Maxim Kalashnikov

Measuring cellular structure at submicrometer scale with light scattering spectroscopy

We present a novel instrument for imaging the angular distributions of light backscattered by biological cells and tissues. The intensities in different regions of the image are due to scatterers of different sizes. We exploit this to study scattering from particles smaller than the wavelength of light used, even when they are mixed with larger particles. We show that the scattering from subcellular structure in both normal and cancerous human cells is best fitted to inverse power-law distributions for the sizes of the scattering objects, and propose that the distribution of scattering objects may be different in normal versus cancerous cells.

Determination of particle size by using the angular distribution of backscattered light as measured with low-coherence interferometry

We employ a novel interferometer to measure the angular distribution of light backscattered by a turbid medium. Through comparison of the measured data with the predictions of Mie theory, we are able to determine the size of the scatterers comprising the medium with subwavelength precision. As the technique is based on low-coherence interferometry, we are able to examine the evolution of the angular distribution of scattered light as it propagates into the medium. The effects of multiple scattering as a function of penetration depth in the medium are analyzed. We also present various considerations for extending this technique to determining structural information in biological tissues, such as the effects of a distribution of particle sizes and the need to average out speckle contributions.

Assessing epithelial cell nuclear morphology by using azimuthal light scattering spectroscopy

We describe azimuthal light scattering spectroscopy (phi/LSS), a novel technique for assessing epithelial-cell nuclear morphology. The difference between the spectra measured at azimuthal angles phi = 0 degrees and phi = 90 degrees preferentially isolates the single backscattering contribution due to large (approximately 10 microm) structures such as epithelial cell nuclei by discriminating against scattering from smaller organelles and diffusive background. We demonstrate the feasibility of using phi/LSS for cancer detection by showing that spectra from cancerous colon tissue exhibit significantly greater azimuthal asymmetry than spectra from normal colonic tissues.

Assessing light scattering of intracellular organelles in single intact living cells

This report presents a model-independent method of assessing contributions to the light scattering from individual organelles in single intact cells. We first measure the 3D index map of a living cell, and then modify the map in such a way so as to eliminate contrast due to a particular intracellular organelle. By calculating and comparing the light scattering distributions calculated from the original and modified index maps using the Rytov approximation, we extract the light scattering contribution from the particular organelle of interest. The relative contributions of the nucleus and nucleolus to the scattering of the entire cell are thus determined, and the applicability of the homogeneous spherical model to non-spherical and heterogeneous organelles in forward scattering is evaluated.

Imaging and measurement of cell structure and organization with submicron accuracy using light scattering spectroscopy

Light scattering spectroscopy (LSS) is a promising optical technique developed for quantitative characterization of tissue morphology as well as in vivo detection and diagnosis of disease such as early cancer. LSS employs a wavelength dependent component of light scattered by epithelial cells and other tissues to obtain information about subcellular structure. We present two novel modalities of LSS, LSS imaging and scattering angle sensitive LSS (a/LSS). LSS imaging provides quantitative information about the epithelial cell nuclei, such as nuclear size, degree of pleomorphism, hyperchromasia, and amount of chromatin. It allows mapping these histological properties over wide areas of epithelial lining. We show that LSS imaging can be used to detect precancerous lesions in optically accessible organs. Using a/LSS, which enables characterization of tissue components with sizes smaller than the wavelength of light, we show that the number of subcellular components with the sizes between 30 nm and few microns scales with the size according to an inverse power-law. We show that the size distribution exponent is an important parameter characterizing tissue organization, for example the balance between stochasticity and order, and has a potential to be applicable for early cancer diagnosis and characterization.

Assessing the contribution of cell body and intracellular organelles to the backward light scattering

We report a method of assessing the contribution of whole cell body and its nucleus to the clinically most relevant backward light scattering. We first construct an experimental system that can measure forward scattering and use the system to precisely extract the optical properties of a specimen such as the refractive index contrast, size distribution, and their density. A system that can simultaneously detect the backscattered light is installed to collect the backscattering for the same specimen. By comparing the measured backscattering spectrum with that estimated from the parameters determined by the forward scattering experiment, the contribution of cell body and nucleus to the backward light scattering is quantitatively assessed. For the HeLa cells in suspension, we found that the cell body contributes less than 10% and cell nucleus on the order of 0.1% to the total backscattering signal. Quantitative determination of the origin of backscattered light may help design a system that aims for detecting particular structure of biological tissues.

Rapid phenotypic stress-based microfluidic antibiotic susceptibility testing of Gram-negative clinical isolates

Bacteremia is a life-threatening condition for which antibiotics must be prescribed within hours of clinical diagnosis. Since the current gold standard for bacteremia diagnosis is based on conventional methods developed in the mid-1800s—growth on agar or in broth—identification and susceptibility profiling for both Gram-positive and Gram-negative bacterial species requires at least 48–72 h. Recent advancements in accelerated phenotypic antibiotic susceptibility testing have centered on the microscopic growth analysis of small bacterial populations. These approaches are still inherently limited by the bacterial growth rate. Our approach is fundamentally different. By applying environmental stress to bacteria in a microfluidic platform, we can correctly assign antibiotic susceptibility profiles of clinically relevant Gram-negative bacteria within two hours of antibiotic introduction rather than 8–24 h. The substantial expansion to include a number of clinical isolates of important Gram-negative species—Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa—reported here underscores the broad utility of our approach, complementing the method’s proven utility for Gram-positive bacteria. We also demonstrate that the platform is compatible with antibiotics that have varying mechanisms of action—meropenem, gentamicin, and ceftazidime—highlighting the versatility of this platform.

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step - bacterial growth - is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane (PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

Optimization of Stress-Based Microfluidic Testing for Methicillin Resistance in Staphylococcus aureus Strains

The rapid evolution of antibiotic resistance in bacterial pathogens is driving the development of innovative, rapid antibiotic susceptibility testing (AST) tools as a way to provide more targeted and timely antibiotic treatment. We have previously presented a stress-based microfluidic method for the rapid determination of antibiotic susceptibility in methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). In this method, stress is used to potentiate the action of antibiotics, and cell death is measured as a proxy for susceptibility. The method allows antibiotic susceptibility to be determined within an hour from the start of the antibiotic introduction. However, the relatively low dynamic range of the signal (2–10% cell response) even with high antibiotic concentrations (10–50 µg/mL) left room for the method’s optimization. We have conducted studies in which we varied the flow patterns, the media composition, and the antibiotic concentration to increase the cell death response and concordantly decrease the required antibiotic concentration down to 1–3 µg/mL, in accordance with the Clinical and Laboratory Standards Institute’s (CLSI) guidelines for AST breakpoint concentrations.

Continuous-flow, microfluidic, qRT-PCR system for RNA virus detection

AbstractOne of the main challenges in the diagnosis of infectious diseases is the need for rapid and accurate detection of the causative pathogen in any setting. Rapid diagnosis is key to avoiding the spread of the disease, to allow proper clinical decisions to be made in terms of patient treatment, and to mitigate the rise of drug-resistant pathogens. In the last decade, significant interest has been devoted to the development of point-of-care reverse transcription polymerase chain reaction (PCR) platforms for the detection of RNA-based viral pathogens. We present the development of a microfluidic, real-time, fluorescence-based, continuous-flow reverse transcription PCR system. The system incorporates a disposable microfluidic chip designed to be produced industrially with cost-effective roll-to-roll embossing methods. The chip has a long microfluidic channel that directs the PCR solution through areas heated to different temperatures. The solution first travels through a reverse transcription zone where RNA is converted to complementary DNA, which is later amplified and detected in real time as it travels through the thermal cycling area. As a proof of concept, the system was tested for Ebola virus detection. Two different master mixes were tested, and the limit of detection of the system was determined, as was the maximum speed at which amplification occurred. Our results and the versatility of our system suggest its promise for the detection of other RNA-based viruses such as Zika virus or chikungunya virus, which constitute global health threats worldwide. Graphical abstractPhotograph of the RT-PCR thermoplastic chip