Biological cells are composed primarily of water; and as such are challenging to image without staining since the induced intensity modulation of transmitted or reflected light is typically insufficient to permit acceptable contrast for optical imaging. This issue may be resolved with the aid of exogenous contrast agents, but this often has a deleterious effect on the cell and precludes in vivo imaging. A unique approach to this problem is afforded by the phase contrast microscope in which optical-path differences in transmitted light is exploited as a contrast mechanism for qualitative imaging. In recent years however, several quantitative phase imaging techniques have been developed which allow for diffraction limited endogenous-contrast imaging with excellent temporal resolution. We hereby present a laser scanning technique for quantitative phase imaging which achieves sub-diffraction limited resolution at the expense of temporal resolution. This instrument is based on a stabilized fiber interfometer which is incorporated into a near-field scanning optical microscope (NSOM) for tri-modal imaging. Our latest results will focus on modifications made to this system to facilitate imaging in a liquid environment. A simple approach for achieving stable shear-force feedback operation in a liquid will be presented. Acquired high resolution images of white blood cells revealed detailed sub-cellular features. Images of fibroblast cells in air and in a liquid environment confirm the efficacy of the feedback operation in a liquid. Moreover, we demonstrate cell refractometry capability without the need for ad hoc modifications. These results clearly highlight the unique potential of this instrument for the study of living cells.
Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) have shown the potential for noninvasive assessment of oral precancers and cancers. We have explored a combination of these nonlinear optical microscopic imaging techniques with widefield fluorescence imaging to assess morphometry similar to that of pathologic evaluation as well as information from endogenous fluorophores, which are altered with neoplastic transformation. Widefield fluorescence revealed areas of interest corresponding to sites with precancers or early tumors, generally resulting in a decrease in green emission or increase in red emission. Subsequent microscopy revealed significant differences in morphology between normal, dysplastic/neoplastic mucosa for all layers. Combination of a widefield and a microscopic technique provides a novel approach for tissue morphometric analysis along with large area assessment of tissue autofluorescence properties.
Multiphoton autofluorescence microscopy (MPAM) offers the ability to assess morphometry similar to that of
pathologic evaluation as well as biochemical information from endogenous fluorophores which are altered with
neoplastic transformation. In this study the spectroscopic properties of normal and neoplastic oral epithelium were
evaluated toward the goal of identifying image/spectroscopic based indicators of neoplastic transformation using
nonlinear optical microscopy.
Results indicated measureable differences between normal, dysplasia, and SCC that could be helpful in delineating
between the three conditions. In particular, a blue shift in autofluorescence emission was experienced for dysplasia
relative to normal. However, in the case of SCC the epithelial emission experienced a significant red shift relative to
both dysplasia and normal and displayed in an additional red peak that was not present in either normal or dysplastic
mucosa. Results were consistent with published results for SCC in the single-photon literature. The study
demonstrates that multiphoton autofluorescence spectroscopy may reveal features of oral mucosa that can be useful for
differentiating normal and neoplastic mucosa. When combined with morphometry provided by MPAM, a potentially
powerful technique for imaging of the oral cavity could be developed which provides both morphometric and
The survival rate for individuals diagnosed with oral cancer is correlated with the stage of detection. Thus the
development of novel techniques for the earliest possible detection of malignancies is of critical importance. Single
photon (1P) autofluorescence spectroscopy has proven to be a powerful diagnostic tool in this regard, but 2P (two
photon) spectroscopy remains essentially unexplored. In this investigation, a spectroscopic system was incorporated into
a custom-built 2P laser scanning microscope. Oral cancer was induced in the buccal pouch of Syrian Golden hamsters by
tri-weekly topical application of 9,10-dimethyl-1,2-benzanthracene (DMBA).Three separated sites where investigated in
each hamster at four excitation wavelengths from 780 nm to 890 nm. A Total of 8 hamsters were investigated (4 normal
and 4 DMBA treated). All investigated sites were imaged via 2p imaging, marked for biopsy, processed for histology
and H&E staining, and graded by a pathologist. The in vivo emission spectrum for normal, mild/high grade dysplasia and
squamous cell carcinoma is presented. It is shown that the hamsters with various stages of dysplasia are characterized by
spectral differences as a function of depth and excitation wavelength, compared to normal hamsters.
Over the last few years, several novel quantitative phase imaging techniques have been developed for the study of
biological cells. However, many of these techniques are encumbered by inherent limitations including 2π phase
ambiguities and diffraction limited spatial resolution. In addition, subsurface information in the phase data is not
exploited. We hereby present a novel quantitative phase imaging system without 2 π ambiguities, which also allows for
subsurface imaging and cell refractometry studies. This is accomplished by utilizing simultaneously obtained shear-force
topography information. We will demonstrate how the quantitative phase and topography data can be used for subsurface
and cell refractometry analysis and will present results for a fabricated structure and a malaria infected red blood cell.