Nonlinear microscopy to investigate brown and white adipocytes in three-dimensional tissue-mimicking environments

in comparison with natural adipose tissue

A Paul1, S Chen2, E Peris3, P Micallef3, M Haugh2, C Brännmark3, S Heilshorn2, I Wernstedt Asterholm3, A Enejder1

1 Chemical Biology, Chalmers University of Technology, Gothenburg. 2 Material Science and Engineering, Stanford University, California

3 Department of Metabolic Physiology, Gothenburg University.

References 1. Brännmark C, Paul A, Ribeiro D, et al. Increased Adipogenesis of Human Adipose-Derived Stem Cells on Polycaprolactone

Fiber Matrices, PLoS ONE. 2014, 9(11).

Introduction

The interest in brown adipose tissue (BAT) has increased during the last years due to its ability to turn energy (triglycerides) into heat, i.e. "wasting" energy. Hijacking this feature might be a way to treat obesity and reduce the energy stores in the white adipose tissue (WAT). We use nonlinear microscopy techniques to understand cell-matrix and cell-cell interactions in the in vivo adipose tissue and in in vitro systems to aid the development of better in vitro cell culture systems.

The research leading to these results has received funding from the People Program (Marie Curie Actions) of the European Union's Seventh Framework Program FP7/2007-2013/ under REA grant agreement nº[607842]. This poster reflects only the author’s views and the Union is not liable for any use that may be made of the information contained herein.

PDMS

Picosecond pulsed laser system: (HighQ/APE 7 ps, 76 MHz): Stokes: 1064 nm, 30 mW, Pump/Probe: 817 nm, 20 mW

contact: alexandra.paul@chalmers.se

ADSC in tissue-mimicking cell growth environments

polymer fibers

protein hydrogels

Multimodal Nonlinear

Microscopy

40x

Funded by the

European Union

Experimental Outline

Results In vitro

Results In vivo

White adipose tissue Brown adipose tissue

Multimodal Nonlinear Microscopy

3 2 1 0

S0

S1 fluorescence

internal conversion

excitation 1 photon, 2 photons

0 1 2

Two-photon excited fluorescence (TPEF)

unstained • Cytosolic components (flavins, NADH/NADPH) • Protein vesicles stained • Mitochondria (Rho123)

3 2 1 0

S0 excitation

SHG

Second Harmonic Generation (SHG)

unstained • Collagen • Myosin • Microtubuli 3

2 1 0

S0

probe

stokes

pump CARS

Coherent Anti-Stokes Raman Scattering (CARS)

unstained • lipids • proteins

β3 adrenergic receptor agonist (β3 ago)

Reactive Oxygen Species

N-Acetylcysteine (NAC)

White adipose tissue Beige adipose tissue

Male C57BL6 mice, 6 weeks old – treatment: 1 week: H2O or NAC, 10 days: vehicle or β3

• Multimodal nonlinear microscopy can be used to assess lipid and mitochondrial density and thus metabolic states of adipose tissue

• NAC preconditioning results in reduction of β3 adrenergic receptor agonist induced browning of white adipose tissue

Elastin-like polypeptide hydrogels • Imprinted with micropatterns • Chemically crosslinked • Stromal vascular fraction from rat seeded on top

aligned unaligned

5 μm 5 μm

CARS (lipids), TPEF (mitochondria)

CARS (protein hydrogel), TPEF (calcein)

40 µm 40 µm

CARS (protein hydrogel), TPEF (actin)

• Multimodal nonlinear microscopy can be used on hydrated protein hydrogels

• Micro-topography (from 4 – 0.6 μm) influences cell alignment

Electrospun polycaprolactone fibers [1] • Human adipose derived stem cells seeded on top • Differentiated to white adipocytes for 14 days

aligned unaligned

30 µm 30 µm

2D

aligned

unaligned

• More mature-like adipocytes for aligned matrices