Andrew C. Millard

Three-Dimensional High-Resolution Second-Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues

We find that several key endogenous protein structures give rise to intense second-harmonic generation (SHG)-nonabsorptive frequency doubling of an excitation laser line. Second-harmonic imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architecture. Unlike fluorescence, SHG suffers no inherent photobleaching or toxicity and does not require exogenous labels. Unlike polarization microscopy, SHIM provides intrinsic confocality and deep sectioning in complex tissues. In this study, we demonstrate the clarity of SHIM optical sectioning within unfixed, unstained thick specimens. SHIM and two-photon excited fluorescence (TPEF) were combined in a dual-mode nonlinear microscopy to elucidate the molecular sources of SHG in live cells and tissues. SHG arose not only from coiled-coil complexes within connective tissues and muscle thick filaments, but also from microtubule arrays within interphase and mitotic cells. Both polarization dependence and a local symmetry cancellation effect of SHG allowed the signal from species generating the second harmonic to be decoded, by ratiometric correlation with TPEF, to yield information on local structure below optical resolution. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures that is enhanced by the intrinsic chirality of the protein helices.

Second harmonic generation imaging of endogenous structural proteins.

We show that structural protein arrays consisting largely of collagen, myosin, and tubulin, and their associated proteins can be imaged in three dimensions with high contrast and resolution by laser-scanning second harmonic generation (SHG) microscopy. SHG is a nonlinear optical scheme and this form of microscopy shares several common advantages with multiphoton excited fluorescence, namely, intrinsic three-dimensionality and reduced out-of-plane photobleaching and phototoxicity. SHG does not arise from absorption and in-plane photodamage considerations are therefore also greatly reduced. In particular, structural protein arrays that are highly ordered and birefringent produce large SHG signals without the need for any exogenous labels. We demonstrate that thick tissues including muscle and bone can be imaged and sectioned through several hundred micrometers of depth. Combining SHG with two-photon excited green fluorescent protein (GFP) imaging allows inference of the molecular origin of the SHG contrast in Caenorhabditis elegans sarcomeres. Symmetry and organization of microtubule structures in dividing C. elegans embryos are similarly studied by comparing the endogenous tubulin contrast with that of GFP::tubulin fluorescence. It is found that SHG provides molecular level data on radial and lateral symmetries that GFP constructs cannot. The physical basis of SHG is discussed and compared with that of two-photon excitation as well as that of polarization microscopy. Due to the intrinsic sectioning, lack of photobleaching, and availability of molecular level data, SHG is a powerful tool for in vivo imaging.

Macromolecular structure of cellulose studied by second-harmonic generation imaging microscopy

The macromolecular structure of purified cellulose samples is studied by second-harmonic generation (SHG) imaging microscopy. We show that the SHG contrast in both Valonia and Acetobacter cellulose strongly resembles that of collagen from animal tissues, both in terms of morphology and polarization anisotropy. Polarization analysis shows that microfibrils in each lamella are highly aligned and ordered and change directions by 90 degrees in adjacent lamellae. The angular dependence of the SHG intensity fits well to a cos2 theta distribution, which is characteristic of the electric dipole interaction. Enzymatic degradation of Valonia fibers by cellulase is followed in real time by SHG imaging and results in exponential decay kinetics, showing that SHG imaging microscopy is ideal for monitoring dynamics in biological systems.

Direct measurement of the voltage sensitivity of second-harmonic generation from a membrane dye in patch-clamped cells

We report what is to our knowledge the first optical imaging of voltage-clamped cells by second-harmonic generation. For the membrane-staining styryl dye di-4-ANEPPS, we determined the sensitivity of second-harmonic generation to be 18%/100 mV at an excitation wavelength of 850 ns. This sensitivity is significantly better than the optimal 10%/100 mV under fluorescence and further establishes the importance of second-harmonic generation for the functional imaging of membrane potential in living cells.

Second harmonic imaging microscopy.

Publisher Summary The chapter provides information on the second harmonic imaging microscopy. Over the past three decades, the physical phenomenon of optical second harmonic generation (SHG) has been used to study interfaces between materials and has been adapted for the purposes of microscopy. SHG is a nonlinear optical process that can take place in a microscope that uses illumination from ultrafast (near-infrared) laser light. One of the first demonstrations of SHG from a biological specimen was of bacteriorhodopsin in a membrane preparation. More recently, SHG microscope images are obtained when one leaflet of the lipid bilayer of the cell membrane is stained with a dye that enhances SHG. SHG is a less efficient process than 2PF but can be significantly resonance enhanced. However, because excited fluorescence (2PF) still results in some photobleaching at the focus, the best wavelengths for SHG are on the edges of two-photon excitation bands, enhancing SHG while reducing absorption.

Wavelength- and time-dependence of potentiometric non-linear optical signals from styryl dyes

Second harmonic generation (SHG) imaging microscopy is an important emerging technique for biological research, complementing existing one- and two-photon fluorescence (2PF) methods. A non-linear phenomenon employing light from mode-locked Ti:sapphire or fiber-based lasers, SHG results in intrinsic optical sectioning without the need for a confocal aperture. Furthermore, as a second-order process SHG is confined to loci lacking a center of symmetry, a constraint that is readily satisfied by lipid membranes with only one leaflet stained by a dye. Of particular interest is “resonance-enhanced” SHG from styryl dyes in cellular membranes and the possibility that SHG is sensitive to transmembrane potential. We have previously confirmed this, using simultaneous voltage-clamping and non-linear imaging of cells to find that SHG is up to four times more sensitive to potential than fluorescence. In this work, we have extended these results in two directions. First, with a range of wavelengths available from a mode-locked Ti:sapphire laser and a fiber-based laser, we have more fully investigated SHG and 2PF voltage-sensitivity from ANEP and ASTAP chromophores, obtaining SHG sensitivity spectra that are consistent with resonance enhancements. Second, we have modified our system to coordinate the application of voltage-clamp steps with non-linear image acquisition to more precisely characterize the time dependence of SHG and 2PF voltage sensitivity, finding that, at least for some dyes, SHG responds more slowly than fluorescence to changes in transmembrane potential.

Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light

Nonlinear optical phenomena, such as two-photon fluorescence (2PF) and second harmonic generation (SHG), in combination with voltage sensitive dyes, can be used to acquire high-resolution spatio temporal maps of electrical activity in excitable cells and tissue. Developments in 1064-nm fiber laser technology have simplified the generation of high-intensity, long-wavelength, femtosecond light pulses, capable of penetrating deep into tissue. To merge these two advances requires the design and synthesis of new dyes that are optimized for longer wavelengths and that produce fast and sensitive responses to membrane potential changes. In this work, we have systematically screened a series of new dyes with varying chromophores and sidechains that anchor them in cell membranes. We discovered several dyes that could potentially be used for in vivo measurements of cellular electrical activity because of their rapid and sensitive responses to membrane potential. Some of these dyes show optimal activity for SHG; others for 2PF. This regulated approach to dye screening also allows significant insight into the molecular mechanisms behind both SHG and 2PF. In particular, the differing patterns of sensitivity and kinetics for these two nonlinear optical modalities indicate that their voltage sensitivity originates from differing mechanisms.

Second harmonic generation imaging of endogenous structural proteins

We find that several key endogenous structural proteins including collagen, acto-myosin, and tubulin give rise to intense second harmonic generation (SHG) and that these structures can be imaged in intact tissues on a laser-scanning microscope. Because SHG is a non-resonant process, this modality suffers little inherent photobleaching or toxicity. In this study we demonstrate the clarity of SHG optical sectioning within unfixed, unstained thick specimens, including fish scales, C. elegans, and mouse muscle, where penetration into tissue upwards of 600 microns was achieved. The simultaneous use of SHG and two-photon excited GFP fluorescence allows for the inference of the molecular isoform that gives rise to SHG from the myofilament lattice in C. elegans. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures. SHG polarization anisotropy is also used to determine the extent of dipolar order and radial symmetry in the helical structures. Comparisons are drawn between SHG and other forms of microscopy including polarization and fluorescence microscopy, highlighting the advantages and disadvantages.

Second harmonic generation imaging of skeletal muscle tissue and myofibrils

Second Harmonic Generation (SHG) imaging microscopy is used to examine the morphology and structural properties of intact muscle tissue. Using biochemical and optical analysis, we characterize the molecular structure underlying SHG from the complex muscle sarcomere. We find that SHG from isolated myofibrils is abolished by extraction of myosin, but is unaffected by removal or addition of actin filaments. We thus determined that the SHG emission arises from domains of the sarcomere containing thick filaments. By fitting the SHG polarization anisotropy to theoretical response curves, we find an orientation for the harmonophore that corresponds well to the pitch angle of the myosin rod α-helix with respect to the thick filament axis. Taken together, these data indicate that myosin rod domains are the key structures giving rise to SHG from striated muscle. Using SHG imaging microscopy, we have also examined the effect of optical clearing with glycerol to achieve greater penetration into specimens of skeletal muscle tissue. We find that treatment with 50% glycerol results in a 2.5 fold increase in achievable SHG imaging depth. Fast Fourier Transform (FFT) analysis shows quantitatively that the periodicity of the sarcomere structure is unaltered by the clearing process. Also, comparison of the SHG angular polarization dependence shows no change in the supramolecular organization of acto-myosin complexes. We suggest that the primary mechanism of optical clearing in muscle with glycerol treatment results from the reduction of cytoplasmic protein concentration and concomitant decrease in the secondary inner filter effect on the SHG signal. The pronounced lack of dependence of glycerol concentration on the imaging depth indicates that refractive index matching plays only a minor role in the optical clearing of muscle.