In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Summary

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Abstract

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

Introduction

Raman microscopy^1,2 is emerging as a powerful label-free method to image biological tissues based on the characteristic frequencies of various chemical bonds in biomolecules. Owing to its non-invasive and well-adaptive imaging capability, Raman microscopy has been widely used for imaging lipid-enriched components in biological tissues like myelin sheath^3,4,5, adipocytes^6,7, and lipid droplets^8,9,<....

Protocol

All animal procedures performed in this work are conducted according to the guidelines of the Laboratory Animal Facility of the Hong Kong University of Science and Technology (HKUST) and have been approved by the Animal Ethics Committee of HKUST. Safety training for laser handling is required to set up and operate the TPEF-SRS microscope. Always wear laser safety goggles with appropriate wavelength range when dealing with laser.

1. Setup of the TPEF-SRS microscope (for setup schematic se.......

Discussion

In this protocol, the basic setup of the TPEF-SRS microscope is described in detail. For SRS imaging, the pump and Stokes beams are temporally and spatially overlapped inside the OPO. However, this overlapping can be disrupted after passing through the microscope system. Therefore, both spatial and temporal optimization of the colocalization of the pump and Stokes beams is necessary and critical to achieving optimal SRS imaging. The temporal delay between the pump and Stokes beam is related to the optical path diffe.......

Materials

Name	Company	Catalog Number	Comments
#2 Forceps	Dumont	11223-20	For laminectomy
10X objective	Nikon	CFI Plan Apo Lambda 10X
25X objective	Olympus	XLPLN25XSVMP2
Burn cream	Betadine
Camera	Sony	α6300
Current amplifier	Stanford research	SR570
Current photomultiplier modules	Hamamatsu	H11461-01
D2 665 nm long-pass dichroic mirror	Semrock	FF665-Di02-25x36	For directing epi-fluorescence signal to the detection module
D3 700 nm short-pass dichroic mirror	Edmund	69-206	For separating SRS from TPEF detection path
Depilating cream	Veet
FS1 975 nm short-pass filter	Edmund	86-108	For blocking stokes beam
FS1 Bandpass filter	Semrock	FF01-850/310	For blocking stokes beam
Fs2 Bandpass filter	Semrock	FF01-525/50	For selecting YFP signal
Fs2 Shortpass filter	Semrock	FF01-715/SP-25	For blocking fs excitation laser beam
Half-wave plate	Thorlabs	SAHWP05M-1700
High-speed photodetector	MenloSystems	FPD 310-F	For checking Stokes beam modulation
Iodine	Betadine
IR Scope	FJW	FIND-R-SCOPE Infrared Viewer 2X Kit Model 84499C2X
Iris	Thorlabs	CPA1
L1	Thorlabs	AC254-060-B-ML
L10	Thorlabs	LA4052-A
L2	Thorlabs	LA1422-B
L3	Thorlabs	AC254-050-B
L4	Thorlabs	AC254-060-B-ML
L7			f=100 mm, AB coating
L8	Thorlabs	LA4874-A
L9	Thorlabs	AC254-035-B-ML
Lock-in amplifier	APE
Mirror	Thorlabs	PF10-03-P01
Motorized flipper	Thorlabs	MFF101/M
multifunctional acquisition card	National Instrument	PCIe-6363
Oscilloscope	Tektronix	TDS2012C
Photodiode	APE		For detecting SRS signal
Picosecond laser source	APE	picoEmerald
Polarizing beam splitter	Thorlabs	CCM1-PBS252/M
Power meter	Newport	843-R
Saline	Braun
Scan lens L5	Thorlabs	SL50-CLS2
Scanning mirror	Cambridge Technology	6215H
Silicone gel	World Precision Inc.	KWIK-SIL
Ti:sapphire fs laser	Coherent	Chameleon Ultra II
Tube lens L6	Thorlabs	TTL200-S8