Ultra-high Information-content Chemical Imaging with Broadband Coherent Anti-Stokes Raman and Two-photon Fluorescence Lifetime Microscopy

Summary

This article demonstrates amultimodal imaging platform combining broadband coherent anti-Stokes Raman scattering (BCARS), two-photon excitation fluorescence (TPEF), and two-photon fluorescence lifetime imaging microscopy (2p-FLIM), enabling ultra-high information-content chemical bioimaging.

Abstract

Raman fingerprint spectroscopy and fluorescence lifetime imaging are emerging tools for studying metabolic profiles of biological specimens. While Raman fingerprint spectroscopy detects intrinsic molecular vibrations that reflect the molecular composition and chemical environment of a sample, fluorescence lifetime imaging measures changes in the excited-state lifetime of fluorophores that are sensitive to their microenvironments. Here, we present a multimodal imaging platform combining broadband coherent anti-Stokes Raman scattering (BCARS) and two-photon fluorescence lifetime imaging (2p-FLIM) microscopy that can acquire biologically relevant Raman fingerprint spectra and fluorescence lifetime signals in vivo and simultaneously. The tremendous chemical information obtained from spatially co-registered BCARS and 2p-FLIM images allows us to characterize the subtle differences between sub-cellular compartments and verify the potential false-positive results generated by fluorescence imaging alone. This is demonstrated by directly comparing the BCARS, 2p-FLIM, and two-photon excitation fluorescence (TPEF) signals simultaneously obtained from the same dye-stained organelle in the live, intact C. elegans expressing a green fluorescent protein (GFP) marker. In this work, we introduce the BCARS/2p-FLIM/TPEF setup scheme, the image acquisition steps, data processing, and representative results showing that the cross-modality imaging method enables rigorous characterization and in vivo detection at sub-cellular resolution. This protocol provides a framework for simultaneous chemical and fluorescence lifetime imaging to improve the accuracy of biological interpretation in complex living systems.

Introduction

One challenge in biological studies is to accurately detect biological building materials in their native states and within the intact architecture of cells and tissues. Raman spectroscopy shows unprecedented potential for detecting intrinsic metabolites and chemical species within biological specimens and has been recently recognized as an emerging tool for metabolic profiling of biological specimens¹. By analyzing the peaks of various metabolic groups shown in Raman fingerprint spectra, increasing evidence shows that the fingerprint features can be used to detect the early stages of diseases such as cancer^2,....

Protocol

1. C . elegans sample preparation and Nile Red Vital staining

NOTE: C. elegans has been heavily used for multiple imaging techniques due to its whole-body transparency. The internal organs and tissues can be easily imaged in the intact animal without dissection. In addition, ethics approval is not required for research involving C. elegans, as they are not considered vertebrates and are not subject to the same ethical regulations as higher animals like rodents. This section describes the synchronization of C. elegans worms and the preparation of Nile Red-stained samples for fluorescence ima....

Results

Figure 5 shows the measured spectra of the narrowband probe and the broadband supercontinuum (SC) pulses used for BCARS excitation. The probe pulse, generated via second harmonic generation of a ~1530 nm signal from a home-built optical parametric oscillator (OPO, Figure 3), is centered at 764.2 nm with a full width at half maximum (FWHM) of 0.2 nm, providing the spectral resolution at ~6.8 cm^-1 in the resulting Raman spectra. The SC pulse, p.......

Discussion

This work demonstrates the architecture of BCARS and 2p-FLIM/TPEF imaging platform in detail. While a dual-output 1030 nm laser is used in our current setup, most high-power fs 1030 nm fiber lasers can be used as a master laser source for building a BCARS microscope. By carefully choosing the length and period of OPO/SHG PPLN crystals, generating a few-ps probe pulse with a sub-ps OPO 1030 nm pump is achievable. Although a relatively high laser repetition rate (100 MHz) or a relatively lower pulse peak power is demonstra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Camera	Hamamatsu	ORCA-Fusion	sCMOS, 2304×2304 pixels
Collection objective	Olympus	LUMPLFLN60XW	Water immersion, 1.0 NA
DAQ	Diligent	MCC USB-1208HS	data aquisition card
Delay line	Newport	DL225	Mechanical delay for probe pulse
Dichroic	Semrock	FF925-Di01	combining probe and SC
Dichroic	Semrock	FF705-Di01	detecting epi-mode fluorescence
Dichroic	Thorlabs	DMLP550R	seperating the FLIM signal and the TPEF signal
Excitation objective	Olympus	UPLSAPO60XWIR	Water immersion, 1.2 NA
Filter	Semrock	FF01-1326/SP-25	Reject the fundamental laser after SHG
Filter	Semrock	FF01-758/SP	Reject the fundamental laser before BCARS camera
Filter	Thorlabs	FESH0650	Reject the fundamental laser before the fluorescence modality
FLIM PMT	PicoQuant	PMA Hybrid	FLIM single photon detector
FPGA	Redpitaya	STEMlab 125-14	Scan synchronization and triggering
Galvo	Thorlabs	GVS101	For scanning and descanning
Grating	Coherent	LightSmyth 1040nm	1000 Groves/mm, pre-chirp for SC
Half-wave plate	Thorlabs	AHWP05M-980	Polarization control
K-cube	Thorlabs	KPA101	PSD driver for the auto-aligner
Laser	Prospective Instruments	FSX-Dual	Dual-output femtosecond laser
Laser	Coherent	Mira-900	Femtosecond laser for fluorescence excitation
PBS	Thorlabs	PBS255	Beam combination for SC and probe
PCF	Thorlabs	LMA-PM-5	10 cm photonic crystal fiber for SC generation
PD	Thorlabs	PDA100A2	OPO feedback loop
Piezo mirror mount	Thorlabs	POLARIS-K1S2P	Piezoelectric Adjuster for the auto-aligner
Pre-amp	EdmundOptics	59-179	TPEF pre-amplifier
Prism	Lambda Research optics	EQP-30SF10	SC compression
PSD	Thorlabs	PDQ80A	Position Sensitive Detector for the auto-aligner
Spectrometer	Teledyne Princeton Instruments	IsoPlane 160	300 g/mm grating, centered at 700 nm
Stage	ASI	MS-2000	Motorized 3-axis stage
TCSPC	PicoQuant	MultiHarp 150	Photon counting device for FLIM
TPEF PMT	Hamamatsu	H9305-03	TPEF detector
Tube Lens	Thorlabs	TL200-2P2	Used in descanning system