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

Abstract

In multiphoton fluorescence laser-scanning microscopy, ultrafast laser pulses [i.e., light pulses having pulse width <or=1 ps (1 ps=10(-12) s)] are commonly employed to circumvent the low-multiphoton absorption cross-sections of common fluorophores. Because of the broad overlapping two-photon absorption spectra of fluorophores and the large spectral bandwidth of a short pulse, simultaneous excitation of many fluorophores is common, which justifies a persistent demand for selective excitation of individual fluorophores. We describe the use of pulse-pair excitation with possibilities of controlling molecular fluorescence in laser-scanning microscopy and compare it with coherent control using pulse sequence [De and Goswami, "Coherent control in multiphoton fluorescence imaging," Proc. SPIE 7183, 71832B (2009)].

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

“…However, control can also be achieved beyond coherence time scales using stimulated emission, as recently shown by our group. 11,12 Extending the previous work using pulse-pair excitation, here we demonstrate how 20-to 50-GHz pulse-train excitation can selectively suppress fluorescence from one particular fluorophore by stimulated emission, which is otherwise not possible using a pulse-pair scheme.…”

“…For pulse-pair excitation, we used a collinear MachZehnder-type interferometer described elsewhere and repeated our earlier experiment. 11 We generated the pulse train with a Fabry-Perot etalon made of a pair of 50/50 beamsplitters 3 mm thick [Castech (Hamilton, New Zealand) BSP-254-030-780]: Due to multiple reflections between the coated surfaces, each pulse generates a pulse train as shown in Fig. 1(a); the intensity of the successive pulses decreases in geometric progression.…”

Selective two-photon fluorescence suppression by ultrafast pulse-pair excitation: control by selective one-color stimulated emission

“…1(a)]. This is precisely the reason for our observation of fluorescence suppression over a long time scale (several tens of picoseconds, corresponding to experimentally maximum accessible time delay accessible with the delay stage 13–15 ).…”

“…(incoherent) population dynamics, where the only “control knob” is the time delay between the pairs. Earlier our group reported selective TPF suppression using a pulse-pair excitation scheme 13 which was explained based on selective stimulated emission by a time-delayed second pulse following the excitation pulse; 14 the control is achieved by simultaneous two-photon absorption (TPA) by two different fluorophores followed by selective one-photon stimulated emission for one particular fluorophore. Here we further explore the mechanistic detail of such one-color control scheme in depth.…”

Towards controlling molecular motions in fluorescence microscopy and optical trapping: a spatiotemporal approach

“…Based on our earlier observation [58,59], we used pulse-pair excitation ( Figure 6(b), (c)) employing a Mach-Zehnder type interferometer for selective fluorescence suppression [60,61]. Here, the first pulse causes a TPA for both DAPI and Texas Red; the time-delayed second pulse causes similar TPA as the first pulse does but, in addition, may dump the population by (one-photon) stimulated emission if the red edge of fluorescence coincides with the excitation wavelength.…”