A multimodal system combining surface sensitive sum frequency generation (SFG) vibrational spectroscopy and total-internal reflection fluorescence (TIRF) microscopy for surface and interface study was developed. Interfacial molecular structural information can be detected using SFG spectroscopy while interfacial fluorescence signal can be visualized using TIRF microscopy from the same sample. As a proof of concept experiment, SFG spectra of fluorescent polystyrene (PS) beads with different surface coverage were correlated with TIRF signal observed. Results showed that SFG signals from the ordered surfactant methyl groups were detected from the substrate surface, while signals from PS phenyl groups on the beads were not seen. Additionally, a lipid monolayer labeled using lipid-associated dye was deposited on a silica substrate and studied in different environments. The contact with water of this lipid monolayer caused SFG signal to disappear, indicating a possible lipid molecular disorder and the formation of lipid bilayers or liposomes in water. TIRF was able to visualize the presence of lipid molecules on the substrate, showing that the lipids were not removed from the substrate surface by water. The integration of the two surface sensitive techniques can simultaneously visualize interfacial molecular dynamics and characterize interfacial molecular structures in situ, which is important and is expected to find extensive applications in biological interface related research.
Three-dimensional digital holographic microscopic phase imaging of objects that are thicker than the wavelength of the imaging light is ambiguous and results in phase wrapping. In recent years, several unwrapping methods that employed two or more wavelengths were introduced. These methods compare the phase information obtained from each of the wavelengths and extend the range of unambiguous height measurements. A straightforward dual-wavelength phase imaging method is presented which allows for a flexible tradeoff between the maximum height of the sample and the amount of noise the method can tolerate. For highly accurate phase measurements, phase unwrapping of objects with heights higher than the beat (synthetic) wavelength (i.e. the product of the original two wavelengths divided by their difference), can be achieved. Consequently, three-dimensional measurements of a wide variety of biological systems and microstructures become technically feasible. Additionally, an effective method of removing phase background curvature based on slowly varying polynomial fitting is proposed. This method allows accurate volume measurements of several small objects with the same image frame.
Coherent anti-Stokes Raman scattering (CARS) microscopy is a powerful technique to image the chemical composition
of complex samples in biophysics, biology and materials science. CARS is a four-wave mixing process. The application
of a spectrally narrow pump beam and a spectrally wide Stokes beam excites multiple Raman transitions, which are
probed by a probe beam. This generates a coherent directional CARS signal with several orders of magnitude higher
intensity relative to spontaneous Raman scattering. Recent advances in the development of ultrafast lasers, as well as
photonic crystal fibers (PCF), enable multiplex CARS. In this study, we employed two scanning imaging methods. In
one, the detection is performed by a photo-multiplier tube (PMT) attached to the spectrometer. The acquisition of a
series of images, while tuning the wavelengths between images, allows for subsequent reconstruction of spectra at each
image point. The second method detects CARS spectrum in each point by a cooled coupled charged detector (CCD)
camera. Coupled with point-by-point scanning, it allows for a hyperspectral microscopic imaging. We applied this
CARS imaging system to study biological samples such as oocytes.
Digital holography records the superposition of the object and reference waves. The subsequent reconstruction of both
amplitude and phase of the optical field is done by numerically propagating the optical field along the direction
perpendicular to the hologram plane in accordance with the laws of diffraction. Phase changes undergone by a light
wave passing through or reflecting from objects can be converted to the optical thickness or height measurements,
providing the three dimensional structural information about the object. Our dual wavelength phase imaging method
allows three dimensional measurements of a wide variety of biological systems and microstructures.