Almar Palonpon

Label-free Raman observation of cytochrome c dynamics during apoptosis

We performed label-free observation of molecular dynamics in apoptotic cells by Raman microscopy. Dynamic changes in cytochrome c distribution at the Raman band of 750 cm-1 were observed after adding an apoptosis inducer to the cells. The comparison of mitochondria fluorescence images and Raman images of cytochrome c confirmed that changes in cytochrome c distribution can be distinguished as release of cytochrome c from mitochondria. Our observation also revealed that the redox state of cytochrome c was maintained during the release from the mitochondria. Monitoring mitochondrial membrane potential with JC-1 dye confirmed that the observed cytochrome c release was associated with apoptosis.

Raman and SERS microscopy for molecular imaging of live cells

Raman microscopy is a promising technology for visualizing the distribution of molecules in cells. A challenge for live-cell imaging using Raman microscopy has been long imaging times owing to the weak Raman signal. Here we present a protocol for constructing and using a Raman microscope equipped with both a slit-scanning excitation and detection system and a laser steering and nanoparticle-tracking system. Slit scanning allows Raman imaging with high temporal and spatial resolution, whereas the laser beam steering system enables dynamic surface-enhanced Raman imaging using gold nanoparticles. Both features enable mapping of the distributions of molecules in live cells and visualization of cellular transport pathways. Furthermore, its utility can be expanded to small-molecule imaging by using tiny Raman-active tags such as alkyne. For example, DNA synthesis in a cell can be visualized by detecting 5-ethynyl-2′-deoxyuridine (EdU), a deoxyuridine derivative with an alkyne moiety. We describe the optics, hardware and software to construct the Raman microscope, and discuss the conditions and parameters involved in live-cell imaging. The whole system can be built in ∼8 h.

Imaging of EdU, an alkyne-tagged cell proliferation probe, by Raman microscopy.

Click-free imaging of the nuclear localization of an alkyne-tagged cell proliferation probe, EdU, in living cells was achieved for the first time by means of Raman microscopy. The alkyne tag shows an intense Raman band in a cellular Raman-silent region that is free of interference from endogenous molecules. This approach may eliminate the need for click reactions in the detection of alkyne-labeled molecules.

Molecular imaging of live cells by Raman microscopy.

Raman microscopy represents an emerging class of tools for molecular imaging of live cells because of the rich information obtained by detecting molecular vibrations. Recently, several Raman imaging techniques based on the parallel detection of Raman spectra have been developed, which can achieve high spatial and temporal resolution suitable for live cell imaging. When combined with tiny Raman tags in the cellular silent region, Raman microscopy has capability to map the distribution of specific target small molecules with minimum perturbation from the tag. Here we review these recent advances in cell imaging techniques based on spontaneous Raman scattering and highlight its potential for the observation and analysis of biological functions.

Structured line illumination Raman microscopy

In the last couple of decades, the spatial resolution in optical microscopy has increased to unprecedented levels by exploiting the fluorescence properties of the probe. At about the same time, Raman imaging techniques have emerged as a way to image inherent chemical information in a sample without using fluorescent probes. However, in many applications, the achievable resolution is limited to about half the wavelength of excitation light. Here we report the use of structured illumination to increase the spatial resolution of label-free spontaneous Raman microscopy, generating highly detailed spatial contrast from the ensemble of molecular information in the sample. Using structured line illumination in slit-scanning Raman microscopy, we demonstrate a marked improvement in spatial resolution and show the applicability to a range of samples, including both biological and inorganic chemical component mapping. This technique is expected to contribute towards greater understanding of chemical component distributions in organic and inorganic materials.

Dual-polarization Raman spectral imaging to extract overlapping molecular fingerprints of living cells

Raman spectral imaging is gaining more and more attention in biological studies because of its label-free characteristic. However, the discrimination of overlapping chemical contrasts has been a major challenge. In this study, we introduce an optical method to simultaneously obtain two orthogonally polarized Raman images from a single scan of the sample. We demonstrate how this technique can improve the quality and quantity of the hyperspectral Raman dataset and how the technique is expected to further extend the horizons of Raman spectral imaging in biological studies by providing more detailed chemical information. The dual-polarization Raman images of a HeLa cell.

High-speed Raman imaging of cellular processes.

Raman scattering microscopy provides information about the distribution and chemical state of molecules in live cells without any labeling or modification. In recent years, the imaging speed of Raman microscopy has improved greatly owing to the development of instruments that can perform parallel acquisition of Raman spectra from multiple points. In this article, we review recent advances in high-speed hyperspectral Raman imaging and its application to observe various biological processes such as cell mitosis, apoptosis and cell differentiation. Furthermore, we discuss the recent progress in Raman tags for the specific observation of bioactive small molecules in complex biological systems, including the development of organelle-specific probes, imaging of lipid rafts in an artificial monolayer membrane and application as a structure-sensitive tag.

Direct Evidence of Chemical Contribution to Surface-enhanced Hyper-Raman Scattering

We report on halide-ion-assisted chemical effect in surface-enhanced hyper-Raman scattering of crystal violet (CV) adsorbed on single silver aggregate. A dramatic increase in spectral intensity was observed in the presence of halide ions as compared to their absence. By measuring the hyper-Rayleigh scattering from single aggregate treated with and without halide ions, we established that it was chemical effect, rather than electromagnetic effect that was responsible for this strong enhancement. We attribute the enhancement to a charge transfer mechanism between CV and metal surface mediated by the halide ions similar to surface-enhanced Raman scattering.

Raman imaging of alkyne as a small tag for biological molecules

Role of small molecules such as drugs or metabolites in cells is commonly studied by fluorescence microscopy in which a fluorescent label is attached to the molecule. However, fluorescent labels are typically large that often interfere with the normal cellular function of the molecule. To avoid the use of bulky fluorescent labels, we introduce a technique that uses a simple small chemical tag called alkyne consisting of two carbons connected by a triple bond. The alkyne-tagged molecule is imaged using Raman microscopy that detects the strong Raman signal from the CC triple bond stretching vibration (~2120 cm-1). Because the alkyne signal is located in the silent region of the cell (1800-2700 cm-1), it does not interfere with any intrinsic cellular Raman signals. Here, we demonstrate this technique by showing Raman images of an alkyne-tagged component of DNA in a living cell using a slit-scanning confocal Raman microscope. This fast imaging technique is based on a line-shaped focus illumination and simultaneous detection of the Raman spectra from multiple points of the sample. Using this microscope, we obtained time-course Raman images of the incorporation of EdU in the DNA of HeLa cells in just several tens of minutes.

Nonlinear fluorescence imaging by photoinduced charge separation

Manipulation of the optical property of fluorescent probes has been a powerful strategy to establish super-resolution microscopy. We describe a new strategy to realize a probe with a nonlinear fluorescence response by using photoinduced charge separation. In this scheme, the first photon is used for the generation of the charge-separated state and the second photon is for fluorescence excitation. This stepwise two-photon absorption was confirmed by detection of a second-order nonlinear fluorescence response. Transient absorption spectra studies and simulation indicate that fluorescence is emitted through the photophysical pathways we proposed. Fluorescence imaging of biological cells showed marked improvements in image contrast and resolution, demonstrating the usefulness of the fluorescent probe in laser scanning confocal microscopy.