Optical phase microscopy is widely adopted for quantitative imaging of optical density in transparent cells and tissues that lack absorption contrast. Fundamentally, the phase information of the sample is contained in the wavefront of the probe beam, often detected by interferometry-based techniques. Here, a novel approach has been developed based on the phase-sensitive second harmonic signals that are generated after the sample. A deep learning algorithm is developed for efficient recovery of the original phase images. Inheriting the advantages of the second harmonic imaging, our second harmonic phase imaging is a label-free technique with a demonstrated phase sensitivity of 1/100 wavelength and high robustness against noises, facilitating applications in biological imaging and remote sensing.
Hydrogen bonding plays an essential role in biological processes. In this report we apply hyper-Raman scattering spectroscopy to probe the effects of the alkyl groups on hydrogen bonding in mixtures of DMSO-methanol. We characterize the dependence of hyper-Raman spectra on concentration and observe suppression of the hyper-Raman responses of the methanol alkyl group at intermediate concentrations. In addition, small frequency shifts in the vibrational frequencies of DMSO and methanol were detected. These results provide new insights into the nature of the hydrogen bonding in solution and into the details of the hydrogen bond’s interaction with the alkyl groups.
Optical phase microscopy is widely adopted for quantitative imaging of optical density in transparent cells and tissues, yet lacks the chemical selectivity. To address this challenge, a bond-selective transient phase imaging (BTSP) technique was developed, in which a transient change in phase induced by infrared excitation of molecular vibrations was detected by a diffraction phase microscope. BTSP achieved chemically selective phase imaging of live cells. We further demonstrated an IR-pump visible-probe phase microscopy based on second harmonic generation after the sample, enabled by deep learning. The phase-sensitive information is encoded into the second harmonic signal, which is decoded using a deep learning algorithm. It presents a label-free technique featured by high phase sensitivity and high robustness against noises, which has promising applications in biological and medical imaging and remote sensing.
The optical activity of Raman scattering provides insight into the absolute configuration and conformation of chiral molecules. Applications of Raman optical activity (ROA) are limited by long integration times due to a relatively low sensitivity of the scattered light to chirality (typically 10-3 to 10-5). We apply ROA techniques to hyper-Raman scattering using incident circularly polarized light and a right-angle scattering geometry. We explore the sensitivity of hyper- Raman scattering to chirality as compared to spontaneous Raman optical activity. Using the excitation wavelength at around 532 nm, the photobleaching is minimized, while the hyper-Raman scattering benefits from the electronic resonant enhancement. For S/R-2-butanol and L/D-tartaric acid, we were unable to detect the hyper-Raman optical activity at the sensitivity level of 1%. We also explored parasitic thermal effects which can be mitigating by varying the repetition rate of the laser source used for excitation of hyper-Raman scattering.
Dimethyl sulfoxide (DMSO) is a biologically important solvent in part due to its dual miscibility with hydrophilic and hydrophobic molecules. Binary solutions of DMSO-water display non-ideal thermodynamics properties such as high viscosity and low freezing point due to hydrogen bonding. The unusual properties of DMSO-water solutions have been exploited to disrupt the formation of secondary structures of proteins during polymerase chain reaction assays and to act as a cryoprotectant for tissues. The exact coordination of the DMSO and water molecules remains unknown. Hyper- Raman scattering was employed for the first time to investigate binary systems of DMSO with water (H2O). As a part of this study, hyper-Raman and Raman spectra of pure solutions were first acquired and compared against existing Raman and IR spectroscopic data. Then the corresponding measurements were taken with deuterated DMSO-d6 and heavy water (D2O) to validate the analysis and to isolate overlapping spectral features. The permissive selection rules of hyper- Raman scattering provide new insight into disruptions of the self-hydrogen bonded networks of DMSO and water and the establishment of hydrogen bonded networks.
Raman spectroscopy is an essential optical tool for molecular fingerprints. The vibrational modes of biologically important molecules including proteins, nucleic acids and lipids have been studied to provide insight into their structure as well as insight into the metabolic processes and biomarker expression of cells. To explore hyper-Raman scattering as a complementary technique to Raman scattering, we build a laser system that can perform Raman and hyper-Raman scattering studies using a single setup. Using three amplification stages we are able to generate 8 ps, 1064 nm pulses at repetition rates up to 30 MHz. Converting the 1064 nm source laser to 532 nm, we achieve fast hyper-Raman detection and collect our spectrum with a commercial spectrometer and CCD. Using a single optical setup, we collect and compare Raman spectra at 532 nm to hyper-Raman spectra at 266 nm for water, ethanol and L-tartaric acid. Furthermore, we observe changes in the hyper-Raman peak intensities of an aqueous L-tartaric acid solution when selecting different laser repetition rates highlighting the need to control laser power and repetition rate to identify and mitigate thermal effects in biomolecules.