Imaging chromophores with undetectable fluorescence by stimulated emission microscopy

Min, Wei; Lü, Sijia; Chong, Shasha; Roy, Rahul; Holtom, Gary R.; Xie, X. Sunney

doi:10.1038/nature08438

Cited by 240 publications

(223 citation statements)

References 24 publications

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“…Various optical techniques have been developed to improve the spatial resolution beyond the diffraction limit (1)(2)(3) and to study, e.g., protein folding and adhesion complexes in living cells (4,5). Though powerful, most optical methods rely on molecular components that emit light (fluoresce) and the spatial resolution cannot yet rival that of electron-based techniques that allow focusing down to the atomic scale (6).…”

mentioning

confidence: 99%

Biological imaging with 4D ultrafast electron microscopy

Flannigan

Barwick

Zewail

2010

Proc. Natl. Acad. Sci. U.S.A.

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Advances in the imaging of biological structures with transmission electron microscopy continue to reveal information at the nanometer length scale and below. The images obtained are static, i.e., time-averaged over seconds, and the weak contrast is usually enhanced through sophisticated specimen preparation techniques and/or improvements in electron optics and methodologies. Here we report the application of the technique of photon-induced near-field electron microscopy (PINEM) to imaging of biological specimens with femtosecond (fs) temporal resolution. In PINEM, the biological structure is exposed to single-electron packets and simultaneously irradiated with fs laser pulses that are coincident with the electron pulses in space and time. By electron energy-filtering those electrons that gained photon energies, the contrast is enhanced only at the surface of the structures involved. This method is demonstrated here in imaging of protein vesicles and whole cells of Escherichia coli, both are not absorbing the photon energy, and both are of low-Z contrast. It is also shown that the spatial location of contrast enhancement can be controlled via laser polarization, time resolution, and tomographic tilting. The high-magnification PINEM imaging provides the nanometer scale and the fs temporal resolution. The potential of applications is discussed and includes the study of antibodies and immunolabeling within the cell.evanescent | nanoscale | biostructure T he development and application of imaging techniques for the visualization of biological structures continues to advance our understanding of such systems. Various optical techniques have been developed to improve the spatial resolution beyond the diffraction limit (1-3) and to study, e.g., protein folding and adhesion complexes in living cells (4, 5). Though powerful, most optical methods rely on molecular components that emit light (fluoresce) and the spatial resolution cannot yet rival that of electron-based techniques that allow focusing down to the atomic scale (6). Force probe microscopies have been used to image surfaces of cells and porosomes with high enough spatial resolution (7), and pulsed X-ray sources, such as synchrotrons and free-electron lasers, hold promise for femtosecond (fs) diffraction studies of individual biological macromolecules (8). Biological imaging with electron microscopy goes back to the 1960s and has since then been advanced to enable structural mapping (9) of biological macromolecules and cells (10-12), including viruses and molecular machines such as the ribosome (6, 13).The visualization of biological structures with an electron microscope presents unique challenges, especially when considering the inherent weak contrast associated with such structures. This weak contrast, which is primarily because of the low-Z (atomic number) elemental composition of biological specimens and the need for thin samples, is often addressed by employing sophisticated specimen preparation techniques (e.g., ultrathin sectioning and staining) and by i...

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mentioning

confidence: 99%

Biological imaging with 4D ultrafast electron microscopy

Flannigan

Barwick

Zewail

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…The first single-molecule optical detection experiment was achieved by absorption using a doublemodulation technique, but had to be carried out at cryogenic conditions, under which the absorption cross-section of the zero-phonon line is 10 6 times larger. 21 Indirect detection schemes combined with modulation techniques have been developed for absorption-based detection, [22][23][24][25] 4 including ground-state depletion microscopy 15,26 and photothermal microscopy. 11,13,14,16,[27][28][29][30] However, most studies using these methods have reported single-wavelength imaging without yielding spectroscopic information.…”

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confidence: 99%

Single-Particle Absorption Spectroscopy by Photothermal Contrast

et al. 2015

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Removing effects of sample heterogeneity through single-molecule and single-particle techniques has advanced many fields. While background free luminescence and scattering spectroscopy is widely used, recording the absorption spectrum only is rather difficult. Here we present an approach capable of recording pure absorption spectra of individual nanostructures. We demonstrate the implementation of single-particle absorption spectroscopy on strongly scattering plasmonic nanoparticles by combining photothermal microscopy with a supercontinuum laser and an innovative calibration procedure that accounts for chromatic aberrations and wavelength-dependent excitation powers. Comparison of the absorption spectra to the scattering spectra of the same individual gold nanoparticles reveals the blueshift of the absorption spectra, as predicted by Mie theory but previously not detectable in extinction measurements that measure the sum of absorption and scattering. By covering a wavelength range of 300 nm, we are furthermore able to record absorption spectra of single gold nanorods with different aspect ratios. We find that the spectral shift between absorption and scattering for the longitudinal plasmon resonance decreases as a function of nanorod aspect ratio, which is in agreement with simulations.

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“…STED competes effectively with this nonradioactive decay, providing a new contrast mechanism which has been investigated in the vascular network of a nude mouse ear [241]. In addition, STED has been implemented in nanofluidic studies [242,243] with reported spatial resolutions better than 70 nm; and in protein flow cytometry research [244].…”

Section: Stimulated Emission Depletion Microscopymentioning

confidence: 99%

‘Go with the flow ’: A review of methods and advancements in blood flow imaging

Daly

Leahy

2012

Journal of Biophotonics

108

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Physics has delivered extraordinary developments in almost every facet of modern life. From the humble thermometer and stethoscope to X‐Ray, CT, MRI, ultrasound, PET and radiotherapy, our health has been transformed by these advances yielding both morphological and functional metrics. Recently high resolution label‐free imaging of the microcirculation at clinically relevant depths has become available in the research domain. In this paper, we present a comprehensive review on current imaging techniques, state‐of‐the‐art advancements and applications, and general perspectives on the prospects for these modalities in the clinical realm. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Imaging chromophores with undetectable fluorescence by stimulated emission microscopy

Cited by 240 publications

References 24 publications

Biological imaging with 4D ultrafast electron microscopy

Biological imaging with 4D ultrafast electron microscopy

Single-Particle Absorption Spectroscopy by Photothermal Contrast

‘Go with the flow ’: A review of methods and advancements in blood flow imaging

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