Fluorescence advantages with microscopic spatiotemporal control

Abstract

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.

Keywords: Fluorescence imaging; femtosecond pulses; microscopy; multi-photon process; nanoparticle; spatiotemporal control; two-photon excitation.

Figure 1

Excitation scheme for commonly employed two-color pump-probe studies involving stimulated emission. The excitation and stimulated emission are shown as upward and downward thin arrows, respectively, while fluorescence is shown as broad downward arrow. Small black arrows pointing downwards indicate vibrational relaxation.

Figure 2

Excitation scheme in our one-color pulse-pair and pulse-train methods described here (a)–(c). The excitation and stimulated emission are shown as upward and downward thin arrows, respectively, while fluorescence is shown as broad downward arrow; the colors are chosen to specify the different wavelengths. Little downward black arrows indicate either vibrational relaxation (solid) or internal conversion (broken).

Figure 3

Change in the relative fluorescence intensity of (a) DAPI versus Texas Red and (b) DAPI versus Mito-tracker Red under one-color pulse-pair excitation. (c) Corresponding fluorescence suppression image of Texas Red relative to DAPI as a function of time delay between pulses under one-color pulse-pair excitation (similar suppression results for pulse train for this dye pair).

Figure 4

Comparing the effect of pulse train with pulse pair: Change in the relative fluorescence intensity of DAPI versus Mito-tracker Red under one-color (a) pulse-pair excitation and (b) pulse train excitation. (c) Corresponding fluorescence suppression image of Mito-tracker Red relative to DAPI as a function of time delay between the trains of pulse under one-color excitation (no suppression for pulse pair excitation for this dye pair).

Figure 5

Two-photon fluorescence for trapping of (a) 100nm particles and (b) 16 nm Q-dots. Inset in (a) shows the fluorescence from the 100 nm particle against a complete dark background. The red line in the both the panels is a guideline for the trapped fluorescence. The step in (a) represents the sequential trapping of two particles.