Background signals in stimulated Raman scattering microscopy and current solutions to avoid them
Background signals in stimulated Raman scattering microscopy and current solutions to avoid them
Genchi, Luca; Laptenok, Sergey P.; Liberale, Carlo
2023-12-31 00:00:00
ADVANCES IN PHYSICS: X 2023, VOL. 8, NO. 1, 2176258 https://doi.org/10.1080/23746149.2023.2176258 REVIEW Background signals in stimulated Raman scattering microscopy and current solutions to avoid them a a a,b Luca Genchi , Sergey P. Laptenok and Carlo Liberale Biological and Environmental Science and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia; Computer, Electrical and Mathematical Sciences and Engineering Division (CEMSE), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia ABSTRACT ARTICLE HISTORY Received 21 December 2022 Stimulated Raman scattering (SRS) microscopy has gained Revised 18 January 2023 popularity in recent years due to its linearity to molecule Accepted 27 January 2023 concentration and laser intensity, and to the lack of the nonresonant background that affects its analogous techni- KEYWORDS que, coherent anti-Stokes Raman scattering. However, SRS is Coherent Raman scattering not a background-free technique. In fact, there are other microscopy; stimulated optical processes – nonlinear transient scattering and non- Raman scattering; label-free microscopy; multi-photon linear transient absorption – that can be detrimental to the microscopy contrast and sensitivity of SRS microscopy. In this review, we provide a description of these competing optical processes and present an up-to-date description of current solutions to minimize their effect on SRS measurements. 1. Introduction Label-free microscopy methods based on vibrational spectroscopy are poised to become fundamental tools for scientific and bio-medical applica- tions [1–4]. In fact, the vibrational signatures of molecular compounds can CONTACT Carlo Liberale carlo.liberale@kaust.edu.sa Biological and Environmental Science and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 L. GENCHI ET AL. be used to gain qualitative and quantitative information in a variety of different applications and studies, such as point-of-care medicine [5,6], guided surgery [7], rapid histology [8], intraoperative diagnosis [9,10] and endoscopy [11], as well as fundamental biology [2,12], pharmaceutics [13,14], cultural heritage [15–18], food industry [19–23], plant science [24–27] and environmental pollution analysis [28–32]. The use of visible or near-IR wavelengths makes spontaneous Raman micro-spectrometry of particular interest for biological applications, thanks to the absence of absorption from water and the possibility of achieving sub- micrometer spatial resolution. However, measurements of spontaneous Raman scattering are usually time-demanding due to the randomized phase of molecular vibrations in the probed volume, which creates an incoherent scattering process. Coherent Raman scattering (CRS) [33,34] microscopy overcomes the acquisition speed limitations of spontaneous Raman imaging by using two synchronized and frequency-detuned laser beams called pump and Stokes beams. In fact, when the frequency differ- ence between the pump and Stokes photons is in resonance with one of the Raman-active molecular vibrations in the specimen, the molecules in the target volume oscillate in phase – i.e. ‘coherent’ oscillations – and the Raman scattering is greatly enhanced [34]. CRS microscopy is implemented in two main modalities, coherent anti- Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy [34]. In particular, SRS microscopy has gained popularity in recent years [3,35,36] thanks to the linear dependence of the signal on molecular concentration and on excitation fields’ intensities, allowing to perform quantitative measurements [4,37–39]. Contrary to the CARS sig- nal, the SRS signal is not affected by a non-vibrationally resonant back- ground contribution generated by the ubiquitous electronic four-wave- mixing nonlinear optical effect (this specific background signal is com- monly referred to as the ”non-resonant background” (NRB) in the litera- ture). For this reason, SRS microscopy has been often referred to as a background-free CRS modality. However, there are other competing effects – in particular, cross-phase modulation (XPM), two-photon absorp- tion (TPA), and thermal lensing (TL) – that generate ubiquitous and spatially uneven non-vibrationally resonant background signals in SRS measurements, reducing contrast and sensitivity [40,41]. Several applica- tions of SRS microscopy would be hindered by the presence of those back- ground signals, motivating the continuous development of novel strategies to minimize their contribution to the measured signal. Here, after presenting a qualitative description of the most important competing effects generating background signals in SRS microscopy, we review the state of the art of current approaches for mitigating or ADVANCES IN PHYSICS: X 3 suppressing them. We then discuss future perspectives and requirements for a gold standard solution for truly background-free SRS microscopy. 2. SRS signal generation and detection In SRS microscopy two laser beams – pump beam with frequency w and Stokes beam with w – are focused in the specimen to coherently drive molecular vibrations in the focal volume. In fact, when the spectral detuning =w w is in resonance with a Raman-active vibrational mode w of p- s v molecules in the target volume, the sample acts as a nonlinear medium and mediates an exchange of energy between the two laser beams causing an intensity loss (stimulated Raman loss, SRL) and an intensity gain (stimu- lated Raman gain, SRG) in the pump and Stokes beams respectively. These intensity variations ∆ I are: p,s ð3Þ ΔI / I I Imðχ ðΩÞÞ (1) P P S ð3Þ ΔI / I I Imðχ ðΩÞÞ (2) S S P where I and I are the intensities of the pump and Stokes beams before the P s � � ð3Þ interaction, and Im X the imaginary part of the Raman-resonant third- order susceptibility (which is linearly proportional to the Raman cross- section σ , hence to the spontaneous Raman spectrum [34]). The informa- tion contained in SRL and SRG is completely symmetrical, therefore the detection of either the pump or the Stokes beam can be used to retrieve the SRS signal. Typical Raman cross sections of molecules of interest in biology are [42] σ � 10 cm /sr, which is several orders of magnitude smaller than the typical cross sections associated with other optical processes. For instance, the electronic absorption cross section of some chromophores can be up to 10 orders of magnitude higher. As a consequence, the relative intensity variations that the beams experience due to the SRS process can be as small as parts per million. For this reason, the minimization of noise sources is critical for the sensitive detection of the SRS signal. In short, there are three major sources of noise in SRS microscopy [43]: the additive noise due to the detection circuitry, the laser shot noise, due to the quantum nature of the light, and the laser Flicker noise, also called ”pink” or 1/f noise. The latter features a power spectral density inversely proportional to the frequency and is the dominant source of noise at low frequencies. For this reason, a lock-in detection scheme with a high modulation frequency in the order of 1ʹs-10ʹs MHz is typically implemented to reach the shot-noise limit (Figure 1). To implement this scheme, the majority of SRS microscopy 4 L. GENCHI ET AL. power line noise and harmonics demodulation frequency interval 1/f noise white noise modulation f frequency Figure 1. Sketch of typical laser noise intensity profile (grey). To avoid the small SRS signal being drowned by the larger laser noise at low frequencies, it is modulated at high frequency (red line), and measured with a lock-in detection scheme (yellow). Figure 2. Intensities of beams in IM-SRS with SRL detection configuration: (left) before, and (right) after a Raman-resonant sample. The high-frequency intensity modulation of the Stokes beam is transferred to the pump beam through the SRS process in the sample. setups proposed to date use an intensity modulation (IM-SRS) approach, which uses an acousto or electro-optic modulator (AOM or EOM, respec- tively) to modulate the intensity of either the pump or the Stokes beam. The modulation is eventually transferred to the other beam through the SRS process occurring in the sample, and a single photodiode placed after the specimen detects the intensity of the probed beam and the SRS signal (either SRL or SRG) is typically extracted using a lock-in amplifier. As a simplifying example, in a SRL configuration (Figure 2) the intensity loss ∆ I of the pump beam is retrieved as a differential measurement between the Stokes-ON and Stokes-OFF amplitudes. Apart from lock-in amplifier detection, the SRS signal can be retrieved by use of tuned amplifiers [44] or boxcar averaging [45]. When the excess laser noise is still the dominating noise factor at these high modulation frequencies, such as when using fiber-based laser sources, it is possible to reduce its contribution significantly by using a balanced ADVANCES IN PHYSICS: X 5 detection scheme [46–50]. Additionally, a multiplexing scheme based on the Hadamard transform can be used to reduce the additive noise [51,52]. Whereas the spontaneous Raman scattering signal intrinsically provides the whole vibrational spectrum at once, in SRS microscopy the signal is typically probed at a single vibrational frequency at a time. To obtain hyperspectral images – i.e. where a vibrational spectrum is obtained at each pixel of the image – the SRS signal has to be measured at several different vibrational frequencies. When the probed frequency range is –1 particularly large, spanning several hundreds of cm , we have so-called broadband SRS measurements [53]. The vibrational spectrum of the major- ity of chemical compounds of biological relevance has two main frequency –1 intervals of interest: the so-called fingerprint (600–1800 cm ) and CH- –1 stretching (2700–3100 cm ) regions. The former, in particular, features several vibrational bands from aromatic amino acids, amide groups, sec- ondary protein structures, and stretching or deformation of bonds formed by carbon atoms with nitrogen, and other carbon atoms [53]. The spectral overlap of Raman bands from different molecules makes difficult their identification and quantification within compounds by relying on measure- ments at single wavenumbers. For this reason, probing the SRS signal across the whole fingerprint-to-CH-stretch spectral range is crucial for reliable discrimination of different molecular species [30,53]. A high spectral reso- –1 lution, capable of resolving the typical 10 cm bandwidth of Raman bands in the fingerprint region in biological specimens, is also needed to allow chemical specificity [53]. 3. Background signals in IM-SRS The IM-SRS configuration has been widely used to date due to its relative ease of implementation. However, the applied intensity modulation lends itself to introducing other contributions to the demodulated signal that, although not related to molecular vibrations, are indistinguishable from the real SRS contribution. These spurious additional signals arise from compet- ing processes – two-photon absorption [54,55] (TPA) and non-linear tran- sient scattering [56] – which are vibrationally non-resonant, i.e. not chemical specific, and spatially non-uniform in heterogeneous samples. Therefore, they don’t provide any chemical information, while instead reducing the contrast, and creating artifacts in SRS imaging that could hinder interesting applications [41,57]. In the following, we describe in more detail the specific characteristics of these competing effects and how they lead to background signals in SRS microscopy. 6 L. GENCHI ET AL. ω ω P P ω ω S S One-color TPA Two-color TPA Figure 3. Jablonski diagram of the different TPA processes that could happen during a SRS measurement. While the concomitant absorption of two Stokes or pump photons is not affecting the SRS signal due to the modulation scheme, the simultaneous absorption of one pump and one Stokes (two-color TPA, TCTPA) gives a spurious signal in SRS measurements. 3.1. Two-photon absorption The TPA process consists of the quasi-simultaneous absorption of two photons by a molecule, promoting one of its electrons from the ground state to a higher energy level (excited state). This occurs via the absorption of either two photons with the same frequency (single-color two-photon absorption), or two photons with different frequencies (two-color two- photon absorption, TCTPA), as sketched in Figure 3. In the IM-SRS scheme, the single-color TPA process does not create a modulated signal recovered by the lock-in amplifier, hence it does not add a spurious contribution to the pure SRS signal. Instead, the TCTPA only occurs when both beams are simultaneously present, hence the related variation in the power of the probed beam is modulated with the same frequency as the modulated beam. This results in a background signal that is recovered by the lock-in amplifier and is not distinguishable from the pure SRS signal [26,58]. When modulating the Stokes/pump beam and detecting the pump/Stokes beam, the signal amplitude variation due to the TCTPA is: ΔI α