Raman microscopy enables us to obtain molecular information in biological samples but has suffered from low signal-to-noise ratio (SNR) due to low of Raman scattering cross-section. Here we developed a cryo-stat equipped Raman microscope for low temperature measurement, allowing long time accumulation of Raman signals. We confirmed the SNR improvement in Raman imaging of cryofixed HeLa cells without photodamage under long time observation at low temperature. The reduction of photobleaching in resonant Raman scattering of carotenoid and cytochrome significantly increases the SNR, demonstrated by 7-color high SNR Raman imaging with multiple Raman tags, including EdU, MitoBADY, and alkyne-tagged Coenzyme Q (AltQ2). AltQ2 is a mobile small molecule that cannot be fixed by chemical fixation.
Raman microscopy provides a variety of insights into molecular composition, chemical state, and environmental conditions in biological samples. However, biological imaging with Raman microscopy have faced challenges such a low signal-to-noise ratio, mainly due to the low scattering efficiency of Raman scattering. To overcome this limitation, we developed a cryo-Raman microscope integrated with a cryostat capable of rapid freezing of biological samples and low-temperature Raman imaging. The spatiotemporal cryofixation of biological samples allows long exposure measurements to accumulate signals without photodamage. We observed both reduction of photobleaching in resonant Raman scattering of cytochromes in cryofixed HeLa cells, and the preservation of redox states of cytochromes in rat heart tissue by cryofixation.
Spontaneous Raman flow cytometers and cell sorters typically have cell or particle throughputs less than 5 event/s due to long signal integration times. Here we report an up to 10x increase in throughput by coupling constant illumination along the flow path with time delay integration, a technique that counteracts sample motion by matching it to the charge transfer velocity of a CCD device. This allows longer signal integration times while also multiplexing acquisition along the flow path. We demonstrate high spectral bandwidth (600 – 3200 cm-1) Raman acquisitions from flowing particles and mammalian cells at throughputs up to 50 events/s.
Here, we performed photoinitiator-free two-photon polymerization (TPP) to fabricate 3D cell micro-scaffolds. By using a visible femtosecond pulsed laser as excitation, TPP is induced in deep UV-absorbing moieties without the use of photo-initiators. We fabricated 3D micro-structures of biocompatible materials and glycidyl methacrylate protein without photoinitiators. By using Raman/Brillouin spectroscopy, we quantitatively investigated the molecular and mechanical properties of the 3D structures, which are important factors for cell functions and growth on scaffolds. We seeded cells on the 3D scaffolds and observed their attachment and proliferation without causing numerous cell deaths due to the absence of potentially cytotoxic photoinitiators.
Simultaneous observation of cytotoxicity and drug distribution at the effector site will bring new insights into understanding mechanism of action of drug. Here, we demonstrated the application of Raman microscopy to visualize drug-induced cytotoxicity and drug distribution as well as its mechanism of action at a real-time and single-cell level. The drug concentration and treatment-dependent decrease of reduced cyt c was found as an early marker of cell apoptosis which is more sensitive than the conventional cell viability assay. This method allows for us to screen the drug cytotoxicity and approach the mechanism at the same time, thereby reducing the cost and time of drug screening.
One major obstacle hampering the utilization of human induced pluripotent stem cells (hiPS) derived cells from bench to bedside is the safety concern of residual undifferentiated hiPS cells. To ensure the clinical use of differentiated cells, a method which can monitor the regenerative processes and assess the cell population without harming and modifying those cells is very critical in this field. Raman microscopy has emerged as a powerful tool in label-free observation and discrimination of cell types without external labels. Cell proliferation, differentiation, and maturation all trigger molecular changes that can be detected via Raman, enabling non-destructive characterization of cell and tissue constructs. By using Raman microscopy, we are aiming at establishing a non-invasive and quantitative evaluation method to monitor hepatic differentiation. In the future, this technique would be an invaluable tool in the quality control and safety assessment of hiPS-derived cell products.
KEYWORDS: Raman spectroscopy, Raman scattering, Live cell imaging, Microscopy, Molecules, Signal detection, Resonance enhancement, Molecular spectroscopy, Microscopes, Signal to noise ratio
Resonance Raman scattering is useful for improving a signal-to-noise ratio and a data acquisition speed in Raman imaging. However, the detection of non-resonance Raman scattering is often hindered by resonance signals and fluorescent background. To aid this dilemma in using resonance Raman scattering, we have developed a confocal Raman microscope with dual-wavelength excitation. Living HeLa cells were measured simultaneously at two different excitation wavelengths. At 532 nm excitation, cytochromes were detected by the resonance effect. At 660 nm excitation, non-resonance signals from proteins and lipids were obtained without any clear influence from cytochromes and fluorescent background.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.