CC BY
34B-I-6
Macroscopic Time- and Spectrally Resolved Fluorescence
Imaging
V. Shcheslavskiy 12, M. Shirmanova 2, J. Lagarto 3'4, D. Yuzhakova 2, A. Mozherov 2, F.S.
Pavone 3'4'5, R. Cicchi 34 and W. Becker 1
1-Becker&Hickl GmbH, Nunsdorfer Ring 7-9, 12277Berlin, Germany 2-Privolzhskiy Research Medical University, Minin and Pozharsky Sq. 10/1, 603005 Nizhny Novgorod, Russia 3-1National Institute of Optics, National Research Council (INO-CNR), Via Nello Carrara 1, 50019, Sesto
Fiorentino, Italy
4-European Laboratory for Non-linear Spectroscopy (LENS), Via Nello Carrara 1, 50019, Sesto Fiorentino,
Italy
5-Department of Physics, University of Florence, Via G. Sansone 1, 50019, Sesto Fiorentino, Italy
vis@becker-hickl. de
Fluorescence lifetime imaging (FLIM) and optical spectroscopy techniques, such as fluorescence time-resolved spectroscopy and Raman spectroscopy are increasingly recognized as a valuable approach for its ability not only to assess structural information, but also to interrogate certain biochemical processes in living organisms and provide information on their functional characteristics [1,2]. In particular, multimodal approaches can enhance the specificity of optical measurements to deliver complimentary and multidimensional information from tissues, and, therefore, there has been much investigation in recent years focusing on increasing the practicality and versatility of multimodal instruments.
Usually FLIM is associated with Fluorescence Lifetime Imaging Microscopy. Consequently, it is often believed that FLIM can only be obtained from microscopic objects. In general, this is not true: FLIM can be combined with any optical technique that scans the sample with a focused beam of light. Centimeter sized objects can be scanned by placing them directly in the image plane of a confocal scan head. Here, we describe a macroscopic confocal laser scanning FLIM system applied for the whole tumor imaging. By placing a dispersive element between the macroscopic scanner and array of the detectors one can realize a macroscopic spectrally resolved FLIM. This arrangement provides an important opportunity not available in the conventional systems using bandpass filters: simultaneous observations of multiple fluorophores. We integrate a 16 channel spectral detector working in the single photon counting mode in the confocal macroscanner and demonstrate the capability of the system on the dispersed on the dish cells labelled with different fluorophores.
Finally, we report the development of a novel fiber-based system to realize co-registered simultaneous acquisition of fluorescence lifetime data and Raman spectra from the same area. Fluorescence lifetime measurements by means of time-correlated single photon counting are realized with periodic out-of-phase external illumination of the field of view, enabling acquisition of data under bright illumination of the specimen. Raman measurements in the near-infrared are realized asynchronously. We present a detailed characterization of this technique and validate its potential to report intrinsic contrast. Fiber-based fluorescence lifetime and Raman maps report complementary structural, compositional and molecular contrast in biological tissues with diverse compositional features.
The work related to spectral FLIM was supported by RSF, contract 20-65-46018.
[1] V.I. Shcheslavskiy, M.V. Shirmanova, A. Jelzow, and W. Becker, Multiparametric time-correlated single photon counting luminescence microscopy, Biochemistry (Mosc) 84, S51-S68 (2019).
[2] K. Suhling, L.M. Hirvonen, J. Levitt, P.-H. Chung, C. Tredigo, A. Marois et al, Advanced Time-correlated single photon counting applications (Springer), Chapter 3 (2015).
