Reflective objectives (ROs) can reduce chromatic aberration across a wide wavelength range in multiphoton microscopy (MPM). However, a systematic characterization of the performance of ROs has not been carried out. In this paper, we analyze the performance of a 0.5 numerical-aperture (NA) RO and compare it with a 0.55 NA standard glass objective (SO), using two-photon fluorescence (TPF) and second-harmonic generation (SHG). For experiments extending ~1 octave in visible and NIR wavelengths, the SO introduces defocusing errors of ~25% for TPF images of sub-diffraction fluorescent beads and ~10% for SHG images of collagen fibers. For both imaging systems, the RO provides a corresponding error of ~4%. This work highlights the potential usefulness of ROs for multimodal MPM applications.
Second-harmonic generation (SHG) microscopy is a label-free imaging technique to study collagenous materials in extracellular matrix environment with high resolution and contrast. However, like many other microscopy techniques, the actual spatial resolution achievable by SHG microscopy is reduced by out-of-focus blur and optical aberrations that degrade particularly the amplitude of the detectable higher spatial frequencies. Being a two-photon scattering process, it is challenging to define a point spread function (PSF) for the SHG imaging modality. As a result, in comparison with other two-photon imaging systems like two-photon fluorescence, it is difficult to apply any PSF-engineering techniques to enhance the experimental spatial resolution closer to the diffraction limit. Here, we present a method to improve the spatial resolution in SHG microscopy using an advanced maximum likelihood estimation (AdvMLE) algorithm to recover the otherwise degraded higher spatial frequencies in an SHG image. Through adaptation and iteration, the AdvMLE algorithm calculates an improved PSF for an SHG image and enhances the spatial resolution by decreasing the full-width-at-halfmaximum (FWHM) by ~20%. Similar results are consistently observed for biological tissues with varying SHG sources, such as gold nanoparticles and collagen in porcine feet tendons. By obtaining an experimental transverse spatial resolution of ~400 nm, we show that the AdvMLE algorithm brings the practical spatial resolution closer to the theoretical diffraction limit. Our approach is suitable for adaptation in micro-nano CT and MRI imaging, which has the potential to impact diagnosis and treatment of human diseases.
KEYWORDS: Luminescence, Nanoplasmonics, Silver, Confocal microscopy, Metals, Resonance enhancement, 3D image processing, Near field optics, 3D image enhancement, Surface plasmons
We have created an enhanced cell-imaging platform for 3D confocal fluorescence cell imaging where fluorescence
sensitivity is amplified for more than 100 folds especially for cell membrane and cytoplasm. The observed
unprecedented three-dimensional fluorescence intensity enhancement on the entire cell microstructure including cell
membrane 10 μm above the substrate surface is attributed to a novel far field enhancement mechanism, nanoplasmon
coupled optical resonance excitation (CORE) mechanism which permits enhanced surface plasmon on the substrate
being evanescently coupled to Whispering Gallery mode optical resonance inside the spheroidal cell membrane
microcavity. Theoretical model of the hypothesis is explained using coupled mode theory. In addition, preliminary result
has been provided for the observation of early stage of transfection in a cancer cell.
Fourier transform-second-harmonic generation (FT-SHG) imaging is used to quantitatively assess the structural
organization of collagen fibers in tendonitis-induced horse tendons. Fiber orientation, isotropy, and the ratio of forward
to backward SHG signal (F/B ratio) are used to differentiate the fiber organization between the normal and diseased
horse tendons. Each second-harmonic generation (SHG) image is divided into several smaller regions of interest (ROI)
and the aforementioned quantitative metrics are calculated across the whole grid. ROIs are further labeled as dark (no or
minimal presence of fibers), isotropic (random fiber organization), or anisotropic (regular fiber organization) regions.
Results show that the normal tendon possesses minimal isotropic regions and small standard deviations in the histograms
of orientation and F/B ratio, indicating an intact and highly regular fiber organization. However, the tendonitis-induced
horse tendons possess higher number of dark and isotropic regions, and larger standard deviations of the measured
parameters, suggesting significantly disoriented and disorganized collagen fibers. This type of quantification would be
highly beneficial in diagnosing and determining the stage of tendonitis in clinical settings. Not limited to tendonitis, the
technique could also be applied to other diseases that structurally affect collagen fibers. The advantage of FT-SHG over
the conventional polarization microscopy is also discussed.
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