Mapping metabolic changes by noninvasive, multiparametric, high-resolution imaging using endogenous contrast

Liu, Zhiyi; Pouli, Dimitra; Alonzo, Carlo; Varone, Antonio; Karaliota, Sevasti; Quinn, Kyle P.; Münger, Karl; Karalis, Katia; Georgakoudi, Irene

doi:10.1126/sciadv.aap9302

Cited by 152 publications

(177 citation statements)

References 99 publications

(157 reference statements)

Supporting

Mentioning

165

Contrasting

Order By: Relevance

“…Here, we show that metabolic autofluorescence imaging can monitor macrophage heterogeneity during polarization and migration within a 3D microfluidic model of the TME. Metabolic autofluorescence imaging enables non-destructive monitoring of metabolic changes in individual, live cells within 3D in vitro cultures and in vivo tumors 31,33,34,[39][40][41][42]50,51,80 . Additionally, 3D microfluidic culture systems, such as the Stacks microfluidic device used in this study, provide simple platforms to recapitulate dynamic Representative heatmaps depicting changes in redox ratio with z-plane.…”

Section: Discussionmentioning

confidence: 99%

Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

Heaster

Humayun

et al. 2020

Preprint

View full text Add to dashboard Cite

Macrophages within the tumor microenvironment (TME) exhibit a spectrum of pro-tumor and antitumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate twophoton autofluorescence imaging of NAD(P)H and FAD to non-destructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours post-stimulation for mouse macrophages (RAW 264.7) stimulated with IFN-γ or IL-4 plus IL-13 in 2D culture, validating that autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse PyVMT or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor co-cultures exhibited significantly different FAD mean lifetimes and greater migration than mono-cultures at 24, 48, and 72 hours post-seeding. In co-cultures with primary human cancer cells, actively-migrating monocyte-derived macrophages had greater redox ratios (NAD(P)H/FAD intensity) compared to passively-migrating monocytes at 24 and 48 hours post-seeding, reflecting metabolic heterogeneity in this subpopulation of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free autofluorescence imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and imaging system provides unique insights into spatiotemporal tumorimmune crosstalk within the 3D TME.

show abstract

Section: Discussionmentioning

confidence: 99%

Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

Heaster

Humayun

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…The beige adipocytes were distinguished from white adipocytes based on their multilocular morphologies, and then their cytoplasmic autofluorescence signals were extracted for FLT fitting. The rise in free NADH fraction in brown and beige fat may be attributed to the thermogenesiselevated glycolysis which produces cytosolic NADH primarily in the free form ( Figure S1) [36]. Figure 2B,C shows the ratios of short-and long-FLT components of NADH and FAD fluorescence in each type of adipocyte, respectively.…”

Section: In Vivo Flim Imaging Of Adipose Tissuesmentioning

confidence: 98%

“…Figure 2B,C shows the ratios of short-and long-FLT components of NADH and FAD fluorescence in each type of adipocyte, respectively. Since the fluorescence quantum yield of NADH increase upon binding to proteins [50], the mitochondrial NADH generated in β-oxidation, which is mainly in protein-bound forms [36], has stronger fluorescence and would also contribute to the ORR increase in thermogenic adipocytes. As shown in Figure 2B and Table S1, the a 1 /a 2 values of the NADH FLT in the brown and beige fat increase by 42.4% and 46.3%, respectively, after NE administration.…”

Section: In Vivo Flim Imaging Of Adipose Tissuesmentioning

confidence: 99%

“…In our recent work, the ORR was used to monitor the thermogenesis of BAT in vivo [14]. NADH and FAD exhibit distinct fluorescence lifetimes (FLTs) in free and protein-bound forms, and the free-to-bound ratios of NADH or FAD can be used to discriminate different cellular metabolic states [33][34][35][36]. The result suggests that redox ratio imaging could probe the accelerated β-oxidation and enhanced metabolic activity in thermogenic adipocytes.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

In vivo study of metabolic dynamics and heterogeneity in brown and beige fat by label‐free multiphoton redox and fluorescence lifetime microscopy

Wei

Qin

et al. 2019

Journal of Biophotonics

View full text Add to dashboard Cite

In this work, the metabolic characteristics of adipose tissues in live mouse model were investigated using a multiphoton redox ratio and fluorescence lifetime imaging technology. By analyzing the intrinsic fluorescence of metabolic coenzymes, we measured the optical redox ratios of adipocytes in vivo and studied their responses to thermogenesis. The fluorescence lifetime imaging further revealed changes in protein bindings of metabolic coenzymes in the adipocytes during thermogenesis. Our study uncovered significant heterogeneity in the cellular structures and metabolic characteristics of thermogenic adipocytes in brown and beige fat. Subgroups of brown and beige adipocytes were identified based on the distinct lipid size distributions, redox ratios, fluorescence lifetimes and thermogenic capacities. The results of our study show that this label-free imaging technique can shed new light on in vivo study of metabolic dynamics and heterogeneity of adipose tissues in live organisms. K E Y W O R D Sbeige fat, fluorescence lifetime, optical redox ratio, thermogenesis

show abstract

“…The presented method allows to investigate the molecular content of plaque regions and to apply different morphological analysis to images of intrinsic SHG/TPEF emitters. Given the many recent applications of automated analysis of SHG and TPEF images , such approach could pave the way for fully automated diagnosis. Fast multiphoton imaging of large tissue sections could detect specific regions of interest (ROIs), which thereafter could be analyzed through fluorescence lifetime for obtaining more specific information.…”

Section: Introductionmentioning

confidence: 99%

Improved label‐free diagnostics and pathological assessment of atherosclerotic plaques through nonlinear microscopy

Baria

Nesi

Santi

et al. 2018

Journal of Biophotonics

View full text Add to dashboard Cite

Coronary heart disease is the most common type of heart disease caused by atherosclerosis. In fact, an arterial wall lesion centered on the accumulation of cholesterol-rich lipids and the accompanying inflammatory response generates a plaque, whose rupture may result in a thrombus with fatal consequences. Plaque characterization for assessing the severity of atherosclerosis is generally performed through standard histopathological examination based on hematoxylin/eosin staining, which is operator-dependent and requires relatively long procedures. In this framework, nonlinear optical microscopy is a valid, label-free alternative to standard diagnostic methods. We combined second-harmonic generation (SHG), two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy in a multimodal scheme for obtaining morphological and molecular information on human carotid ex vivo specimens affected by atherosclerosis. In this study, discrimination between different tissues within the atherosclerotic plaque was achieved based on both lifetime, TPEF-to-SHG ratio, and image pattern analysis. The presented methodology aims to be a starting point for future fully automated and fast characterization of atherosclerotic biopsies; moreover, it could be extended to the study of other tissues and pathologies. Combined TPEF/SHG mapping of a carotid specimen affected by atherosclerosis.

show abstract

Mapping metabolic changes by noninvasive, multiparametric, high-resolution imaging using endogenous contrast

Abstract: Two-photon imaging provides noninvasive, label-free, quantitative assays of metabolic changes at the single-cell or tissue level.

Cited by 152 publications

References 99 publications

Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

In vivo study of metabolic dynamics and heterogeneity in brown and beige fat by label‐free multiphoton redox and fluorescence lifetime microscopy

Improved label‐free diagnostics and pathological assessment of atherosclerotic plaques through nonlinear microscopy

Contact Info

Product

Resources

About