Eric R. Tkaczyk

Kinetic properties of ASC protein aggregation in epithelial cells.

Apoptosis‐associated speck‐like protein with CARD domain (ASC), an adaptor protein composed of caspase recruitment and pyrin domains, can efficiently self‐associate to form a large spherical structure, called a speck. Although ASC aggregation is generally involved with both inflammatory processes and apoptosis, the detailed dynamics of speck formation have not been characterized. In this report, speck formation in HeLa cells transfected with ASC is examined by time‐lapse live‐imaging by confocal laser scanning microscopy. The results show that ASC aggregation is a very rapid and tightly regulated process. Prior to speck formation, soluble ASC aggregation is a low probability event, and the affinity of ASC subunits for one another is very low. Following a speck nucleation event, the affinity for further addition of ASC subunits increases dramatically, and aggregation is a highly energetically favorable reaction (Gibbs free energy ∼ −40 kJ/mol). This leads to a rapid depletion of soluble ASC, making it highly unlikely that a second speck will form inside the same cell and assuring that speck formation is “all or none,” with a well‐defined end point. Comparison with kinetic models of the aggregation process indicates diffusion, instead of active transport, is the dominant process for speck growth. Though speck formation and aggresome formation share some properties, we show that the two processes are distinct. J. Cell. Physiol. 222: 738–747, 2010.

Multiphoton flow cytometry strategies and applications

A handful of research teams around the world have recently begun to utilize multiphoton techniques in cytometry, especially for in vivo applications. These approaches offer similar enhancements to flow cytometry as the multiphoton phenomenon brought to the field of microscopy at the turn of the 20th century, with at least six advantages over single‐photon excitation. Here, we review the published literature on multiphoton cytometry in vivo or in vitro from the initial experiments in 1999 to present. Multiphoton cytometry instrumentation set‐ups vary from adapted multiphoton microscopy to a dedicated system, with laser pulse power and repetition rate serving as important variables. Two‐beam geometry enables quantitation of cell size. Labeling strategies include conjugated fluorophore targeting, with folate and/or dendrimer platforms. With two‐color measurement, ratiometric labeling is also possible, where one dye serves as a trigger to indicate the amount of excitation a cell receives, and another informs of cellular function. With two‐color labeling, geometric fluorophore distribution proves important in theory and experiment for detection sensitivity curves and detected event intensity correlation. The main biological achievements to date using this young technology are reviewed, with emphasis on real‐time monitoring of minute‐by‐minute and long‐term cell dynamics as well as the clinically significant surveillance of circulating tumor cells. For this goal, minimally invasive two‐photon flow cytometry with a fiber probe may overcome the primary issue of sample volume. The technique of multicolor, multiphoton flow cytometry greatly enhances the capabilities of flow cytometry to investigate the dynamics of circulating cells in cancer and other important diseases, and may in the future benefit from advances in microscopy such as super‐resolution imaging, coherent control, and bioluminescence.

Quantitative two-photon flow cytometry—in vitro and in vivo

Flow cytometry is a powerful technique for quantitative characterization of fluorescence in cells. Quantitation is achieved by ensuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow. Two-photon excitation has the advantages that it enables simultaneous excitation of multiple dyes and achieves a very high SNR through simplified filtering and fluorescence background reduction. We demonstrate that two-photon excitation in conjunction with a targeted multidye labeling strategy enables quantitative flow cytometry even under conditions of nonuniform flow, such as may be encountered in simple capillary flow or in vivo. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling cells with targeted nanoparticles containing multiple fluorophores enables normalization of the fluorescence signal and thus quantitative measurements under nonuniform excitation. Flow cytometry using two-photon excitation is demonstrated for detection and differentiation of particles and cells both in vitro in a glass capillary and in vivo in the blood stream of live mice. The technique also enables us to monitor the fluorescent dye labeling dynamics in vivo. In addition, we present a unique two-beam scanning method to conduct cell size measurement in nonuniform flow.

Vectorial laws of refraction and reflection using the cross product and dot product.

We demonstrate that published vectorial laws of reflection and refraction of light based solely on the cross product do not, in general, uniquely determine the direction of the reflected and refracted waves without additional information. This is because the cross product does not have a unique inverse operation, which is explained in this Letter in linear algebra terms. However, a vector is in fact uniquely determined if both the cross product (vector product) and dot product (scalar product) with a known vector are specified, which can be written as a single equation with a left-invertible matrix. It is thus possible to amend the vectorial laws of reflection and refraction to incorporate both the cross and dot products for a complete specification with unique solution. This enables highly efficient, unambiguous computation of reflected and refracted wave vectors from the incident wave and surface normal.

Control of the blue fluorescent protein with advanced evolutionary pulse shaping

We demonstrate optical coherent control of the two-photon fluorescence of the blue fluorescent protein (BFP), which is of interest in investigations of protein-protein interactions. In addition to biological relevance, BFP represents an interesting target for coherent control from a chemical perspective due to its many components of highly nonexponential fluorescence decay and low quantum yield resulting from excited state isomerization. Using a genetic algorithm with a multiplicative (rather than ratiometric) fitness parameter, we are able to control the ratio of BFP fluorescence to second-harmonic generation without a considerable drop in the maximized signal. The importance of linear chirp and power-scaling on the discrimination process is investigated in detail.

MICROFLUIDIC DROPLET CONSISTENCY MONITORING AND ENCAPSULATED CELL DETECTION VIA LASER EXCITATION

Microfluidic droplets formed in emulsions are used in a variety of analytical techniques and hold great potential for future scientific and commercial applications. Our experiments merge quantitative quality engineering methods into the microdroplet field. We present a unique microdroplet generation and consistency monitoring system with laser optics excitation and detection. Our setup analyzes each droplet with sub-millisecond signal resolution and single photon accuracy, and is compatible with process control methods. To demonstrate the consistency of microdroplet generation over time, we measure and examine the mean frequency of aqueous plug-shaped droplet (microplug) formation in oil phase, as well as the mean length of plugs, and the interval between consecutive droplets. We also demonstrate the detection of cancer cells encapsulated within aqueous microdroplets in continuous oil phase flow. Two-channel optical monitoring allows for the simultaneous and independent inspection of both microdroplet generation and identification of green fluorescent protein-labelled cancer cells within the droplets. Increased accuracy and consistency are central to many established and developing microfluidic technologies. A systematic, quantitative approach as demonstrated with our experiments may be essential in the development of advanced microfluidic concepts that require exacting reproducibility and would greatly benefit from incorporated automated measurement techniques for process control.

Control of Two-photon Fluorescence of Common Dyes and Conjugated Dyes

We present a comprehensive study of the selective excitation of two-photon fluorescence from various pairs of dyes and dyes in different conjugation states with tailored pulse shapes found with a genetic algorithm (GA). We investigate a number of biologically important dyes, and include dyes conjugated to trastuzumab (Herceptin®) and to a poly(amidoamine) dendrimer. We consider in detail the ability of tailored pulse shaping to discriminate dyes with significant spectral overlap. Our procedure for adaptive pulse shaping includes power-law and chirp-scaling checks to prevent trivial convergences. The GA uses a multiplicative fitness parameter in a graded search method that converges on pulse shapes that not only differentiate two-photon processes, but do so in a high signal regime. We consider the results in terms of not only the absolute maximum ratio of discrimination achieved, but also present the evolutionary course of the GA and compare the improvement to a quantitative measure of the noise level. We also implement a time-domain acousto-optic measurement of two-photon excitation cross-section spectra. The results show that the ability to discriminate dyes is determined almost entirely by their differences in two-photon excitation cross section.

Extended cavity laser enhanced two-photon flow cytometry

We demonstrate enhanced sensitivity in two-photon flow cytometry with an extended cavity laser excitation source. At low power, the home-built 20-MHz oscillator was able to detect a significantly larger fraction, in either phosphate buffered saline (PBS) or whole blood, of green fluorescent protein (GFP)-expressing MCA-207 cells cross-labeled with the membrane-binding lipophilic dye DiD. A geometrical model is used to explain unique features of the signals resulting from the different spatial distribution of DiD and GFP. These unique features include sub-square law scaling of unsaturated two-photon signal, a sigmoidal sensitivity curve for detection under varying powers for cell detection thresholds as low as a single photon, and uncorrelated signal strengths in two detection channels.

Pre-administration of PepFect6-microRNA-146a nanocomplexes inhibits inflammatory responses in keratinocytes and in a mouse model of irritant contact dermatitis.

The skin is a difficult to access tissue for efficient delivery of large and/or charged macromolecules, including therapeutic DNA and RNA oligonucleotides. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. MiR-146a is known as anti-inflammatory miRNA that inhibits multiple factors from the nuclear factor (NF)-κB pathway in various cell types, including keratinocytes. In this study, PF6 was shown to form unimodal nanocomplexes with miR-146a mimic that entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets from the NF-κB pathway and the genes known to be activated by NF-κB, C-C motif ligand (CCL)5 and interleukin (IL)-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24 and C-X-C motif ligand 1 (CXCL1) in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases.

Physicochemical properties of blue fluorescent protein determined via molecular dynamics simulation

Blue fluorescent protein (BFP) is a mutant of green fluorescent protein (GFP), where the chromophore has been modified to shift the emitted fluorescence into the blue spectral region. In this study, MD calculations were performed with the GROMACS simulation package and AMBER force field to investigate the dependence of BFPs physicochemical properties on temperature and applied pressure. The MD approach enabled us to calculate the compressibility of protein itself, separately from the nontrivial contribution of the hydration shell, which is difficult to achieve experimentally. The computed compressibility of BFP (3.94 x10(-5) MPa(-1)) is in agreement with experimental values of globular proteins. The center-of-mass diffusion coefficient of BFP and its dependence on temperature and pressure, which plays an important role in its application as a probe for intracellular liquid viscosity measurement, was calculated and found to be in good agreement with photobleaching recovery experimental data. We have shown that decreased temperature as well as applied pressure increases the water viscosity, but the concomitant decrease of the BFP diffusion coefficient behaves differently from Stokes-Einstein formula. It is shown that the number of hydrogen bonds around the protein grows with pressure, which explains the aforementioned deviation. Pressure also reduces root mean square (RMS) fluctuations, especially those of the most flexible residues situated in the loops. The analysis of the RMS fluctuations of the backbone Calpha atoms also reveals that the most rigid part of BFP is the center of the beta-barrel, in accord with temperature B factors obtained from the Protein Data Bank.