A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.
Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule’s vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.
1. Generation of CARS and DR-CARS Signals
In order to generate CARS and DR-CARS signals simultaneously, three tunable and synchronized short pulsed laser sources are required.
2. The Use of Three Short Pulsed Lasers
The use of three short pulsed lasers results in the production of several CARS signals, from the various combinations of two lasers, as well as 3-Color CARS and DR-FWM signals from the combination of all three lasers.
3. Sample Preparation
In order to obtain clear, reproducible images some care must be taken in preparing the sample.
4. Sample Analysis
In order to properly take advantage of the doubly-resonant enhancement effect the Raman spectra of both Raman-resonant substances must be known.
5. Image Processing
Extracting additional information based on these three images now requires some fairly simple image processing.
6. Representative Results
Figure 1: Diagram of DR-CARS microscopy system as described above.
Figure 2: White light image of a C. elegans worm in deuterated glucose solution prepared on a glass coverslip and ready for imaging.
Figure 3: Raman spectrum of modified oleic acid (an unsaturated fatty acid) that includes an alkyne modification (a carbon triple-bond carbon group). The strong CH resonances at 2845 cm-1 and the alkyne resonance at 2100 cm-1 are both well isolated from the fingerprint region (the region of densely packed peaks), making them ideal markers for coherent Raman imaging.
Figure 4: Typical spectrum of coherent Raman signals generated when three short-pulsed lasers are overlapped within the sample. The arrows point to the process(es) responsible for each signal as represented by energy diagrams. In the diagrams shown here dashed arrows indicate photons from the laser and the OPO’s and solid arrows indicate the resulting signal. The solid horizontal lines indicate the energy of the Raman vibration and give a visual representation that, in DR-CARS, mixing the same 3 input photons simultaneously probes two different Raman vibrations.
Figure 5: Typical results from imaging C. elegans worms using DR-CARS and CARS. The top row used the three signals indicated in Figure 4 to image a worm in a solution of deuterated glucose. In the second row the images were appropriately normalized and in the third row the difference images were produced by subtracting each of the CARS images from the DR-CARS image.
Raman spectroscopy and Raman-based imaging are powerful emerging tools in the bio sciences. Currently, this is particularly true for the in vivo and in vitro study of cellular metabolism and metabolic disorders in processing and storage of lipids. Most bio-macromolecules contain a large number of similar, mostly carbon-based molecular bonds, so that the Raman spectra obtained from cells and organisms are typically a convolution of contributions from lipids, proteins, nucleic acids, sugars, etc. Lipids are relatively easy to isolate from these complex spectra, because of their tendency to form dense droplets or bilayers and because they contain extended chains with a large number of aliphatic CH bonds. Our ability to isolate specific proteins, amino acids, RNA, or DNA within the complex cellular environment is, however, very limited. This is particularly true if these molecules of interest are only present at μM concentrations and below. Here, the ability to probe weak Raman resonances utilizing our newly introduced DR-CARS difference imaging technique provides a potentially powerful approach for their chemical microanalysis and imaging. Admittedly, the most complicated portion of this protocol is the alignment and synchronization of the laser system. When starting from scratch, the synchronization of the pulses, i.e. ensuring that the pulses are overlapped in time despite the different paths they take can be facilitated by the use of a pulse autocorrelator. Once spatial and temporal overlap is achieved, CARS and DR-CARS signals should be readily detectable. However, the first alignment is often crude, resulting in weak signals. The best practice for aligning this system well is to initially generate weak signals and then to improve the signal strength by gently tweaking the mirrors along each path and adjusting the temporal overlap using the delay stages. Although the spectrometer/monochromator acts as a very efficient baffle for room light the cleanest results can be achieved by operating the system with the room lights turned off and curtains or lens tubing to minimize background introduced by the various other light sources (e.g computer monitors, indicator lights, LEDs, etc.).
Our particular setup utilizes single-photon counting avalanche photo-diode (APD) detectors and time-correlated single photon counting (TCSPC) hardware for detection 5. This enables us to detect extremely weak signals with relatively low noise but many groups have found photo-multiplier tubes (PMT’s) with variable gain advantageous when making similar measurements. The advantage of PMT’s is that they offer variable gain and have a much larger detection area which can simplify alignment of the detector. Additionally, our setup utilizes piezo stages to translate the objective in order to achieve beam scanning. The advantage of this is that we have the ability to return to any spot within the previously scanned image with a high degree of accuracy and take additional measurements including spontaneous Raman spectra. Other groups have been successful utilizing scanning mirror assemblies, or even entire confocal scanning units such as the Olympus FluoView system, which offers much faster imaging but is limited in its ability to precisely return to arbitrary locations within an image.
Tuning the lasers to match the Raman resonance is also a critical step that may require some optimization. Although the Raman peaks may be known the maximum spectral peak intensity obtained from DR-CARS and CARS does not necessarily correspond to the maximum of the spontaneous Raman peak. This is due to the intrinsic interference of signals generated by four-wave mixing leading to a non-resonant background signal and CARS, which distorts CARS spectra relative to spontaneous Raman spectra. The spectral location of the peak of the CARS signal can be calculated, but a more practical approach is to tune the OPOs in several, small spectral steps across the expected location of the Raman resonance. This process should yield a clear maximum. In fact, for the greatest sensitivity from DR-FWM both resonances must be tuned to this maximum.
One last potential problem of the DR-CARS approach has to also be discussed, i.e. the DR-CARS signal will depend on a homogeneous distribution of the Raman-active amplifying molecule. For most biological objects, this could well be the broad OH resonance from water, which is abundant and almost omnipresent. Water is, however, excluded from hydrophobic regions with a cell, such as lipid droplets, leading a distortion of the signals obtained when utilizing the water resonance to amplify lipid modes. In our example, we have used a solution of deuterated glucose to generate an easily detectable and abundant signal for our biological sample. Similarly, deuterated water or deuterated biological buffers, such as d-HEPES could be used. In our example, the lipid droplets within the C. elegans worm were small enough to always contain both, the deuterated glucose solution and lipids within the focused laser spot of our system. This, however, is not generally true. A particular example would be adipocytes, which generate rather large lipid droplets within their cytoplasm. This means, any experiment conducted with the DR-CARS technique requires careful preparation and control experiments to verify the results.
The authors have nothing to disclose.
We would like to thank Iwan Schie and Sebastian Wachsmann-Hogiu for their contributions in developing the DR-CARS technique. Tyler Weeks acknowledges support by the Lawrence Scholar Program from Lawrence Livermore National Laboratory. Thomas Huser is grateful for support from the American Heart Association through the Grant-in-Aid program. This work was also supported in part by funding from the National Science Foundation. The Center for Biophotonics, an NSF Science and Technology Center, is managed by the University of California, Davis, under Cooperative Agreement No. PHY 0120999. Support is also acknowledged from the UCD Clinical Translational Science Center under grant number UL1 RR024146 from the National Center for Research Resources (NCRR).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
60X water immersion objective | Olympus | UPLSAPO 60XW | ||
Inverted Microscope | Olympus | IX-71SIF-3 | ||
Pockels Cell | ConOptics | 350-160 | ||
Picotrain pump Laser | HighQ | IC-1064-10000 | ||
Optical Parametric Oscillator | APE | Levante IR | ||
1.5 Glass cover slips | Fisher Scientific | 12-545-102 25cm-1 | ||
Half-wave plates | Thor Labs | AHWP05M-980 | ||
Polarizing Beam Splitter Cubes | Thor Labs | PBS052 or PBS053 | ||
Spectrometer/Monochromator | PI Acton | Spectra Pro 2300i | ||
CCD Camera | PI Acton | PIXIS: 100B | ||
Avalanche Photo Diode | Perkin Elmer | SPCM-AGR-14-12691 | ||
XYZ Piezo Stage | Physik Instruments | P 733-2CL P 721.CDQ |
This is a combination of an XY stage and a Z objective holder | |
Dichroic Mirrors | Semrock | Ff01-720/SP-25 LPD01-633RS-25 |
These specific dichroics are not critical, any set with the appropriate transmission/reflection characteristics will be sufficient. | |
Dichroic Mirror | Chroma | Z830rdc | To combine the different near-infrared laser beams | |
TCSPC board | PicoQuant | Timeharp 200 | ||
Symphotime Imaging Software | PicoQuant | |||
Matlab | Mathworks |