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.

High-throughput line-illumination Raman microscopy with multislit detection.

Raman microscopy is an emerging tool for molecular imaging and analysis of living samples. Use of Raman microscopy in life sciences is, however, still limited because of its slow measurement speed for spectral imaging and analysis. We developed a multiline-illumination Raman microscope to achieve ultrafast Raman spectral imaging. A spectrophotometer equipped with a periodic array of confocal slits detects Raman spectra from a sample irradiated by multiple line illuminations. A comb-like Raman hyperspectral image is formed on a two-dimensional detector in the spectrophotometer, and a hyperspectral Raman image is acquired by scanning the sample with multiline illumination array. By irradiating a sample with 21 simultaneous illumination lines, we achieved high-throughput Raman hyperspectral imaging of mouse brain tissue, acquiring 1108800 spectra in 11.4 min. We also measured mouse kidney and liver tissue as well as conducted label-free live-cell molecular imaging. The ultrafast Raman hyperspectral imaging enabled by the presented technique will expand the possible applications of Raman microscopy in biological and medical fields.

Single-cell Raman microscopy with machine learning highlights distinct biochemical features of neutrophil extracellular traps and necrosis.

The defining biology that distinguishes neutrophil extracellular traps (NETs) from other forms of cell death is unresolved, and techniques which unambiguously identify NETs remain elusive. Raman scattering measurement provides a holistic overview of cell molecular composition based on characteristic bond vibrations in components such as lipids and proteins. We collected Raman spectra from NETs and freeze/thaw necrotic cells using a custom built high-throughput platform which is able to rapidly measure spectra from single cells. Principal component analysis of Raman spectra from NETs clearly distinguished them from necrotic cells despite their similar morphology, demonstrating their fundamental molecular differences. In contrast, classical techniques used for NET analysis, immunofluorescence microscopy, extracellular DNA, and ELISA, could not differentiate these cells. Additionally, machine learning analysis of Raman spectra indicated subtle differences in lipopolysaccharide (LPS)-induced as opposed to phorbol myristate acetate (PMA)-induced NETs, demonstrating the molecular composition of NETs varies depending on the stimulant used. This study demonstrates the benefits of Raman microscopy in discriminating NETs from other types of cell death and by their pathway of induction.

Quantitative Drug Dynamics Visualized by Alkyne-Tagged Plasmonic-Enhanced Raman Microscopy.

Visualizing live-cell uptake of small-molecule drugs is paramount for drug development and pharmaceutical sciences. Bioorthogonal imaging with click chemistry has made significant contributions to the field, visualizing small molecules in cells. Furthermore, recent developments in Raman microscopy, including stimulated Raman scattering (SRS) microscopy, have realized direct visualization of alkyne-tagged small-molecule drugs in live cells. However, Raman and SRS microscopy still suffer from limited detection sensitivity with low concentration molecules for observing temporal dynamics of drug uptake. Here, we demonstrate the combination of alkyne-tag and surface-enhanced Raman scattering (SERS) microscopy for the real-time monitoring of drug uptake in live cells. Gold nanoparticles are introduced into lysosomes of live cells by endocytosis and work as SERS probes. Raman signals of alkynes can be boosted by enhanced electric fields generated by plasmon resonance of gold nanoparticles when alkyne-tagged small molecules are colocalized with the nanoparticles. With time-lapse 3D SERS imaging, this technique allows us to investigate drug uptake by live cells with different chemical and physical conditions. We also perform quantitative evaluation of the uptake speed at the single-cell level using digital SERS counting under different quantities of drug molecules and temperature conditions. Our results illustrate that alkyne-tag SERS microscopy has a potential to be an alternative bioorthogonal imaging technique to investigate temporal dynamics of small-molecule uptake of live cells for pharmaceutical research.

Label-free molecular imaging of living cells.

Optical signals based on Raman scattering, coherent anti-Stokes Raman scattering (CARS), and harmonic generation can be used to image biological molecules in living cells without labeling. Both Raman scattering and CARS signals can be used to detect frequencies of molecular vibrations and to obtain the molecular distributions in samples. Second-harmonic optical signals can also be generated in structured arrays of noncentrosymmetric molecules and can be used to detect structured aggregates of proteins, such as, collagen, myosin and tubulin. Since labeling techniques using chemical and biological reactions may cause undesirable changes in the sample, label-free molecular imaging techniques are essential for observation of living samples.

Raman microscopy for dynamic molecular imaging of living cells.

We demonstrate dynamic imaging of molecular distribution in unstained living cells using Raman scattering. By combining slit-scanning detection and optimizing the excitation wavelength, we imaged the dynamic molecular distributions of cytochrome c, protein beta sheets, and lipids in unstained HeLa cells with a temporal resolution of 3 minutes. We found that 532-nm excitation can be used to generate strong Raman scattering signals and to suppress autofluorescence that typically obscures Raman signals. With this technique, we reveal time-resolved distributions of cytochrome c and other biomolecules in living cells in the process of cytokinesis without the need for fluorescent labels or markers.

Deep-UV biological imaging by lanthanide ion molecular protection.

Deep-UV (DUV) light is a sensitive probe for biological molecules such as nucleobases and aromatic amino acids due to specific absorption. However, the use of DUV light for imaging is limited because DUV can destroy or denature target molecules in a sample. Here we show that trivalent ions in the lanthanide group can suppress molecular photodegradation under DUV exposure, enabling a high signal-to-noise ratio and repetitive DUV imaging of nucleobases in cells. Underlying mechanisms of the photodegradation suppression can be excitation relaxation of the DUV-absorptive molecules due to energy transfer to the lanthanide ions, and/or avoiding ionization and reactions with surrounding molecules, including generation of reactive oxygen species, which can modify molecules that are otherwise transparent to DUV light. This approach, directly removing excited energy at the fundamental origin of cellular photodegradation, indicates an important first step towards the practical use of DUV imaging in a variety of biological applications.

Deep ultraviolet resonant Raman imaging of a cell.

We report the first demonstration of deep ultraviolet (DUV) Raman imaging of a cell. Nucleotide distributions in a HeLa cell were observed without any labeling at 257 nm excitation with resonant bands attributable to guanine and adenine. Obtained images represent DNA localization at nucleoli in the nucleus and RNA distribution in the cytoplasm. The presented technique extends the potential of Raman microscopy as a tool to selectively probe nucleic acids in a cell with high sensitivity due to resonance.

Deep UV resonant Raman spectroscopy for photodamage characterization in cells.

We employed deep UV (DUV) Raman spectroscopy for characterization of molecular photodamage in cells. 244 nm light excitation Raman spectra were measured for HeLa cells exposed to the excitation light for different durations. In the spectra obtained with the shortest exposure duration (0.25 sec at 16 µW/µm(2) irradiation), characteristic resonant Raman bands of adenine and guanine at 1483 cm(-1) and tryptophan and tyrosine at 1618 cm(-1) were clearly visible. With increasing exposure duration (up to 12.5 sec), these biomolecular Raman bands diminished, while a photoproduct Raman band at 1611 cm(-1) grew. By exponential function fitting analyses, intensities of these characteristic three bands were correlated with sample exposure duration at different intensities of excitation light. We then suggest practical excitation conditions effective for DUV Raman observation of cells without photodamage-related spectral distortion.