Mark D. Modell

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

This article reports the development of an optical imaging technique, confocal light absorption and scattering spectroscopic (CLASS) microscopy, capable of noninvasively determining the dimensions and other physical properties of single subcellular organelles. CLASS microscopy combines the principles of light-scattering spectroscopy (LSS) with confocal microscopy. LSS is an optical technique that relates the spectroscopic properties of light elastically scattered by small particles to their size, refractive index, and shape. 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. Scanning the confocal volume across the sample creates an image. CLASS microscopy approaches the accuracy of electron microscopy but is nondestructive and does not require the contrast agents common to optical microscopy. It provides unique capabilities to study functions of viable cells, which are beyond the capabilities of other techniques.

Multispectral scanning during endoscopy guides biopsy of dysplasia in Barrett's esophagus

Esophageal cancer is increasing in frequency in the United States faster than any other cancer. Barretts esophagus, an otherwise benign complication of esophageal reflux, affects approximately three million Americans and precedes almost all cases of esophageal cancer. If detected as high-grade dysplasia (HGD), most esophageal cancers can be prevented. Standard-of-care screening for dysplasia uses visual endoscopy and a prescribed pattern of biopsy. This procedure, in which a tiny fraction of the affected tissue is selected for pathological examination, has a low probability of detection because dysplasia is highly focal and visually indistinguishable. We developed a system called endoscopic polarized scanning spectroscopy (EPSS), which performs rapid optical scanning and multispectral imaging of the entire esophageal surface and provides diagnoses in near real time. By detecting and mapping suspicious sites, guided biopsy of invisible, precancerous dysplasia becomes practicable. Here we report the development of EPSS and its application in several clinical cases, one of which merits special consideration.

Confocal light absorption and scattering spectroscopic microscopy

We have developed a novel optical method for observing submicrometer intracellular structures in living cells, which is called confocal light absorption and scattering spectroscopic (CLASS) microscopy. It combines confocal microscopy, a well-established high-resolution microscopic technique, with light-scattering spectroscopy. CLASS microscopy requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales of the order of 100 nm.

Optical, real-time monitoring of the glomerular filtration rate.

An easy and accurate assessment of the renal function is a critical requirement for detecting the initial functional decline of the kidney induced by acute or chronic renal disease. A method for measuring the glomerular filtration rate is developed with the accuracy of clearance techniques and the convenience of plasma creatinine. The renal function is measured in rats as the rate of clearance determined from time-resolved transcutaneous fluorescence measurements of a new fluorescent glomerular filtration agent. The agent has a large dose-safety coefficient and the same space distribution and clearance characteristics as iothalamate. This new approach is a convenient and accurate way to perform real-time measurements of the glomerular filtration rate to detect early kidney disease before the renal function becomes severely and irreversibly compromised.

Single Gold Nanorod Detection Using Confocal Light Absorption and Scattering Spectroscopy

Gold nanorods have the potential to be employed as extremely bright molecular marker labels for fluorescence, absorption, or scattering imaging of living tissue. However, samples containing a large number of gold nanorods usually exhibit relatively wide spectral lines. This linewidth limits the use of the nanorods as effective molecular labels, since it would be rather difficult to image several types of nanorod markers simultaneously. In addition, the observed linewidth does not agree well with theoretical calculations, which predict significantly narrower absorption and scattering lines. The discrepancy could be explained by apparent broadening because of the contribution of nanorods with various sizes and aspect ratios. We measured native scattering spectra of single gold nanorods with the confocal light absorption and scattering spectroscopy system, and found that single gold nanorods have a narrow spectrum as predicted by the theory, which suggests that nanorod-based molecular markers with controlled narrow aspect ratios, and to a lesser degree size distributions, should provide spectral lines sufficiently narrow for effective biomedical imaging.

Observation of plasmon line broadening in single gold nanorods

Attempts to realize the important potential of gold nanorods as extremely bright molecular markers have been limited by the broad spectroscopic linewidths usually observed. We identify the origin of this broadening as inhomogeneous broadening due to the extreme sensitivity of the surface plasmon resonance to the nanorod aspect ratio. Using confocal light scattering spectroscopic microscopy, we observed the narrow homogeneously broadened plasmon lines of single gold nanorods and obtained the first quantitative measurements of this homogeneous broadening. We show that homogeneous broadening can be predicted from first principals.

Light-scattering spectroscopy differentiates fetal from adult nucleated red blood cells: may lead to noninvasive prenatal diagnosis

Present techniques for prenatal diagnosis are invasive and present significant risks of fetal loss. Noninvasive prenatal diagnosis utilizing fetal nucleated red blood cells (fNRBC) circulating in maternal peripheral blood has received attention, since it poses no risk to the fetus. However, because of the failure to find broadly applicable identifiers that can differentiate fetal from adult NRBC, reliable detection of viable fNRBC in amounts sufficient for clinical use remains a challenge. In this Letter we show that fNRBC light-scattering spectroscopic signatures may lead to a clinically useful method of minimally invasive prenatal genetic testing.

Diagnostic imaging of esophageal epithelium with clinical endoscopic polarized scanning spectroscopy instrument

This letter reports the development of an endoscopic polarized scanning spectroscopy (EPSS) instrument compatible with existing endoscopes. This instrument uses light scattering spectroscopy (LSS). In proof-of-principle studies using a single-point instrument, LSS has successfully demonstrated the ability to identify pre-cancer in the epithelial tissues of five different organs, including Barretts esophagus (BE). The EPSS instrument can provide real time in vivo information on the location of otherwise invisible high grade dysplasia (HGD), a predictor of adenocarcinoma, and thus can serve as a guide for biopsy. It should greatly reduce the time and labor involved in performing screening and obtaining diagnoses, cause less patient discomfort and ensure that fewer biopsies are required for the reliable location of pre-cancerous lesions.

Studying cell dynamics and function with CLASS microscopy

Confocal light absorption and scattering spectroscopic (CLASS) microscopy is a novel optical technique for observing submicron intracellular structures in living cells. It allows monitoring nondestructively cell function and cell dynamics in vivo and in real time. CLASS microscopy, having accuracy well beyond the diffraction limit, does not require cell fixation as the electron microscopy. In addition, it provides not only size information but also information about the biochemical and physical properties of the cell. CLASS microscopy can also visualize multiple compartments inside of living cell without employing exogenous molecular markers which are required by fluorescence microscopy and which can affect normal cell functioning. Recently we improved our CLASS microscope by utilizing the full power output of the supercontinuum laser and used it to study apoptosis in live cells.

Low-coherent autocorrelation interferometry of layered media

Anew technique of low-coherent interferometry is discussed. In the method, the optical field reflected from the layered medium is directed into a longitudinal share scanning interferometer for autocorrelation analysis. The object under analysis is outside the interferometer and the reference beam is not used. Theory, results of numerical simulation, and experimental data are presented.