyl sulfoxide are recorded and the lifetime of the fluorescing species is reported. The pH dependence of the absorption spectrum confirms the presence of the anthocyanins.

Selective suppression of two-photon fluorescence in laser scanning microscopy by ultrafast pulse-train excitation.

Selective excitation of a particular fluorophore in the presence of others demands clever design of the optical field interacting with the molecules. We describe the use of 20- to 50-GHz pulse-train excitation leading to two-photon absorption, followed by successive one-photon stimulated emission as a potential technique in the context of controlling two-photon molecular fluorescence, with applications in microscopy.

Towards Stable Trapping of Single Macromolecules in Solution.

The implementation of high instantaneous peak power of a femtosecond laser pulse at moderate time-averaged power (~10 mW) to trap latex nanoparticles, which is otherwise impossible with continuous wave illumination at similar power level, has recently been shown [De, A. K., Roy, D., Dutta, A. and Goswami, D. "Stable optical trapping of latex nanoparticles with ultrashort pulsed illumination", Appd. Opt., 48, G33 (2009)]. However, direct measurement of the instantaneous trapping force/stiffness due to a single pulse has been unsuccessful due to the fleeting existence (~100 fs) of the laser pulse compared with the much slower time scale associated with the available trapping force/stiffness calibration techniques, as discussed in this proceeding article. We also demonstrate trapping of quantum dots having dimension similar to macromolecules.

Stable optical trapping of latex nanoparticles with ultrashort pulsed illumination.

Here we report how ultrafast pulsed illumination at low average power results in a stable three-dimensional (3D) optical trap holding latex nanoparticles which is otherwise not possible with continuous wave lasers at the same power level. The gigantic peak power of a femtosecond pulse exerts a huge instantaneous gradient force that has been predicted theoretically earlier and implemented for microsecond pulses in a different context by others. In addition, the resulting two-photon fluorescence allows direct observation of trapping events by providing intrinsic 3D resolution.

A systematic study on fluorescence enhancement under single-photon pulsed illumination.

We present a detailed study on fluorescence enhancement by 'stroboscopic' illumination with light pulses having duration ranging from few milliseconds to sub-picoseconds. We show how a delicate balance between pulse width and pulse repetition rate can result in an unprecedented fluorescence enhancement that has immediate applications in fluorescence imaging.

Exploring the nature of photo-damage in two-photon excitation by fluorescence intensity modulation.

We investigate the relative photo-damage effects during one- and two-photon excitations and demonstrate that there exist fundamental differences in the damage induced by a high repetition rate laser as compared to that of a CW laser. This difference is evident from the degree of enhanced fluorescence intensity achieved by blanking the excitation with an optical chopper. Such an enhancement in fluorescence intensity provides better signal-to-noise ratio that could have immediate applications in multiphoton imaging of live specimens.

Coherent Control in Multiphoton Fluorescence Imaging.

In multiphoton fluorescence laser-scanning microscopy ultrafast laser pulses, i.e. light pulses having pulse-width ≤ 1picosecond (1 ps = 10-12s), are commonly used to circumvent the low multiphoton absorption cross-sections of common fluorophores. Starting with a discussion on how amplitude modulation of ultrashort pulse-train enhances the two-photon fluorescence providing deep insight into laser-induced photo-thermal damage, the effect of controlling time lag between phase-locked laser pulses on imaging is described. In addition, the prospects of laser pulse-shaping in signal enhancement (by temporal pulse-compression at the sample) and selective excitation of fluorophores (by manipulating the phase and/or amplitude of different frequency components within the pulse) are discussed with promising future applications lying ahead.

Three-dimensional image formation under single-photon ultra-short pulsed illumination.

The major thrust of modern day fluorescence laser-scanning microscopy have been towards achieving better and better depth resolution embodied by the invention and subsequent development of confocal and multi-photon microscopic techniques. However, each method bears its own limitations: in having sufficient background fluorescence and photo-damage resulting from out-of-focus illumination for the former, while low multi-photon absorption cross-sections of common fluorophores for the latter. Here we show how the intelligent choice of single-photon ultrashort pulsed illumination can circumvent all these shortcomings by exemplifying the tiny spatial stretch of an ultrashort pulse. Besides achieving a novel way of optical sectioning, this new method offers improved signal-to-noise ratio as well as reduced photo-damage which are crucial for live cell imaging under prolonged exposure to light.

Towards Spatio-Temporal Control in Optical Trapping.

Using both continuous-wave (CW) and high repetition rate femtosecond lasers, we present stable 3-dimensional trapping of 1µm polystyrene microspheres. We also stably trapped 100nm latex nanoparticles using the femtosecond mode-locked laser at a very low average power where the CW lasers cannot trap, demonstrating the significance of the fleeting temporal existence of the femtosecond pulses. Trapping was visualized through dark-field microscopy as well as through a noise free detection using two-photon fluorescence as a diagnostics tool owing to its intrinsic 3-dimensional resolution. Comparison between a Gaussian versus a flat-top Gaussian beam profile demonstrates the importance of laser spatial mode in optical trapping.

Ultrafast pulse-pair control in multiphoton fluorescence laser-scanning microscopy.

In multiphoton fluorescence laser-scanning microscopy, ultrafast laser pulses [i.e., light pulses having pulse width