Rebecca M. Williams

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.

Two-photon molecular excitation provides intrinsic. 3-dimensional resolution for laser-based microscopy and microphotochemistry

With the development of sensitive and specific fluorescent indicators, modern laser scanning microscopies enable visualization and measurement of submicron, dynamic processes inside living cells and tissues. Here we describe the working principles of new, nonlinear laser microscopies based on two‐photon molecular excitation. In these techniques, a pulsed laser produces peak photon densities high enough that when focused into an appropriate medium, excitation by photon energy combinations can occur. For example, two red photons interacting simultaneously with a fluorescent molecule can excite within it a UV electronic transition, one corresponding to twice the energy of each single photon. Because the amount of two‐photon excitation depends on the square of the local illumination intensity, this process exhibits a unique localization to the diffraction‐limited spot of the beam focus. Elsewhere along the beam, excitation of background and photodamage is virtually nonexistent. Focal point localization of two‐photon excitation lends to all visualization, measurement, and photopharmacology studies an intrinsic, three‐dimensional resolution. We describe some preliminary biological applications, specifically, imaging of vital DNA stains in developing cells and embryos, imaging of cellular metabolic activity from NADH autofluorescence, spatially resolved measurements of cytoplasmic calcium ion activity, and optically induced micropharmacology using caged bioeffector molecules.—Williams, R. M., Piston, D. W., Webb, W. W. Two‐photon molecular excitation provides intrinsic three‐dimensional resolution for laser‐based microscopy and microphotochemistry. FASEB J. 8: 804‐813; 1994.

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.

Tuning three-dimensional collagen matrix stiffness independently of collagen concentration modulates endothelial cell behavior

Numerous studies have described the effects of matrix stiffening on cell behavior using two-dimensional synthetic surfaces; however, less is known about the effects of matrix stiffening on cells embedded in three-dimensional in vivo-like matrices. A primary limitation in investigating the effects of matrix stiffness in three dimensions is the lack of materials that can be tuned to control stiffness independently of matrix density. Here, we use collagen-based scaffolds where the mechanical properties are tuned using non-enzymatic glycation of the collagen in solution, prior to polymerization. Collagen solutions glycated prior to polymerization result in collagen gels with a threefold increase in compressive modulus without significant changes to the collagen architecture. Using these scaffolds, we show that endothelial cell spreading increases with matrix stiffness, as does the number and length of angiogenic sprouts and the overall spheroid outgrowth. Differences in sprout length are maintained even when the receptor for advanced glycation end products is inhibited. Our results demonstrate the ability to de-couple matrix stiffness from matrix density and structure in collagen gels, and that increased matrix stiffness results in increased sprouting and outgrowth.

Biophysical control of invasive tumor cell behavior by extracellular matrix microarchitecture

Fibrillar collagen gels, which are used extensively in vitro to study tumor-microenvironment interactions, are composed of a cell-instructive network of interconnected fibers and pores whose organization is sensitive to polymerization conditions such as bulk concentration, pH, and temperature. Using confocal reflectance microscopy and image autocorrelation analysis to quantitatively assess gel microarchitecture, we show that additional polymerization parameters including culture media formulation and gel thickness significantly affect the dimensions and organization of fibers and pores in collagen gels. These findings enabled the development of a three-dimensional culture system in which cell-scale gel microarchitecture was decoupled from bulk gel collagen concentration. Interestingly, morphology and migration characteristics of embedded MDA-MB-231 cells were sensitive to gel microarchitecture independently of collagen gel concentration. Cells adopted a polarized, motile phenotype in gels with larger fibers and pores and a rounded or stellate, less motile phenotype in gels with small fibers and pores regardless of bulk gel density. Conversely, cell proliferation was sensitive to gel concentration but not microarchitecture. These results indicate that cell-scale gel microarchitecture may trump bulk-scale gel density in controlling specific cell behaviors, underscoring the biophysical role of gel microarchitecture in influencing cell behavior.

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.

Quantitative Fluorescence Confocal Laser Scanning Microscopy (CLSM)

The confocal imaging geometry provides a dramatic optical advantage for fluorescence microscopy by discriminating against out-of-focus background with minimal loss of image-forming signal. Significant enhancement of both axial and lateral imaging resolution is also available but only with substantial signal loss. Because of these optical advantages, the confocal laser scanning microscope (CLSM) can clearly image thin optical sections from within thick fluorescence-labeled living specimens. A stack of optical sections is easily combined to reveal three-dimensional (3D) fluorescent marker distributions with diffraction-limited spatial resolution. When bright stable fluorophores are available, cellular dynamics can be measured by recording a time series of CLSM images.

Nuclear import of the retinoid X receptor, the vitamin D receptor, and their mutual heterodimer

The nuclear receptor retinoid X receptor (RXR) can regulate transcription through homotetramers, homodimers, and heterodimers with other nuclear receptors such as the vitamin D receptor (VDR). The mechanisms that underlie the nuclear import of RXR, VDR, and RXR-VDR heterodimers were investigated. We show that RXR and VDR translocate into the nucleus by distinct pathways. RXR strongly bound to importinβ and was predominantly nuclear in the absence of ligand. Importin binding and nuclear localization of RXR were modestly enhanced by its ligand, 9-cis-retinoic acid. On the other hand, VDR selectively associated with importinα. Importin association and correspondingly nuclear import of VDR were markedly augmented by 1,25(OH)2D3. RXR-VDR dimerization inhibited the ability of RXR to bind importinβ and to mobilize into the nucleus using its own nuclear localization signal. In contrast, VDR recruited RXR-VDR heterodimers to importinα and mediated nuclear import of the heterodimers in response to 1,25(OH)2D3. Hence nuclear import of RXR-VDR heterodimers is mediated preferentially by VDR and is controlled by the VDR ligand. The observations reveal a novel mechanism by which an RXR heterodimerization partner dominates the activity of the heterodimers.

Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and full-field laser scanning microscopes

Development of a laser scanning microscope for simultaneous three-dimensional imaging in both a full-field laser scanning mode (FLSM) and a confocal laser scanning mode (CLSM) permits the direct comparison of axial resolution and out-of-focus background rejection as a function of sample thickness for both FLSM and CLSM with varying detector aperture (pinhole) radii. The sample-dependent detector aperture radii that optimize the signal-to-noise ratio (S/N) in the CLSM are experimentally determined. The results verify earlier calculations [Appl. Opt. 33, 603 (1994)]. Using these results, we discuss the practical and theoretical limits on the S/N in the CLSM and compare them with those of a full-field epifluorescence microscope (FEM) that is enhanced by image deconvolution. The specimen volume over which the FLSM exhibits imaging properties that are equivalent to a FEM is calculated in the appendices.

Obesity-dependent changes in interstitial ECM mechanics promote breast tumorigenesis

Obesity leads to fibrotic remodeling of mammary adipose tissue, and the resulting increase in interstitial extracellular matrix stiffness promotes breast tumor malignancy. Fat fibrosis and breast cancer One of the many risk factors for cancer is obesity—but why? Seo et al. examined the cellular, structural, and molecular changes that happen in breast tissue in obese animals and people. They found that obesity induces fibrotic remodeling of the mammary fat pad, leading to changes in extracellular matrix (ECM) mechanical properties, via myofibroblasts and adipose stem cells (ASCs), regardless of ovary function. Through altered mechanotransduction, ECM from obese mice promoted human breast cancer cell growth, as well as the growth of premalignant breast cells (those that have yet to become cancerous). Tissues from obese patients revealed more severe fibrotic remodeling around tumors and higher levels of a key mechanosignaling component, YAP/TAZ, than their lean counterparts. The authors further demonstrated that caloric restriction in obese mice decreased fibrosis in mammary fat, suggesting a therapeutic angle for obesity-related cancers. By linking tumorigenesis to the behavior of fat cells and ECM mechanics, the authors point toward new drug targets for preventing cancer progression. However, a cautionary tale also exists in the use of adipose tissue and cells for patients after mastectomy, as ASCs from obese individuals may have the capacity to promote breast cancer recurrence. Obesity and extracellular matrix (ECM) density are considered independent risk and prognostic factors for breast cancer. Whether they are functionally linked is uncertain. We investigated the hypothesis that obesity enhances local myofibroblast content in mammary adipose tissue and that these stromal changes increase malignant potential by enhancing interstitial ECM stiffness. Indeed, mammary fat of both diet- and genetically induced mouse models of obesity were enriched for myofibroblasts and stiffness-promoting ECM components. These differences were related to varied adipose stromal cell (ASC) characteristics because ASCs isolated from obese mice contained more myofibroblasts and deposited denser and stiffer ECMs relative to ASCs from lean control mice. Accordingly, decellularized matrices from obese ASCs stimulated mechanosignaling and thereby the malignant potential of breast cancer cells. Finally, the clinical relevance and translational potential of our findings were supported by analysis of patient specimens and the observation that caloric restriction in a mouse model reduces myofibroblast content in mammary fat. Collectively, these findings suggest that obesity-induced interstitial fibrosis promotes breast tumorigenesis by altering mammary ECM mechanics with important potential implications for anticancer therapies.