Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging

Abstract

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two-photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy-to-operate platform capable to perform two-photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples.

Third harmonic generation microscopy of cells and tissue organization

“…To enhance current microscopy practice, THG can be combined with other multiphoton-excited fluorescence intensity signals or with fluorescence lifetime imaging (FLIM) within the same excitation and detection setup (Pelegati et al, 2012) (Box 1). For instance, multi-parameter approaches have combined label-free SHG and THG with fluorescence imaging (Fig.…”

Multimodal spectroscopy of single fluorescent nanoprobes : photophysics and characterization

“…Interestingly, the underlying mechanism of blinking varies between emitter classes. Although the exact nature of blinking is still being widely debated [26], both blinking and bleaching already play an essential role in recently developed super-resolution microscopy techniques like STORM, PALM, RESOLFT, and BLINK [27][28][29][30][31][32][33].…”

“…Since it has been shown that blinking statistics depends on excitation power, [26,27] we adjusted the excitation power to yield equal emission intensity for the different excitation wavelengths. The excitation power that was used was kW/cm 2 , comparable to the excitation power typically used in other studies [28][29][30][31][32].…”

“…Moreover, multimodal FLIM systems are already being used for in-vivo diagnostics [28] or are combined with non-linear harmonic generation or multiphoton microscopy [29][30][31][32][33] to resolve more details in biological tissue. In the future, multimodal FLIM is expected to continuously expand, for example, moving towards FRET based microscopy in living cells [34][35][36], and could also be combined with numerous other fluorescence-based techniques [37] to expand the modality of the system, thus providing an increasing set of observables on processes occurring at the nanoscale.…”

“…A number of parameters that characterize single emitter emission are now routinely accessible at ambient temperatures, including emission intensity and polarization [22,23], fluorescence lifetime [24][25][26], and the emission spectrum [27][28][29]. Access to these parameters yields unique insights into distinct properties of single emitters, and enables the determination of the distributions of the relevant experimental parameters, revealing, for example, distinct substates and energetic levels in a heterogeneous population [30,31]. Room temperature excitation spectroscopy of single quantum dots…”