Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Summary

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Abstract

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 µm. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

Introduction

In life science applications, SRS microscopy has emerged as powerful tool for label-free imaging. The basic idea of SRS microscopy is to combine the strength of vibrational contrast and its ability to acquire images in a few seconds.

SRS is a process in which the frequency difference between two laser beams frequencies (pump signal and stokes signal at different frequencies) matches the molecular vibration of an investigated sample, causing stimulated Raman scattering and a significant increase in the Stokes signal. Unlike linear Raman spectroscopy, SRS exhibits a nonlinear dependence on the incoming light fields and produces coherent radia....

Protocol

1. Starting up the laser system

1. Check if the temperature of chillers is maintained at or below 20 °C.
2. Check if the humidity control unit is working properly and humidity is maintained at a value around 40%.
3. Turn on the Ti:Sa laser, strictly following the instructions in the manual.
4. Set the wavelength to 810 nm.
5. Turn on the OPO and the connected mini-computer. Run the application that controls the OPO laser.
6. Select bypass if 100% of the Ti:Sa laser output is required at the exit of the OPO box.
7. Deselect bypass if 20% of the Ti:Sa laser output and the OPO laser....

Results

An example of SRS measurement (i.e., SRS measurement in a single point of the sample) is reported in Figure 7. When the beams are not overlapped in time or space, the obtained result is reported in Figure 8a. In off-resonance, the amplitude of signal measured by LIA is zero, while the phase of signal measured by LIA jumps between negative and positive values. Whereas, when the beams are overlapped in space, moving the delay line in an appropriate range, t.......

Discussion

SRS microscopy has taken label-free imaging to new heights, especially in studies of complex biological structures such as lipids, which are fundamental to cells and cellular architecture. Lipids are involved in multiple physiological pathways such as production of biological membranes, and they serve as biosynthetic precursors and signal transducers¹⁰. Lipids are packaged into specialized intracellular organelles, also called lipid droplets (LDs). Their diameters vary from few tens of nanometers .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acquisation tool	Nikon	Nikon C2Tool	Acquisation supported tool
APE Pulse link control software	APE-	APE Pulse link control software	software control
Autocorrelator	APE	APE PulseCheck USB 50	Autocorrelator
Detector	Thorlabs	Thorlabs DET10A	Photodiode
Detector card	Thorlabs	Thorlabs VRC	IR detector Card
Dichroic mirror	Semrock	Semrock FF875-Di01-25X36	Dichroic mirror
Dichroic mirror	Semrock	FF875-Di01-25x36	Dichroic mirror
EOM	Conoptics	(EOM CONOPTICS 3350-160 KD*P).	Pockels cell
Fast detector	Thorlabs	Thorlabs DET025AL/M	Photodiode
Fast mirror scanning unit	Nikon	C2	Microscpe scanning head
Femtosecond laser Ti:SA	Coherent	Coherent Chameleon Ultra II	Chameleon Ultra II
Function generator	TTi	TG5011 AIM – TTi	Function generator
Inverted optical microscope	Nikon	Eclipse TE-2000-E, Nikon	Eclipse TE-2000-E, Nikon
Lock-in Amplifier	Standford Research System	SR844-200 MHz dual phase	A lock-in amplifier from Stanford Research Systems
Notch filter,	Semrock	NF03-808E-25	Notch filter
Optical delay line	Newport	Newport M-ILS200CC	Tunable optical delay line
Optical Parametric Oscillator	Coherent	Coherent Compact OPO	Coherent Compact OPO
Oscilloscope	WaveRunner	640Zi 4GHz OSC/LeCroy	Digital Oscilloscope
PCI Card	National instrument	NI PCIe 6363	Data acquisation card
Position Sensors Detectors	Newport	Newport Conex PSD9	Position detector sensor
Power meter head	Coherent	PowerMax PM10,	Laser power detector
Translation Stages	Thorlabs	Thorlabs PT1/M	Meachnical Translation Stage with Standard Micrometer