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.
Many styryl dyes have been developed for use in imaging electrophysiology given the fast electrochromism of their fluorescence. The molecular characteristics responsible for electrochromism also make them strong harmonophores. Second harmonic generation (SHG) from a number of these dyes has been investigated for use in a new method of imaging electrochemical activity in neurons, cardiomyocytes and other cells. Depending on the choice of dye and excitation wavelength, the voltage-sensitivity of SHG may be up to four times as large as the voltage-sensitivity of fluorescence (one- or two-photon), as is well characterised by "slow" voltage-switching experiments. While some fluorescent dyes respond to changes in trans-membrane potential by slow mechanisms, such as a change in partitioning between the cell membrane and the extracellular medium, styryl dyes were developed because they respond rapidly, using a fast electrochromic shift of their fluorescence excitation and emission spectra. For use in imaging mammalian nerves, for instance, a response time of the order of a millisecond is necessary. Here we report on our characterisation of the time dependence of the voltage-sensitivity of SHG from styryl dyes using "fast" voltage-switching experiments, as compared with simultaneous two-photon fluorescence imaging for a number of different dyes.
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.
Second harmonic generation (SHG) imaging microscopy is an important emerging technique for biological research, with many advantages over existing one- or two-photon fluorescence techniques. A non-linear phenomenon employing 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. Many important structural proteins such as collagen and cellulose show intrinsic SHG, thus providing access to sub-resolution information on symmetry. However, we are particularly interested here in "resonance-enhanced" SHG from styryl dyes. In general SHG is a combination of a true second-order process and a third-order process dependent on a static electric field, such that SHG from membrane-bound dyes depends on a cell's trans-membrane potential. With simultaneous patch-clamping and non-linear imaging of cells, we have found that SHG is a sensitive probe of trans-membrane potential with sensitivities that are up to four times better than those obtained under optimal conditions using one-photon fluorescence imaging. With the sensitivity of SHG to local electric fields from other sources such as the membrane dipole potential as well as the quadratic dependence of SHG on concentration, we have found that SHG imaging of styryl dyes is also a powerful technique for the investigation of lipid phases and rafts and for the visualization of the dynamics of membrane-vesicle fusion following fertilization of an ovum.
Second harmonic generation (SHG) imaging microscopy is an important emerging technique for biological research, with many advantages over existing one- or two-photon fluorescence techniques. SHG is of growing interest to those in the biomedical community studying structural proteins such as collagen and to those in neuroscience using voltage-sensitive dyes. An important consideration in the application of non-linear phenomena such as SHG to routine microscopy is the complexity of the laser source used for excitation. Almost all applications in ultrafast microscopy currently employ mode-locked Ti:sapphire lasers, and though these systems have improved considerably in recent years, they are still expensive, large and complicated for those with skills outside of ultrafast optics. Here we report on SHG microscopy using a high power femtosecond fiber laser. The Femtopower1060 from Fianium Ltd. is an ultrafast fiber laser operating at 1064nm. With a passively mode-locked master source, a high power fiber amplifier and a built-in pulse compressor, the laser produces high quality pulses shorter than 200fs with a repetition rate of 100MHz and an average power of 1W. The unit is turn-key, air-cooled and maintenance free with a small footprint and proves to be an excellent source for SHG and two-photon microscopy at this wavelength outside the range of most Ti:sapphire systems and without those systems' complexity.
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.
As ultrafast multiphoton microscopes become more useful for biological imaging, a major challenge for researchers is to determine the exposure conditions that provide the best combination of image resolution, contrast and specimen viability. To do this requires an accurate understanding of the spatial and temporal evolution of ultrashort pulses at the focus produced by a microscope objective. The objective itself, however, can significantly alter the pulses. Some effects, such as the broadening of pulses due to group delay dispersion in materials along the path, are understood and partial compensation for them can be made. Other effects, such as radial variations in the propagation time and variations in the pulse width, are less well understood. In this work, we investigate the radially dependent propagation and focusing of ultrashort pulses through a Zeiss CP- Achromat 100X, 1.25 NA, infinity-corrected, oil immersion microscope objective. We also extend to this high numerical aperture case the technique of collinear type II second harmonic generation frequency-resolved optical grating which has previously been used to measure the temporal intensity and phase of ultrashort pulses at the focus of air objectives with lower numerical aperture.
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