Coherent Nonlinear Optical Imaging: Beyond Fluorescence Microscopy

Min, Wei; Freudiger, Christian W.; Lü, Sijia; Xie, X. Sunney

doi:10.1146/annurev.physchem.012809.103512

Cited by 537 publications

(473 citation statements)

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“…Several techniques have been developed to ''boost'' this signal, including resonance Raman scattering, surface-enhanced Raman scattering, 8 and coherent Raman scattering. [9][10][11][12] Each one of these techniques has its own pros and cons in the analysis of single cells, which could be detailed in a separate review of its own, but for brevity, we limit our discussion of Raman scattering in this review solely to the use of spontaneous Raman scattering in the analysis of single cells. We briefly summarize recent advances in instrument development, multivariate statistical analysis of complex hyperspectral data cubes containing Raman spectra, and specific applications that demonstrate the power of Raman spectroscopy for single-cell analysis.…”

Section: Introductionmentioning

confidence: 99%

Methods and Applications of Raman Microspectroscopy to Single-Cell Analysis

2013

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Raman spectroscopy is a powerful biochemical analysis technique that allows for the dynamic characterization and imaging of living biological cells in the absence of fluorescent stains. In this review, we summarize some of the most recent developments in the noninvasive biochemical characterization of single cells by spontaneous Raman scattering. Different instrumentation strategies utilizing confocal detection optics, multispot, and line illumination have been developed to improve the speed and sensitivity of the analysis of single cells by Raman spectroscopy. To analyze and visualize the large data sets obtained during such experiments, sophisticated multivariate statistical analysis tools are necessary to reduce the data and extract components of interest. We highlight the most recent applications of single cell analysis by Raman spectroscopy and their biomedical implications that have enabled the noninvasive characterization of specific metabolic states of eukaryotic cells, the identification and characterization of stem cells, and the rapid identification of bacterial cells. We conclude the article with a brief look into the future of this rapidly evolving research area.

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Section: Introductionmentioning

confidence: 99%

Methods and Applications of Raman Microspectroscopy to Single-Cell Analysis

2013

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“…1) and thereby overcomes the sensitivity or biocompatibility limitations of other Raman imaging modalities 10, 11 . Coupling SRS with strong vibrational tags such as alkynes (C≡C) allows effective imaging of diverse biomolecules 12–15 , but detection sensitivity is still limited to about 15 mM for typical chemical bonds such as C-H and 200 μM for the stronger C≡C bond that leaves many targets (such as metabolites, proteins, RNA, organelles) out of reach 10–13 .…”

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confidence: 99%

Super-multiplex vibrational imaging

Chen

Shi

et al. 2017

Nature

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The ability to directly visualize a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have been used successfully to explore structural-functional relationships in nervous systems, profile RNA in situ, reveal tumor microenvironment heterogeneity or study dynamic macromolecular assembly1–4, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a “color barrier” due to the intrinsically broad (~1500 cm−1) and featureless nature of fluorescence spectra5 that limits the number of resolvable colors to 2 to 5 (or 7-9 if using complicated instrumentation and analysis)6–8. Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width ~10 cm−1) and thus doesn’t suffer this problem, but its feeble signals make many demanding bio-imaging applications impossible. And while surface-enhanced Raman scattering offers remarkable sensitivity and multiplicity, it cannot be readily used to quantitatively image specific molecular targets inside live cells9. Here we show that carrying out stimulated Raman scattering under electronic pre-resonance conditions (epr-SRS) enables imaging with exquisite vibrational selectivity and sensitivity (down to 250 nM with 1-ms) in living cells. We also create a palette of triple-bond-conjugated near-infrared dyes that each display a single epr-SRS peak in the cell-silent spectral window, and that with available fluorescent probes give 24 resolvable colors with potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this super-multiplex optical imaging approach for untangling intricate interactions in complex biological systems.

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“…Multiphoton microscopy generates contrast by making use of nonlinear optical processes which take place when high energy densities are achieved [4,5], a situation which requires the use of short pulsed sources (in the femtosecond range -the shorter the pulse, the higher the energy density). Of the different non-linear effects, the most widely used is two-photon excitation microscopy (2PM), where the fluorescence in the sample is excited through the simultaneous absorption of two photons of lower energy, an event which requires high energy flux.…”

Section: Multiphoton Microscopymentioning

confidence: 99%