We report development of a novel optical imaging technique capable of non-invasively monitoring single subcellular organelles in a living cell without use of exogenous contrast agents common to optical microscopy.

We report the development of a novel optical imaging technique, confocal light scattering spectroscopic microscopy, capable of non-invasively determining the dimensions and other physical properties of single subcellular organelles with 5 nm accuracy.

We report light scattering experiments using aqueous suspensions of subcellular organelles. From the observed spectra, we extract size distributions that are in very agreement with the results of electron microscopy studies.

Introduction: Light Scattering Spectroscopy has been a recently developed as a non-invasive technique capable of sizing the cellular organelles. With this technique, we monitor the heat-induced sub-cellular structural transformations in a human RPE cell culture. Material and Methods: A single layer of human RPE cells (ATCC) was grown on a glass slide. Cells are illuminated with light from a fiber-coupled broadband tungsten lamp. The backscattered (180 degree) light spectra are measured with an optical multichannel analyzer (OMA). Spectra are measured during heating of the sample. Results: We reconstructed the size distribution of sub-micron organelles in the RPE cells and observed temperature-related changes in the scattering density of the organelles in the 200-300nm range (which might be peroxisomes, microsomes or lysosomes). The sizes of the organelles did not vary with temperature, so the change in scattering is most probably due to the change in the refractive indexes. As opposed to strong spectral variation with temperature, the total intensity of the backscattered light did not significantly change in the temperature range of 32-49 °C. Conclusion: We demonstrate that Light Scattering Spectroscopy is a powerful tool for monitoring the temperature-induced sub-cellular transformations. This technique providing an insight into the temperature-induced cellular processes and can play an important role in quantitative assessment of the laser-induced thermal effects during retinal laser treatments, such as Transpupillary Thermal Therapy (TTT), photocoagulation, and Photodynamic Therapy (PDT).

A long-standing impediment for applications of optical techniques in cellular biology is the inability to characterize subcellular structures whose dimensions are much less than about 1 mum. In this paper, we describe a method based on light scattering spectroscopy that can find the size distribution of subcellular organelles as small as 100 nm with an accuracy of 20 nm. We report experiments using aqueous suspensions of subcellular organelles enriched in mitochondria, zymogen granules, and microsomes. From the observed light scattering spectra, we extract size distributions that are in excellent agreement with the results of electron microscopy. Further studies are underway to extract the shapes of organelles in addition to their sizes.

In this work we have studied the dispersion of the disorder-induced (D) and the second-order (G') Raman bands in single wall carbon nanotubes using several laser excitation energies (E-laser) in the range 1.5-3.0 eV. An anomalous step-like behavior was observed in the E-laser dependence of the G'-band frequency. This result is interpreted as a manifestation of the one-dimensional (1D) behavior of the phonon spectrum in carbon nanotubes.

Get PDF Email Share Share with Facebook Tweet This Post on reddit Share with LinkedIn Add to CiteULike Add to Mendeley Add to BibSonomy Get Citation Copy Citation Text E. B. Hanlon, I. Itzkan, and N. W. Kowall, "Optical Spectroscopy For Detection Of Alzheimer’s Disease," in Biomedical Optical Spectroscopy and Diagnostics, T. Li, ed., Vol. 38 of OSA Trends in Optics and Photonics (Optica Publishing Group, 2000), paper SuD1. Export Citation BibTex Endnote (RIS) HTML Plain Text Citation alert Save article

Cultures of the phytoplankton diatom, Pseudonitzschia multiseries, have been harvested under controlled growth conditions ranging from late logarithmic to late stationary phase (17-58 days). The amount of domoic acid (DA) present in the growth media and in the homogenized cells has been determined by HPLC. Defined samples of media, homogenized cells, whole cells, and whole cells in media have been laser excited at 251 nm for the purpose of selectively exciting intense UV resonance Raman spectra from DA in the samples. Neither media nor cell component spectra from algae seriously interfere with DA spectra. The spectral cross sections for the dominant 1652-cm-1 mode of DA have been determined for 242-, 251-, and 257-nm excitation. Maximum sensitivities are achieved with 251-nm excitation because cross sections for DA are a maximum, and interference from other algal components becomes very small. DA concentrations that have been determined with 251-nm excitation by resonance Raman methods correlate closely with values determined independently with HPLC, especially at higher DA concentrations. The UV resonance Raman analysis of DA in phytoplankton algae is shown to be very sensitive and quantitative as well as rapid and nonintrusive.

We describe a new scanning microscopy technique, phase-dispersion microscopy (PDM). The technique is based on measuring the phase difference between the fundamental and the second-harmonic light in a novel interferometer. PDM is highly sensitive to subtle refractive-index differences that are due to dispersion (differential optical path sensitivity, 5 nm). We apply PDM to measure minute amounts of DNA in solution and to study biological tissue sections. We demonstrate that PDM performs better than conventional phase-contrast microscopy in imaging dispersive and weakly scattering samples.

Raman spectroscopy is a potentially important clinical tool for real-time diagnosis of disease and in situ evaluation of living tissue. The purpose of this article is to review the biological and physical basis of Raman spectroscopy of tissue, to assess the current status of the field and to explore future directions. The principles of Raman spectroscopy and the molecular level information it provides are explained. An overview of the evolution of Raman spectroscopic techniques in biology and medicine, from early investigations using visible laser excitation to present-day technology based on near-infrared laser excitation and charge-coupled device array detection, is presented. State-of-the-art Raman spectrometer systems for research laboratory and clinical settings are described. Modern methods of multivariate spectral analysis for extracting diagnostic, chemical and morphological information are reviewed. Several in-depth applications are presented to illustrate the methods of collecting, processing and analysing data, as well as the range of medical applications under study. Finally, the issues to be addressed in implementing Raman spectroscopy in various clinical applications, as well as some long-term directions for future study, are discussed.

The purpose of this study was to investigate whether near-infrared (NIR) fluorescence spectroscopy could be used to detect Alzheimer's disease (AD) by brain tissue autofluorescence. Unfixed temporal cortex specimens from AD cases and age-matched, non-AD controls were frozen at autopsy and then thawed just prior to spectral measurement. Spectra of intrinsic tissue fluorescence induced by 647 nm light were recorded from 650 to 850 nm. We used principal component analysis of the tissue spectra from 17 AD cases and 5 non-AD control cases in a calibration study to establish a diagnostic algorithm. Retrospectively applied to the calibration set, the algorithm correctly classified 23 of 24 specimens. In a prospective study of 19 specimens from 5 AD brains and 2 non-AD control brains, 3 of the 4 control specimens and all AD specimens were correctly diagnosed. Both the excitation light used and the measured brain tissue autofluorescence are at NIR wavelengths that can propagate through skull and overlying tissue. Therefore, our results demonstrate an optical spectroscopic technique that carries direct molecular level information about disease. This is the first step toward a clinical tool that has the potential to be applied to the noninvasive diagnosis of AD in living patients.

The anomalous resonant behavior of the tangential Raman modes of carbon nanotubes has been studied in the critical region of laser energies 1.7-2.2 eV. The special enhancement of the Raman modes is explained by a model that takes into account the transition between the singularities in the one-dimensional density of electronic states for the metallic nanotubes and the distribution of diameters in the sample. The results agree with direct measurements of the electronic density of states for the metallic nanotubes and establish their association with the specially enhanced high frequency, first-order Raman modes. [S0163-1829(98)50848-X].

In agreement with previous results reported for colloidal silver clusters, effective surface-enhanced Raman cross sections of about 10-(16) cm(2) per molecule, corresponding to enhancement factors on the order of 10(14), have also been obtained for molecules attached to colloidal gold clusters. Spatially isolated nearly spherical colloidal gold particles of about 60 nm size show maximum enhancement factors on the order of 10(3) at 514 nm excitation, close to the single plasmon resonance. The enhancement factor increases by eleven orders of magnitude when colloidal gold clusters are formed by aggregation of the gold colloids and when near-infrared excitation is applied. The large effective surface-enhanced Raman cross section has been estimated by a straightforward method based on steady-state population redistribution due to the pumping of molecules to the first excited vibrational state via the strongly enhanced Raman process. Our experimental finding confirms the important role of colloidal clusters for extremely large surface-enhanced Raman scattering (SERS) enhancement factors. Simultaneously, it suggests colloidal gold clusters as a substrate for high-sensitivity surface-enhanced Raman scattering, which can provide an enhancement level sufficient for Raman single molecule detection. Due to its chemical inactivity, gold might have some advantages compared to silver, particularly in biomedical spectroscopy.