We present a clever design concept of using femtosecond laser pulses in microscopy by selective excitation or de-excitation of one fluorophore over the other overlapping one. Using either a simple pair of femtosecond pulses with variable delay or using a train of laser pulses at 20-50 Giga-Hertz excitation, we show controlled fluorescence excitation or suppression of one of the fluorophores with respect to the other through wave-packet interference, an effect that prevails even after the fluorophore coherence timescale. Such an approach can be used both under the single-photon excitation as well as in the multi-photon excitation conditions resulting in effective higher spatial resolution. Such high spatial resolution advantage with broadband-pulsed excitation is of immense benefit to multi-photon microscopy and can also be an effective detection scheme for trapped nanoparticles with near-infrared light. Such sub-diffraction limit trapping of nanoparticles is challenging and a two-photon fluorescence diagnostics allows a direct observation of a single nanoparticle in a femtosecond high-repetition rate laser trap, which promises new directions to spectroscopy at the single molecule level in solution. The gigantic peak power of femtosecond laser pulses at high repetition rate, even at low average powers, provide huge instantaneous gradient force that most effectively result in a stable optical trap for spatial control at sub-diffraction limit. Such studies have also enabled us to explore simultaneous control of internal and external degrees of freedom that require coupling of various control parameters to result in spatiotemporal control, which promises to be a versatile tool for the microscopic world.
Controlling two-photon molecular fluorescence leading to selective fluorophore excitation has been a long sought after goal in fluorescence microscopy. In this letter, we thoroughly explore selective fluorescence suppression through simultaneous two-photon absorption by two different fluorophores followed by selective one-photon stimulated emission for one particular fluorophore. We achieve this by precisely controlling the time delay between two identical ultrafast near infrared laser pulses.
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.
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.
The broad spectral window of an ultra-short laser pulse and the broad overlapping multiphoton absorption spectra of
common fluorophores restrict selective excitation of one fluorophore in presence of others during multiphoton
fluorescence microscopy. Also spatial resolution, limited by the fundamental diffraction limit, is governed by the beam
profile. Here we show our recent work on selective fluorescence suppression using a femtosecond pulse-pair excitation
which is equivalent to amplitude shaping using a pulse shaper. In addition, prospects of laser beam shaping in imaging
are also briefly discussed.
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.