Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Summary

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Abstract

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Introduction

Obesity has become a global health problem threatening one third of the population around the world, and it presents a serious medical concern, given its association with poor mental health¹ and deadly diseases including diabetes², cardiovascular diseases³ and some types of cancer⁴. Study of lipid metabolism is essential to better understand the biological problems behind obesity. Rapid and specific quantification of lipid storage entails the detection of fatty acids and their derivatives, as well as sterol-containing metabolites, with high sensitivity and preferably with s....

Protocol

1. Instrumental setup for Stimulated Raman Scattering Microscopy

NOTE: The SRS microscopy system is built upon a picosecond laser with pump integrated optical parametric oscillator and a confocal laser scanning microscope. The oscillator provides two picosecond pulse trains, including a Stokes beam at 1,064 nm and a pump beam tunable between 700–990 nm. Temporal and spatial overlapping of the two beams are achieved inside the laser. A built-in electro-optic modulator (EOM) is designed spec.......

Discussion

In defense against obesity and its associated metabolic disorders, important research efforts have been implemented to better understand the regulatory mechanisms of lipid homeostasis. For quantitative detection of lipid molecules in biological samples, label-free imaging by SRS microscopy has been proven to be a reliable alternative to biochemical assays and other staining methods. Our group and others have revealed novel biological mechanisms in lipid metabolic regulation by combining the use of C. elegans and.......

Materials

Name	Company	Catalog Number	Comments
A/D converter	Olympus	Analog Unit
Agarose	GeneMate	3119	For making agarose pads
Alignment tool - adapter	Thorlabs	SM1A4	For mounting the tool on scope
Alignment tool - target	Thorlabs	VRC2SM1	For viewing IR laser
Alignment tool - tube	Thorlabs	SM1L40	Length can vary
Autocorrelator (Optional)	APE	pulseCheck
Bandpass filter	minicircuits	BBP-21.4+ if modulated at 20MHz or KR Electronics 2724 if modulated at 8 MHz	For signal with modulation frequency filtering
BNC cables
Dissection microscope	Nikon	SMZ800	For handling and picking worms for imaging
Dodecane	Sigma-Aldrich	44010	Used for calibration of the SRS signal
Filter	Chroma Technology	890/220 CARS	For removing Stokes beam
General purpose laboratory labeling tape	VWR	89097	For making agarose pads
Glass coverslips	VWR	48393-106	For covering worms for imaging
Glass microscope slide	VWR	16004-422	For making agarose pads
Laser scanning microscope	Olympus	FV3000
Lens	Thorlabs	L1: AC254-050-B L2: AC254-075-B	For beam expander
Lock-in amplifier	Zurich	HF2LI
Lowpass filter	minicircuits	BLP-1.9+	For power supply noise suppression
Mirrors	Thorlabs	BB1-E03	For relay and periscope
Objective	Olympus	UPlanSAPO 20x 0.75, UPlanSAPO 60XW 1.20
Photodiode	Thorlabs	FDS1010
Picosecond laser source	APE	picoEmerald
Power supply	TEKPOWER	TP1342U	For photodiode, reversed 50V voltage
Sodium azide	Sigma	S2002	For anaesthesizing the worms
Worm picker	WormStuff	59-AWP