Catherine Greenhalgh

Visualization of mitochondria in cardiomyocytes

The simultaneous detection of third harmonic (THG), and multiphoton excitation fluorescence (MPF) or second harmonic (SHG) from the same focal volume has led us to the development of a nonlinear multimodal microscopic biological imaging tool. The multimodal microscope has been applied for imaging of isolated live cardiomyocytes, and investigation of structural origin of the THG and SHG signals has been performed. By employing the different image contrast mechanisms, differentiation of structures inside a single live adult rat cardiomyocyte has been achieved. Based on structural crosscorrelation image analysis between NAD(P)H fluorescence and THG, and morphology of cardiomyocytes we were able to assign large part of the structure revealed by THG to the mitochondria. The crosscorrelation of THG with fluorescence of tetramethylrhodamine methyl ester (TMRM) labeled cardiomyocytes confirmed the mitochondrial origin of THG. The SHG generated structures were anticorrelated with THG and possessed the characteristic pattern of the myofibrils in the myocyte in accordance with the literature. Possible visualization of mitochondria with THG microscopy appeared due to enhancement of the third harmonic by multilayer arrangement of cristae.

Influence of semicrystalline order on the second-harmonic generation efficiency in the anisotropic bands of myocytes

The influence of semicrystalline order on the second-harmonic generation (SHG) efficiency in the anisotropic bands of Drosophila melanogaster sarcomeres from larval and adult muscle has been investigated. Differences in the semicrystalline order were obtained by using wild-type and mutant strains containing different amounts of headless myosin. The reduction in semicrystalline order without altering the chemical composition of myofibrils was achieved by observing highly stretched sarcomeres and by inducing a loss of viability in myocytes. In all cases the reduction of semicrystalline order in anisotropic bands of myocytes resulted in a substantial decrease in SHG. Second-harmonic imaging during periodic contractions of myocytes revealed higher intensities when sarcomeres were in the relaxed state compared with the contracted state. This study demonstrates that an ordered semicrystalline arrangement of anisotropic bands plays a determining role in the efficiency of SHG in myocytes.

Intermyofilament dynamics of myocytes revealed by second harmonic generation microscopy

Drosophila melanogaster larva myocytes are imaged with second harmonic generation (SHG) microscopy undergoing forced stretching and rhythmic contractions to determine the nature of the SHG signal. During stretching, double peaked SHG profiles of the anisotropic (A-) bands evolve into single peaks with a higher SHG intensity. The dip in the intensity profile at the center of the A-band is attributed to destructive interference from out-of-phase second harmonic radiating myosin molecules that, in the central region of myofilaments, are arranged antiparallel. An intensity increase at the center of the A-band appears during forced stretching due to a small, less than 100 nm, intermyofilament separation of the antiparallel myosin molecules leading to constructive interference of the SHG radiation. In addition, the same phenomenon occurs during periodic contractions of the myocyte, where an SHG intensity increase with the lengthening of sarcomeres is observed. The SHG intensity dependence on sarcomere length can be used for imaging myocyte contractions with low resolution microscopy, and can be applied for the development of diagnostic tools where monitoring of muscle contraction dynamics is required.

Applications of nonlinear microscopy for studying the structure and dynamics in biological systems

Laser scanning nonlinear optical microscopy is used to study structure and dynamics of cellular and sub-cellular structures in vivo. Under tight focusing conditions with a high numerical aperture objective, nonlinear optical signals such as third harmonic generation (THG), second harmonic generation (SHG), and multiphoton excitation fluorescence (MPF) are simultaneously produced. MPF is extensively used in biological imaging. Unfortunately, fluorescence is accompanied by heat dissipation in the sample and photobleaching effects. On the other hand, parametric processes such as SHG and THG are free of photobleaching since they involve only virtual electronic states where there is no transfer of energy into the medium. There are many naturally occurring structures that exhibit harmonic generation effects, and hence, do not require dyes that can potentially disrupt the normal functionality of the system. SHG is efficiently generated in non-centrosymmetric media, such as chiral structures and interfaces. The THG signal is generated due to a break in symmetry at interfaces and can be enhanced by the presence of multilamellar structures, as in the mitochondria or chloroplasts. Many interesting biological processes, such as signal transduction in neurons or ATP synthesis in mitochondria, involve the movement of ions across membranes. THG and SHG are sensitive to changing electric potential gradients, and hence are ideally suited for dynamical investigations of these biological processes. The present work will expose the structural factors and conditions that influence THG and SHG generation efficiencies in biological samples. Examples of visualizing chloroplasts and mitochondria will illustrate the advantages of harmonic generation microscopy for studying structural and functional properties of the in vivo systems.

Time and structural crosscorrelation image analysis of microscopic volumes, simultaneously recorded with second harmonic generation, third harmonic generation, and multiphoton excitation fluorescence microscopy

Our newly developed multimodal microscope enables simultaneous collection of second harmonic generation (SHG), third harmonic generation (THG) and multiphoton excitation fluorescence (MPF) signals. The signals can be generated within different or the same intercellular structures. In comparing two signals, traditional methods of image crosscorrelation analysis using Pearsons coefficient provide a general parameter as to whether the images are similar, however it does not give detailed information about correlation of different structures inside the images. We present here a new technique that employs a pixel by pixel analysis over an entire area or volume that is used to correlate the structures appearing in the images. The result of the analysis reveals structures within the sample that are generated by both nonlinear signals as well as highlighting the structures that are generated by only one of the nonlinear signals. The algorithm provides a means to colocalize different structures revealed by the different nonlinear contrast mechanisms. Structural correlation maps are useful in identifying the origin of structures in one nonlinear contrast mechanism when the origin of structures in another is known. Image analysis has also been exploited for sequences of images taken in time. The intensity fluctuations in time for each pixel reveal regions of intense physiological activity in biological samples. Correlation of time dependent fluctuations from different pixels in the image time series allows construction of the structural map that undergoes similar time behavior or appears out of phase. These structural correlation analysis techniques are demonstrated based on polystyrene beads and cardiomyocytes.

Dynamic investigation of Drosophila myocytes with second harmonic generation microscopy

The functional dynamics and structure of both larval and adult Drosophila melanogaster muscle were investigated with a nonlinear multimodal microscope. Imaging was carried out using a home built microscope capable of recording the multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation signals simultaneously at a scanning rate of up to ~12 frames/sec. The sample was excited by a home built femtosecond Ti:Sapphire laser at 840 nm, or by a Yb-ion doped potassium gadolinium tungstate (Yb:KGW) crystal based oscillator at 1042 nm. There was no observable damage detected in the myocyte after prolonged scanning with either of the lasers. Microscopic second harmonic generation (SHG) appears particularly strong in the myocytes. This allows the fast contraction dynamics of the myocytes to be followed. The larger sarcomere size observed in the larvae myocytes is especially well suited for studying the contraction dynamics. Microscopic imaging of muscle contractions showed different relaxation and contraction rates. The SHG intensities were significantly higher in the relaxed state of the myocyte compared to the contracted state. The imaging also revealed disappearance of SHG signal in highly stretched sarcomeres, indicating that SHG diminishes in the disordered structures. The study illustrates that SHG microscopy, combined with other nonlinear contrast mechanisms, can help to elucidate physiological mechanisms of contraction. This study also provides further insight into the mechanisms of harmonic generation in biological tissue and shows that crystalline arrangement of macromolecules has a determining factor for the high efficiency second harmonic generation from the bulk structures.

Second- and third-harmonic generation and multiphoton excitation fluorescence microscopy for simultaneous imaging of cardiomyocytes

Simultaneous detection of second harmonic generation (SHG), third harmonic generation (THG) and multiphoton excitation fluorescence with ultrafast laser pulses from a Nd:Glass laser was used to image isolated adult rat cardiomyocytes. The simultaneous detection enabled visualization of different organelles of cardiomyocytes, based on the different contrast mechanisms. It was found that SHG signal depicted characteristic patterns of sarcomeres in a myofilament lattice. The regular pattern of the THG signal, which was anticorrelated with the SHG signal, suggested that the third harmonic is generated within mitochondria. By labeling the cardiomyocytes with the mitochondrial dye tetramethylrhodamine methyl ester (TMRM), comparisons could be made between the TMRM fluorescence, THG, and SHG images. The TMRM fluorescence had significant correlation with THG signal confirming that part of the THG signal originates from mitochondria.

Optimization of nonlinear excitation for reducing light-induced changes in photosynthetic systems during imaging with multimodal microscopy

Nonlinear microscopy is a very attractive tool for studying photosynthetic organisms on cellular and subcellular levels. The multimodal microscope can be employed to image photosynthetic structures simultaneously with multiphoton excitation fluorescence (MPF), second harmonic generation (SHG), and third harmonic generation (THG) contrast mechanisms. Although the multimodal nonlinear microscope delivers invaluable information about the structure, spectroscopic properties, and functional dynamics of photosynthetic systems, the prompt light-induced changes of highly light sensitive pigment-protein complexes complicate the extensive study of photosynthetic organisms. In this work, we investigated the extent of light-induced changes in chloroplasts from higher plants by imaging with a Ti:Sapphire femtosecond laser and a Yb-ion doped potassium gadolinium tungstate (Yb:KGW) femtosecond laser. The Ti:Sapphire laser delivered 800 nm wavelength and ~25 fs duration pulses at a 26.7 MHz repetition rate. In comparison, the Yb:KGW laser provided a 1042 nm wavelength, ~200 fs pulses at a repetition rate of 14.6 MHz. The 800 nm pulses predominantly excited chlorophyll pigments via two-photon excitation, while 1042 nm excitation resulted in two-photon absorption of carotenoids. The induced fluorescence quenching, and decrease in SHG and THG signal was much stronger when imaged with a Ti:Sapphire laser. Prolonged imaging of up to tenths of minutes with the Yb:KGW laser did not result in appreciable changes of all three nonlinear signals. The difference in the light-induced changes most probably appears due to the difference in excited state dynamics following chlorophyll or carotenoid excitation. The slow component of MPF and THG changes as well as change in SHG reflects the light-induced macroorganization of the grana, while the fast MPF and THG component is tentatively attributed to the generation of quenchers from chlorophyll molecules. The success in imaging photosynthetic samples for prolonged periods of time with a Yb:KGW laser opens a new window of opportunity for thorough in vivo investigations of photosynthetic structures.

A diode-pumped high power extended cavity femtosecond Yb:KGW laser: from development to applications in nonlinear microscopy

We present the design and development of a diode-pumped high average power femtosecond laser based on a crystal of Yb-ion doped potassium gadolinium tungstate (Yb:KGW) and a semiconductor saturable absorber for passive mode-locking. The laser delivered up to 0.85 W of average power with ~200 fs pulses at a repetition rate of 14.6 MHz, corresponding to a pulse energy of 60 nJ with a peak power of ~300 kW. The developed laser system was used to visualize the structure of muscle cells from Drosophila melanogaster larvae in vivo by acquiring high-resolution images with a nonlinear multimodal scanning microscope, capable of simultaneous detection of two-photon fluorescence, second and third harmonic signals.

Dynamic and Structural Visualization of Muscle Structure in Drosophila with Multimodal Harmonic Generation Microscopy

Multimodal non-linear microscopy was used to investigate the structure and functional dynamics of muscle from larvae and adult Drosophila. Multimodal technology proved to be a powerful tool for a photobleaching free method of physiological investigations.