SyncRGB-FLIM: synchronous fluorescence imaging of red, green and blue dyes enabled by ultra-broadband few-cycle laser excitation and fluorescence lifetime detection

Abstract

We demonstrate for the first time that an ultra-broadband 7 femtosecond (fs) fewcycle laser can be used for multicolor nonlinear imaging in a single channel detection geometry, when employing a time-resolved fluorescence detection scheme. On a multichromophore-labelled cell sample we show that the few-cycle laser can efficiently excite the multiple chromophores over a >400 nm two-photon absorption range. By combining the fewcycle laser excitation with time-correlated single-photon counting (TCSPC) detection to record two-photon fluorescence lifetime imaging microscopy (FLIM) images, the localization of different chromophores in the cell can be identified based on their fluorescence decay properties. The novel SyncRGB-FLIM multi-color bioimaging technique opens the possibility of real-time protein-protein interaction studies, where its single-scan operation translates into reduced laser exposure of the sample, resulting in more photoprotective conditions for biological specimens.

3D Polymer Architectures for the Identification of Optimal Dimensions for Cellular Growth of 3D Cellular Models

“…While in the present study the scaffold design is deliberately kept as simple as possible a more complex 3D design would provide more detailed information about the cellular behavior on the scaffold, where especially the limited scaffold height in the current study could be extended. In this case, confocal imaging would not be ideal since its limited sample penetration capabilities, but could be done with standard or even advanced multiphoton imaging techniques with extended depth penetration [ 35 ]. Multiphoton imaging would also provide the possibility to do label free imaging of the seeded cells via metabolic markers [ 36 ].…”

Analysis of Combination Frequencies Arising from the Ionization of Gases by Multicolor Femtosecond Pulses

“…At present, the processes associated with the generation of secondary radiation in gases under the action of ionizing laser pulses are widely studied in connection with the possibility of creating compact sources of frequency-tunable super-broadband coherent radiation in wide ranges of central wavelengths (100-10,000 nm) [1][2][3][4][5]. Within this range, several different subranges can be distinguished, each of which is relevant for different applied areas; as a rule, such ultrashort pulses are in demand for ultra-precise measurements and spectroscopy [6,7], control of various quantum or chemical processes [8,9], biomedical imaging [10], while the possibility of frequency tuning further expands the range of applicability of such sources. This can be done using the frequency tuning of the ionizing pulse components in optical parametric generators [11][12][13].…”

Characterizing ultrashort laser pulses with second harmonic dispersion scans

“…Since its invention, the d-scan has become a well-established technique in many laboratories around the world. It has been implemented and tested with different target pulse widths and central frequencies, and d-scan-compressed pulses have enabled a variety of applications ranging from pump-probe spectroscopy to biomedical imaging [34][35][36][37].…”