Confocal light absorption and scattering spectroscopic microscopy monitors organelles in live cells with no exogenous labels

Itzkan, Irving; Qiu, Le; Fang, Hui; Zaman, Munir M.; Vitkin, Edward; Ghiran, Ionita; Salahuddin, Saira; Modell, Mark D.; Andersson, Charlotte; Kimerer, Lauren; Cipolloni, Patsy Benny; Lim, Kee-Hak; Freedman, Steven D.; Bigio, Irving J.; Sachs, Benjamin P.; Hanlon, Eugene B.; Perelman, Lev T.

doi:10.1073/pnas.0708669104

Cited by 121 publications

(106 citation statements)

References 26 publications

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“…Recently, confocal light absorption and scattering spectroscopic (CLASS) microscopy, which combines LSS with the principles of confocal microscopy, was developed by Itzkan, Fang, Perelman, and colleagues. 24,25 The multispectral nature of LSS enables it to measure internal cell structures much smaller than the diffraction limit without damaging the cell or requiring exogenous markers, which could affect cell function. CLASS microscopy approaches the accuracy of electron microscopy but is nondestructive and does not require the contrast agents common to traditional optical microscopy.…”

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Spectroscopy of Scattered Light for the Characterization of Micro and Nanoscale Objects in Biology and Medicine

et al. 2014

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The biomedical uses for the spectroscopy of scattered light by micro and nanoscale objects can broadly be classified into two areas. The first, often called light scattering spectroscopy (LSS), deals with light scattered by dielectric particles, such as cellular and sub-cellular organelles, and is employed to measure their size or other physical characteristics. Examples include the use of LSS to measure the size distributions of nuclei or mitochondria. The native contrast that is achieved with LSS can serve as a non-invasive diagnostic and scientific tool. The other area for the use of the spectroscopy of scattered light in biology and medicine involves using conducting metal nanoparticles to obtain either contrast or electric field enhancement through the effect of the surface plasmon resonance (SPR). Gold and silver metal nanoparticles are non-toxic, they do not photobleach, are relatively inexpensive, are wavelength-tunable, and can be labeled with antibodies. This makes them very promising candidates for spectrally encoded molecular imaging. Metal nanoparticles can also serve as electric field enhancers of Raman signals. Surface enhanced Raman spectroscopy (SERS) is a powerful method for detecting and identifying molecules down to single molecule concentrations. In this review, we will concentrate on the common physical principles, which allow one to understand these apparently different areas using similar physical and mathematical approaches. We will also describe the major advancements in each of these areas, as well as some of the exciting recent developments.Index headings: Light scattering; Spectroscopy; Nanoparticle; Surface plasmon; Surface enhanced Raman; SERS. INTRODUCTIONT he spectroscopy of scattered light has played an increasingly important role in biology and medicine because it can provide information about subcellular and extracellular biological structures using native contrast, as well as information about nanoparticles that are employed as the exogenous imaging markers or vehicles for the delivery of therapeutic agents. Light scattering by these microscopic objects is governed by the very basic principles of electromagnetic wave propagation and interaction with matter developed many decades ago. The impressive advances in biomedical instrumentation that have recently been achieved are examples of the successful applications of these fundamental principles. An analogy can be made between the development of biomedical light scattering and the development of other medical imaging modalities, for example the discovery of the very basic physics of X-rays led to the development of computed tomography decades later, while the initial understanding of another basic physical effect, nuclear magnetic resonance, eventually led to magnetic resonance imaging. Thus it can be argued that the field of biomedical optics is primarily concerned with the technological development of new medical applications of electromagnetic wave interactions, described by Maxwell's equations, with exogenous and endogenous...

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Spectroscopy of Scattered Light for the Characterization of Micro and Nanoscale Objects in Biology and Medicine

et al. 2014

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“…The submucosal background can be excluded by one of various gating techniques 12–18 and the smaller organelles have a very different scattering spectral dependence than that of the nuclei. Elastic light scattering can also be used to measure other cellular compartments, such as mitochondria 19 , whose spectra 20 are sufficiently different from that of nuclei to be distinguished 21 . The combination of gating and difference in spectral behavior allows the epithelial nuclear scattering spectrum to be isolated in the processed light scattering spectroscopy (LSS) signal.…”

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Light scattering spectroscopy identifies the malignant potential of pancreatic cysts during endoscopy

et al. 2017

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Pancreatic cancers are usually detected at an advanced stage and have poor prognosis. About one fifth of these arise from pancreatic cystic lesions. Yet not all lesions are precancerous, and imaging tools lack adequate accuracy for distinguishing precancerous from benign cysts. Therefore, decisions on surgical resection usually rely on endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). Unfortunately, cyst fluid often contains few cells, and fluid chemical analysis lacks accuracy, resulting in dire consequences, including unnecessary pancreatic surgery for benign cysts and the development of cancer. Here, we report an optical spectroscopic technique, based on a spatial gating fibre-optic probe, that predicts the malignant potential of pancreatic cystic lesions during routine diagnostic EUS-FNA procedures. In a double-blind prospective study in 25 patients, with 14 cysts measured in vivo and 13 postoperatively, the technique achieved an overall accuracy of 95%, with a 95%confidence interval of 78–99%, in cysts with definitive diagnosis.

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“…Optical detection through coherent light scattering offers a much valued platform for its label-free nature and capacities to extract both morphology and molecular information. Characterization of nucleus by scattered light signals thus attracts active research efforts [2][3][4][5][6][7]. Determination of cellular and nuclear morphology is fundamentally a challenging inverse problem for their complex 3D structures.…”

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Realistic optical cell modeling and diffraction imaging simulation for study of optical and morphological parameters of nucleus

Zhang¹,

Feng²,

Jiang³

et al. 2016

Opt. Express

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Coherent light scattering presents complex spatial patterns that depend on morphological and molecular features of biological cells. We present a numerical approach to establish realistic optical cell models for generating virtual cells and accurate simulation of diffraction images that are comparable to measured data of prostate cells. With a contourlet transform algorithm, it has been shown that the simulated images and extracted parameters can be used to distinguish virtual cells of different nuclear volumes and refractive indices against the orientation variation. These results demonstrate significance of the new approach for development of rapid cell assay methods through diffraction imaging. References and links 1.D. Zink, A. H. Fischer, and J. A. Nickerson, "Nuclear structure in cancer cells," Nat. Rev. Cancer 4, 677-687 (2004 IntroductionNucleus is one of the largest organelles inside eukaryotic cells, provides the site for DNA and RNA synthesis, plays critical roles in cell development. Hence it serves as one of major targets for cell assay by morphology and is especially important for detection of abnormal conditions and cancer diagnosis [1]. Optical detection through coherent light scattering offers a much valued platform for its label-free nature and capacities to extract both morphology and molecular information. Characterization of nucleus by scattered light signals thus attracts active research efforts [2][3][4][5][6][7]. Determination of cellular and nuclear morphology is fundamentally a challenging inverse problem for their complex 3D structures. For example, structural reconstruction requires large amount of measured data per cell and often expensive computation that is too long for rapid assay [8,9]. If one aims at only to distinguish cell types such as cancer from normal or apoptotic from viable cells, however, the goal may be achieved empirically with moderate amount of measured data per cell and powerful algorithms of pattern recognition. In either case, it is very useful to develop realistic optical cell models (OCMs) and accurate simulation tools for forward calculations of measured signals of scattered light. They can be employed, for example, to generate training data for algorithm development in search of the correlations between morphological features of cells and diffraction patterns of coherent light scatter.In this report, we present a numerical approach based on previous studies for establishing realistic OCMs for generating virtual cells and accurate simulation of polarized diffraction image (p-DI) data [9][10][11][12][13]. The new approach takes the advantage of 3D cell morphology and molecular information acquired from the fluorescent confocal images to produce simulated p-DI data that are comparable to the measured ones acquired with a polarization diffraction imaging flow cytometry (p-DIFC) system [14][15][16][17][18][19][20]. To demonstrate the utility of the realistic OCMs, we have investigated the effects of nuclear morphology and refractive index (RI) on di...

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Confocal light absorption and scattering spectroscopic microscopy monitors organelles in live cells with no exogenous labels

Cited by 121 publications

References 26 publications

Spectroscopy of Scattered Light for the Characterization of Micro and Nanoscale Objects in Biology and Medicine

Spectroscopy of Scattered Light for the Characterization of Micro and Nanoscale Objects in Biology and Medicine

Light scattering spectroscopy identifies the malignant potential of pancreatic cysts during endoscopy

Realistic optical cell modeling and diffraction imaging simulation for study of optical and morphological parameters of nucleus

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