Warren R. Zipfel

Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation

Multicolor nonlinear microscopy of living tissue using two- and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermolecular structures produces images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provides crucial information for physiology and pathology. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimers disease and cancer.

Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum

Calcium is a second messenger in virtually all cells and tissues. Calcium signals in the nucleus have effects on gene transcription and cell growth that are distinct from those of cytosolic calcium signals; however, it is unknown how nuclear calcium signals are regulated. Here we identify a reticular network of nuclear calcium stores that is continuous with the endoplasmic reticulum and the nuclear envelope. This network expresses inositol 1,4,5-trisphosphate (InsP3) receptors, and the nuclear component of InsP3-mediated calcium signals begins in its locality. Stimulation of these receptors with a little InsP3 results in small calcium signals that are initiated in this region of the nucleus. Localized release of calcium in the nucleus causes nuclear protein kinase C (PKC) to translocate to the region of the nuclear envelope, whereas release of calcium in the cytosol induces translocation of cytosolic PKC to the plasma membrane. Our findings show that the nucleus contains a nucleoplasmic reticulum with the capacity to regulate calcium signals in localized subnuclear regions. The presence of such machinery provides a potential mechanism by which calcium can simultaneously regulate many independent processes in the nucleus.

Multiphoton microscopy in biological research

From its conception a decade ago, multiphoton microscopy has evolved from a photonic novelty to an indispensable tool for gleaning information from subcellular events within organized tissue environments. Its relatively deep optical penetration has recently been exploited for subcellularly resolved investigations of disease models in living transgenic mice. Its enhanced spectral accessibility enables aberration-free imaging of fluorescent molecules absorbing in deep-UV energy regimes with simultaneous imaging of species having extremely diverse emission spectra. Although excited fluorescence is the primary signal for multiphoton microscopy, harmonic generation by multiphoton scattering processes are also valuable for imaging species with large anharmonic modes, such as collagen structures and membrane potential sensing dyes.

Simultaneous spatial and temporal focusing of femtosecond pulses

We experimentally demonstrate the concept of simultaneous spatial and temporal focusing of femtosecond pulses. Our technique has the potential to significantly reduce background excitation which fundamentally limits the imaging depth in scattering biological specimens.

Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery.

Multiphoton fluorescence photobleaching recovery (MP-FPR) is a technique for measuring the three-dimensional (3D) mobility of fluorescent molecules with 3D spatial resolution of a few microns. A brief, intense flash of mode-locked laser light pulses excites fluorescent molecules via multiphoton excitation in an ellipsoidal focal volume and photobleaches a fraction. Because multiphoton excitation of fluorophores is intrinsically confined to the high-intensity focal volume of the illuminating beam, the bleached region is restricted to a known, three-dimensionally defined volume. Fluorescence in this focal volume is measured with multiphoton excitation, using the attenuated laser beam to measure fluorescence recovery as fresh unbleached dye diffuses in. The time course of the fluorescence recovery signal after photobleaching can be analyzed to determine the diffusion coefficient of the fluorescent species. The mathematical formulas used to fit MP-FPR recovery curves and the techniques needed to properly utilize them to acquire the diffusion coefficients of fluorescently labeled molecules within cells are presented here. MP-FPR is demonstrated on calcein in RBL-2H3 cells, using an anomalous subdiffusion model, as well as in aqueous solutions of wild-type green fluorescent protein, yielding a diffusion coefficient of 8.7 x 10(-7) cm(2)s(-1) in excellent agreement with the results of other techniques.

Multiphoton excitation cross‐sections of molecular fluorophores

Nonlinear excitation of fluorophores through molecular absorption of two or three near-infra-red photons from the tightly focused femtosecond pulses of a mode-locked laser offers the cellular biologist an unprecedented panoply of biomolecular indicators for microscopic imaging and cellular analysis. Measurements of the two-photon excitation spectra of 25 ultra-violet and visible absorbing fluorophores from 690 to 1050 nm reveal useful cross sections for near infra-red excitation, providing an artists palette of emission markers, chemical indicators, and native cellular absorbers for living biological preparations. Measurements of three-photon fluorophore excitation spectra now suggest relatively benign wavelengths to excite deeper UV fluorophores. The inherent optical sectioning capabilities of focused nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background. Measured nonlinear excitation spectra are described and implications to nonlinear microscopy for biological imaging are defined.

Optimization of Pairings and Detection Conditions for Measurement of FRET between Cyan and Yellow Fluorescent Proteins

Detection of Förster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater Förster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET.

Core-shell silica nanoparticles as fluorescent labels for nanomedicine

Progress in biomedical imaging depends on the development of probes that combine low toxicity with high sensitivity, resolution, and stability. Toward that end, a new class of highly fluorescent core-shell silica nanoparticles with narrow size distributions and enhanced photostability, known as C dots, provide an appealing alternative to quantum dots. Here, C dots are evaluated with a particular emphasis on in-vivo applications in cancer biology. It is established that C dots are nontoxic at biologically relevant concentrations, and can be used in a broad range of imaging applications including intravital visualization of capillaries and macrophages, sentinel lymph node mapping, and peptide-mediated multicolor cell labeling for real-time imaging of tumor metastasis and tracking of injected bone marrow cells in mice. These results demonstrate that fluorescent core-shell silica nanoparticles represent a powerful novel imaging tool within the emerging field of nanomedicine.

Delivery of nanojoule femtosecond pulses through large-core microstructured fibers

We investigate femtosecond-pulse propagation through large-core microstructured fibers. Although these fibers are highly multimode, excitation of the fundamental mode is readily achieved, and coupling to higher-order modes is weak even when the fiber is bent or twisted. For prechirped input pulses with energies as large as 3 nJ, pulses as short as 140 fs were produced at the output of the fiber. Such a system could prove to be extremely useful for applications such as in vivo multiphoton microscopy and endoscopy that require delivery of femtosecond pulses and collection of fluorescence.

Conformational changes of calmodulin upon Ca2+ binding studied with a microfluidic mixer

A microfluidic mixer is applied to study the kinetics of calmodulin conformational changes upon Ca2+ binding. The device facilitates rapid, uniform mixing by decoupling hydrodynamic focusing from diffusive mixing and accesses time scales of tens of microseconds. The mixer is used in conjunction with multiphoton microscopy to examine the fast Ca2+-induced transitions of acrylodan-labeled calmodulin. We find that the kinetic rates of the conformational changes in two homologous globular domains differ by more than an order of magnitude. The characteristic time constants are ≈490 μs for the transitions in the C-terminal domain and ≈20 ms for those in the N-terminal domain of the protein. We discuss possible mechanisms for the two distinct events and the biological role of the stable intermediate, half-saturated calmodulin.