Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment

Abstract

Cancer metastasis involves complex cell behavior and interaction with the extracellular matrix by metabolically active cells. To observe invasion and metastasis with sub-cellular resolution in vivo, multiphoton microscopy (MPM) allows imaging more deeply into tissues with less toxicity, compared with other optical imaging methods. MPM can be combined with second harmonic generation (SHG), fluorescent lifetime imaging microscopy (FLIM), and spectral-lifetime imaging microscopy (SLIM). SHG facilitates imaging of stromal collagen and tumor-stroma interactions, including the architecture and remodeling of the tumor microenvironment. FLIM allows characterization of exogenous and endogenous fluorophores, such as the metabolites FAD and NADH to score for metabolic state and provide optical biomarkers. SLIM permits additional identification and separation of endogenous and exogenous fluorophores by simultaneously collecting their spectra and lifetime, producing an optical molecular "fingerprint". Both FLIM and SLIM also serve as an improved method for the assessment of Förster (or fluorescence) resonance energy transfer (FRET). Hence, the use and further development of these approaches strongly enhances the visualization and quantification of tumor progression, invasion, and metastasis. Herein, we review recent developments of multiphoton FLIM and SLIM to study 2D and 3D cell migration, invasion into the tumor microenvironment, and metastasis.

Tissue Optical Clearing, Three-Dimensional Imaging, and Computer Morphometry in Whole Mouse Lungs and Human Airways

“…Our method overcomes significant analysis hurdles and can automatically quantify a labeled structure (nerves in this case) in the context of its 3D tissue compartment. The approach can be readily used to study other sparse and heterogeneous structures, such as rare sites of microchimerism where fetal cells travel into the lung during pregnancy or the earliest micron-sized sites of tumor metastases (43,44). Whole lung microscopic imaging has the potential to fill the gap between large-scale (e.g., CT, MRI) and small-scale (e.g., fluorescence microscopy) imaging data to provide a comprehensive analysis of cellular/ subcellular structures throughout the lung.…”

Intravital microscopy: new insights into metastasis of tumors

“…components exhibit comparable excitation and emission spectra, the resulting fluorescence levels are similar and cannot be used to distinguish tissues. However, as the lifetime of autofluorescence differs between different cell types, autofluorescence lifetime measurements have successfully been used to distinguish healthy tissues from tumor tissues (Galletly et al, 2008;Provenzano et al, 2009). …”

Imaging molecular dynamics in vivo – from cell biology to animal models

“…MPLSM has revolutionized the study of immune cell dynamics (reviewed in Garside and Brewer, 2008), Ca 2+ dynamics (reviewed in Gobel and Helmchen, 2007) and the mechanisms underlying tumor cell invasion of host tissue (reviewed in Provenzano et al, 2009). In a seminal study Ahmed et al used multi-photon microscopy to characterise tissue-specific expression of GFP in the mammary glands of transgenic mice (Ahmed et al, 2002).…”

“…Of particular interest is the use of NADH fluorescence to read out the oxidative state in cells and as a marker of hypoxic regions in tumors (reviewed in Provenzano et al, 2009). It has been established that free NADH has a short fluorescence lifetime of around 400 pseconds, whereas the lifetime increases to between 1000 and 3700 pseconds when it is enzyme bound (Lakowicz et al, 1992b;Niesner et al, 2008).…”