Ignacy Gryczynski

Multiphoton Ligand-Enhanced Excitation of Lanthanides

We describe multiphoton excitation of the lanthanides europium (Eu3+) and terbium (Tb3+) when these ions are complexed with nucleic acids, proteins, and fluorescent chelators. In all cases excitation occurs by multiphoton absorption of the sensitizers. For the nucleotide GDP and an oligonucleotide with several guanines, the sensitized emission of Tb3+ excited at 776 nm indicated a three-photon process. For Tb3+ bound to the wild-type troponin C and a single tryptophan mutant (26W), excitation at 794 nm was also close to a three-photon process. For lanthanide chelators containing various sensitizers, we observed three-photon excitation in the case of methyl anthranilate, a mixuture of two- and three-photon excitation for carbostyril 124, and a two-photon process with a coumarin derivative. In the case of coumarin-sensitized emission of Eu3+ varied from a two- to a three-photon process at wavelengths ranging from 780 to 880 nm. The sensitized luminescence also shows significantly higher photostability compared to the fluorescence from the organic fluorophores alone. These results suggest the use of multiphoton-induced sensitized lanthanide fluorescence in biochemistry and cellular imaging.

2 – Basics of Fluorescence and FRET

This chapter discusses the most important characteristics of fluorescence that plays a fundamental role in understanding the basics and the applications of Forster (fluorescence) resonance (radiationless) energy transfer (FRET). FRET is the transfer of electronic excitation energy between isolated donor D and acceptor A of suitable spectroscopic properties. The donor molecules, typically, emit at shorter wavelengths, which overlap with the absorption spectrum of the acceptor. This energy transfer occurs without the appearance of the photon and is the result of long-range interactions between the D and A dipoles. The most important factors affecting FRET are the overlap integral, the quantum yield of the donor in the absence of the acceptor, and the orientation factor. The quantitative analysis of steady-state and time-resolved FRET measurements provides information on global structures and conformational dynamics, and reveals thermodynamic parameters for conformational transition. This information is essential for the understanding of biological functions of proteins, DNA/RNA, and other biological assemblies that are frequently mediated by transitions between alternative conformations.

Noble-Metal Surfaces for Metal-Enhanced Fluorescence

Noble metal nanoparticles exhibit strong absorption bands, which known as the surface plasmon resonances, result in strong absorption and scattering, and create an enhanced local electromagnetic field near-to the surface of the particles. The surface plasmon resonances are highly dependent on the size and the shape of the metal and the dielectric properties of the surrounding medium. These near field enhancements have given rise to surface-enhanced resonant Raman scattering (SERRS) and metal-enhanced fluorescence (MEF) (Figure 1). Unlike SEERS, the optimal MEF signal occurs at a certain distance from the surface of the metal nanoparticles. The fluorophores in direct contact with the metal surface are typically quenched. Theoretical and experimental work using rough surfaces and particles has suggested that the distance-dependent enhancement fluorescence intensity is more pronounced for low quantum yield fluorophores.1–8 This enhancement is accompanied by a significantly reduced lifetime. The increased fluorescence intensities accompanied by reduced lifetimes suggest an increased radiative decay rate for the fluorophores interacting with the metals.

Multi-pulse based approach to study dynamics of molecular assemblies with subwavelength resolution (Conference Presentation)

The long standing unmet need of optical microscopy has been imaging subcellular structures with nanometer precision with speed that will allow following physiological processes in real time. Herein we presenting a new approach (multi-pulse pumping with time-gated detection; MPP-TGD) to increase image resolution and most importantly to significantly improve imaging speed. Alternative change from single pulse to multiple-pulse excitation within continuous excitation trace (in interleave excitation mode) allows for the instantaneous and specific increase (many-folds) in the intensity of subwavelength sized object labeled with long-lived probes. This permits for quick localization of the object. Such intensity change (blinking) on demand can be done with MHz frequency allowing for ultrafast point localization several hundred folds faster than localization based on single molecule blinking. Much higher speed for super-resolution imaging will pave the way for obtaining real time functional information and probing structural rearrangements at the nanometer scale in-vitro and in-vivo. This will have a critical impact on many biomedical applications and enhance our understanding of many cellular functions.nWe use the microtubules as a model biological system with our new approach to studying microtubule dynamics in real time. The recent work based on single molecule localization microscopy (SMLM) (Mikhaylova et al., 2015) clearly indicates that microtubules are ~25 nm diameter hollow biopolymers that are organized in a closely spaced (about 20-70 nm apart) microtubule bundles. These structures are organized differently between axons and dendrites and their precise organization in different cell compartments is not completely understood.