Stimulated Raman scattering (SRS) microscopy is a label‐free method generating images based on chemical contrast within samples, and has already shown its great potential for high‐sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio‐samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub‐cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto‐optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon‐Hydrogen (CH)‐stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm−1, is demonstrated.
Spontaneous Raman microscopy reveals the chemical composition of a sample in a label-free and non-invasive fashion by directly measuring the vibrational spectra of molecules. However, its extremely low cross section prevents its application to fast imaging. Stimulated Raman scattering (SRS) amplifies the signal by several orders of magnitude thanks to the coherent nature of the nonlinear process, thus unlocking high-speed microscopy applications that provide analytical information to elucidate biochemical mechanisms with subcellular resolution. Nevertheless, in its standard implementation, narrowband SRS provides images at only one frequency at a time, which is not sufficient to distinguish constituents with overlapping Raman bands. Here, we report a broadband SRS microscope equipped with a home-built multichannel lock-in amplifier simultaneously measuring the SRS signal at 32 frequencies with integration time down to 44 µs, allowing for detailed, high spatial resolution mapping of spectrally congested samples. We demonstrate the capability of our microscope to differentiate the chemical constituents of heterogeneous samples by measuring the relative concentrations of different fatty acids in cultured hepatocytes at the single lipid droplet level and by differentiating tumor from peritumoral tissue in a preclinical mouse model of fibrosarcoma.
We present a 3D printed high‐NA catadioptric lens for forward signal collection in laser‐scanning microscopy. The lens can replace expensive and bulky high‐NA microscope objectives or condensers and is so thin to fit inside top‐stage microscope incubators. The lens is particularly beneficial in Stimulated Raman Scattering (SRS), where a high‐NA beam collection is needed to avoid artefacts due to Cross Phase Modulation.
Further details can be found in the article by Andrea Bertoncini, Sergey P. Laptenok, Luca Genchi, Vijayakumar P. Rajamanickam, and Carlo Liberale (https://onlinelibrary.wiley.com/doi/10.1002/jbio.202000219).
Stimulated Raman Scattering (SRS) is a fast chemical imaging technique with remarkable bio-science applications. Cross Phase Modulation (XPM) is a ubiquitous non-linear phenomenon that can create spurious background signals that render difficult a high-contrast imaging in SRS measurements. The XPM-induced signal is usually suppressed using high Numerical Aperture (NA) microscope objectives or condensers to collect the transmitted excitation beam. However, these high-NA optics feature short working distances, hence they are not compatible with stage-top incubators, that are necessary to perform live-cell time-lapse experiments in controlled environments. Here, we show a 3D printed high-NA compact catadioptric lens that fits inside stage-top incubators and allows the collection of XPM-free SRS signals. The lens delivers SRS images and spectra with a quality comparable to a signal collection with a high-NA microscope objective. We also demonstrate the compatibility of the 3D printed lens with other non-linear microscopies usually associated with SRS in multimodal microscopes.
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