International Journal of Molecular Sciences

Review Synchrotron Radiation X-ray Diffraction Techniques Applied to Insect Flight Muscle

Hiroyuki Iwamoto

Japan Synchrotron Radiation Research Institute, SPring-8, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan; [email protected]; Tel.: +81-791-58-2506



Received: 26 April 2018; Accepted: 5 June 2018; Published: 13 June 2018


Abstract: X-ray fiber diffraction is a powerful tool used for investigating the molecular structure of muscle and its dynamics during contraction. This technique has been successfully applied not only to skeletal and cardiac muscles of vertebrates but also to insect flight muscle. Generally, insect flight muscle has a highly ordered structure and is often capable of high-frequency oscillations. The X-ray diffraction studies on muscle have been accelerated by the advent of 3rd-generation synchrotron radiation facilities, which can generate brilliant and highly oriented X-ray beams. This review focuses on some of the novel experiments done on insect flight muscle by using synchrotron radiation X-rays. These include diffraction recordings from single myofibrils within a flight muscle fiber by using X-ray microbeams and high-speed diffraction recordings from the flight muscle during the wing-beat of live insects. These experiments have provided information about the molecular structure and dynamic function of flight muscle in unprecedented detail. Future directions of X-ray diffraction studies on muscle are also discussed.

Keywords: insect flight muscle; X-ray diffraction; synchrotron radiation; X-ray microbeam; time-resolved measurement

1. Introduction Like light microscopy and electron microscopy, X-ray diffraction is one of the techniques used to resolve the fine structure of objects by using the scattering of electromagnetic waves or electron beams. Generally, the spatial resolution obtained in each technique is defined by the diffraction limit, which is comparable to the wavelength of the beams used. Both X-ray beams and the electron beams (used in the electron microscope) have wavelengths comparable to or shorter than the lengths of atomic bonds and, in principle, atomic resolution should be obtained by using both of these techniques. In protein crystallography, such atomic-resolution structures of proteins are routinely obtained. For this purpose, the generation of high-quality protein crystals is essential, but this is very often a difficult process. The resolution of protein 3-D structures obtained by single-particle analyses in cryo-electron microscopy (cryo-EM) used to be worse than 1 nm, but, recently, this has been dramatically changed by the advent of direct detectors, which have nominal pixel sizes equivalent to ~1 Å at the scale of the sample. By using this type of detector, the resolution has improved to 2 to 3 Å [1]. The processing that leads to the determination of such high-resolution structures is enormous and usually tens of thousands of particle images are collected, classified and merged. The focus of this review is not the pursuit of such a high spatial resolution, but is on the non-invasive nature of X-ray diffraction methods that allows observation of biomolecules functioning under physiological conditions. The high penetrability of X-ray beams even allows us to observe the biomolecules in living organisms, and brilliant synchrotron radiation X-ray beams make it possible to do it at a high time resolution. In this study, we describe examples of the application of this technique to muscle, with a special emphasis on insect flight muscle, which has a structure regular enough for

Int. J. Mol. Sci. 2018, 19, 1748; doi:10.3390/ijms19061748 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 1748 2 of 19 productive use of X-ray diffraction recordings. First, the basic principles of X-ray diffraction are briefly described. Next, we describe the instruments needed to record X-ray diffraction patterns especially in synchrotron radiation facilities. Then we describe the latest results of research obtained by using this technique and, lastly, we discuss some future direction for X-ray diffraction studies on muscle.

2. Outline of X-ray Diffraction Principles As for detailed explanation for the principles of X-ray diffraction and its application to muscle, a number of excellent textbooks and reviews have been published [2–14] and the readers who are interested in learning the principles in depth are encouraged to refer to these articles.

2.1. Scattering of X-rays by An Object As in the case of visible light, X-ray photons are scattered by an object. In the atoms that constitute the object, the nuclei (protons and neutrons) have little contribution to the scattering. As such, the scattering by an object is almost equivalent to the scattering by the electrons within the object. Mathematically, the distribution of the scattered photons is the Fourier transform of the object and this Fourier transform is called the structure factor of the object. To be accurate, the intensity of the scattering is proportional to the square of the amplitude of the structure factor. The length in the structure factor is inversely related to the length in the world that we normally observe, which is why the space for the structure factor is called reciprocal space. Reciprocal space is intuitively difficult to comprehend. However, if one performs a Fourier transformation once again (inverse Fourier transformation), the function in the reciprocal space is brought back to real space. The lenses of light and electron microscopes do this directly and the real-space image of the object is restored at a different magnification. The Fourier transform of the object or the structure factor is a complex number and has a real part and an imaginary part. In other words, it has an amplitude and a phase. To restore the real-space image by inverse Fourier transformation, information of both the amplitude and the phase is required. With the lenses used in light microscopes and electron microscopes, the two quantities are preserved and the real-space image of the object can be restored. In the case of X-rays, lenses are not usually used. The intensity distribution of the scattered waves (diffraction pattern) is recorded by various types of detectors, and the patterns are directly analyzed in various ways. Regardless of the type of detectors, they can record only the amplitude of the scattered waves, and the phase information is lost. Without phase information, the real-space image of the object cannot be restored. This is called the phase problem and great efforts have been made to solve the problem in every field of X-ray diffraction studies including protein crystallography. Why are lenses not used for X-ray? The reason is that it is difficult (although not impossible) to fabricate lenses that can be used to create an X-ray microscope. The reasons for this difficulty are as follows: (1) the refractive index of matter for X-rays is very close to 1 (actually very slightly less than 1) so refractive X-ray lenses are not feasible. (2) as for mirrors the condition in which total reflection occurs is very limited for X-rays (the angle of incidence to the mirror must be very small) so that concave mirrors that are used for telescopes cannot be fabricated for X-rays. (3) even if we can fabricate focusing optics for X-rays, the accuracy of fabrication must be at the atomic scale to obtain the spatial resolution expected from the wavelengths of X-rays. Figure1 shows examples of the focusing devices designed for X-rays. The device in Figure1A is called a Fresnel zone plate, and is usually fabricated by micro-lithography or nano-lithography. It is a circular grating consisting of concentric rings of alternating transparent and scattering zones, and the interval between the two neighboring rings are closer as they approach the outer edge. Due to this configuration, the beams scattered by the outer zones are diffracted more strongly towards the center. Therefore, all the diffracted beams are directed towards a single focus. X-ray microscopes using this device have actually been tested (e.g., [15–17]). The spatial resolution is determined by the accuracy of lithography and it is currently around 20 nm. This value is rather disappointing when compared Int. J.Int. Mol. J. Sci.Mol.2018 Sci. 2018, 19,, 174819, x FOR PEER REVIEW 3 of 19 3 of 19 accuracy of lithography and it is currently around 20 nm. This value is rather disappointing when compared to the wavelengths of X-rays (~0.1 nm) and is only slightly better than that of super- to the wavelengths of X-rays (~0.1 nm) and is only slightly better than that of super-resolution light resolution light microscopes [18]. Another type of focusing device is a pair of bent planar mirrors microscopescalled Kirkpatric [18]. Another-Baez (KB) type mirrors of focusing (see Fig deviceure 1B is). a An pair X- ofray bent microscope planar mirrorsusing this called device Kirkpatric-Baez is free of (KB)chr mirrorsomatic (see aberration Figure 1thatB). Anis inherent X-ray microscopein the zone plates using [19 this]. The device spatial is free resolution of chromatic obtained aberration with this that is inherentX-ray microscope in the zone is plates50 nm, [ and19]. this The also spatial depends resolution on the obtainedaccuracy of with fabrication this X-ray of the microscope mirrors. X- isray 50 nm, and thismicroscopes also depends are still on in thethe accuracydevelopmental of fabrication stage and ofit thewill mirrors.take years X-ray to reach microscopes a practical arestage still for in the developmentalbiological use. stage and it will take years to reach a practical stage for biological use.

FigureFigure 1. Schematic 1. Schematic drawings drawings of of some some focusing focusing devices devices forfor X X-ray-ray beams. beams. (A (A) Fresnel) Fresnel zone zone plate. plate. This This is a circularis a circular grating grating with with alternating alternating densities. densities. The The focalfocal distance depends depends on onthe theX-ray X-ray wavelengths wavelengths and whiteand white X-rays X-rays do do not not focus; focus (;B (B)) Kirkpatric-Baez Kirkpatric-Baez (KB) (KB) mirrors mirrors.. These These are are most most commonly commonly used usedfor for focusing X-ray beams in synchrotron radiation facilities, but are rarely used as imaging optics. The focusing X-ray beams in synchrotron radiation facilities, but are rarely used as imaging optics. The focal focal distance does not depend on the X-ray wavelengths and can be changed by altering the bend of distance does not depend on the X-ray wavelengths and can be changed by altering the bend of the the mirrors. The drawings are exaggerated. mirrors. The drawings are exaggerated. There is a third type of lens that can be used for X-ray. It uses a computer as a lens. This type of imagingThere is is a called third typethe lens of- lensless imaging that can, and be usedthis emerging for X-ray. technique It uses awill computer be briefly as explained a lens. This at the type of imagingend of is this called review. the lens-less imaging, and this emerging technique will be briefly explained at the end of this review. 2.2. Analysis of Scattered X-rays in the Reciprocal Space 2.2. AnalysisIn protein of Scattered crystallography, X-rays in the the Reciprocal phase information Space is restored by heavy-atom labeling, multi- wavelength anomalous diffraction (MAD) or single-wavelength anomalous diffraction (SAD) In protein crystallography, the phase information is restored by heavy-atom labeling, methods [20]. In non-crystalline diffraction studies including fiber diffraction and protein solution multi-wavelengthscattering, the scattered anomalous intensities diffraction are usually (MAD) and or traditionally single-wavelength directly analyzed anomalous and interpreted diffraction in (SAD) methodsreciprocal [20]. space. In non-crystalline This means that diffraction researchers studies are requested including to “read” fiber the diffraction diffraction and pattern protein as it solution is. scattering,For detailed the scattered explanation intensities as for “how are usuallyto read” andreaders traditionally are encouraged directly to refer analyzed to the andtextbooks interpreted and in reciprocalreviews space. listed Thisin the means first paragraph that researchers of this section are requested, since only tothe “read” basics the(for diffractionapplication to pattern muscle) as it is. For detailedare described explanation here. as for “how to read” readers are encouraged to refer to the textbooks and reviews listedFirst, ina diffraction the first paragraph pattern (the of thissquare section, of the since structure only the factor basics of (forthe object) application represents to muscle) the are describedspectrum here. of density plotted against the spatial frequency known as the power spectrum. For an object with continuous densities (such as a protein molecule), its power spectrum is continuous. If there are First, a diffraction pattern (the square of the structure factor of the object) represents the spectrum regularly repeating structures, it means that the densities prevail at a certain spatial frequency (or of densityinterval) plotted and show against up as the a peak spatial in the frequency power spectrum. known Therefore as the power, a diffraction spectrum. pattern For is an sensitive object with continuousto periodic densities structures. (such as a protein molecule), its power spectrum is continuous. If there are regularlyUsually repeating an object structures, is three it- meansdimensional, that thewhich densities means prevailits structure at a certainfactor is spatial also three frequency- (or interval)dimensional. and showWhen upits diffraction as a peak inpattern the power is recorded spectrum. by using Therefore, a planar detector, a diffraction one can pattern record is only sensitive to periodicone slice structures. of its structure factor. To collect all the data needed to reconstruct the 3-D structure of the object,Usually it must an object be rotated. is three-dimensional, In the case of a muscle which specimen, means however, its structure all the factor intensities is also of three-dimensional. the structure Whenfactor its diffraction are usually pattern recorded is in recorded a single byexposure using awithout planar the detector, need of one rotating can record the specimen only one. This slice is of its structure factor. To collect all the data needed to reconstruct the 3-D structure of the object, it must be rotated. In the case of a muscle specimen, however, all the intensities of the structure factor are usually recorded in a single exposure without the need of rotating the specimen. This is because a muscle or a muscle fiber contains a large number of myofibrils with random orientations around their long Int. J. Mol. Sci. 2018, 19, 1748 4 of 19

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 19 axes. This situation makes the diffraction pattern rotationally averaged, and it poses some difficulty in reconstructingbecause a the muscle un-rotated or a muscle sarcomeric fiber contains structure. a large number of myofibrils with random orientations around their long axes. This situation makes the diffraction pattern rotationally averaged, and it A useful and important notion in “reading” a diffraction pattern is called the convolution theorem. poses some difficulty in reconstructing the un-rotated sarcomeric structure. The convolutionA useful of twoand functions,important notionf (t) and in g“reading”(t), is expressed a diffraction in the pattern following is called equation. the convolution theorem. The convolution of two functions, f(t) and g(t), is expressed in the following equation. Z ∞ c(t) = ∞ f (τ)g(t − τ)dτ (1) c(t) = −∞f (τ )g(t −τ )dτ (1) ∫−∞ It is easier to understand the meaning of the equation by looking at the illustration in Figure2. It is easier to understand the meaning of the equation by looking at the illustration in Figure 2. Then theThen convolution the convolution theorem theorem states states that thethat Fourier the Fourier transformation transformation of theof the convolution convolution of of two two functions is the productfunctions of is the the Fourierproduct of transforms the Fourier oftransforms the two of functions. the two functions.

Figure 2. Convolution theorem. A small crystal of protein molecules in which the molecules are Figurearranged 2. Convolution in a hexagonal theorem. lattice ( ATop small right) crystal is the convolution of protein of moleculestwo functions in. T whichhe function the defining molecules are arrangedthe in electron a hexagonal density of lattice each molecule (Top right (Top) is left the) and convolution the function of defining two functions. the hexagonal The l functionattice (Top defining the electronmiddle density). The asterisk of each represents molecule the (operationTop left )of and convolution. the function The bottom defining row the represents hexagonal the lattice (Top middlediffraction). The pattern asterisk (Fourier represents transform) theof the operation functions in of the convolution. top row. The Thediffraction bottom pattern row from represents the diffractionthe protein pattern crystal (Fourier is the product transform) of the pattern of the from functions a single in molecule the top from row. the The lattice. diffraction The protein pattern is from meant to be a ryanodine receptor. the protein crystal is the product of the pattern from a single molecule from the lattice. The protein is meant toThe be basic a ryanodine structure receptor. of the sarcomere of a striated muscle, either vertebrate or insect, is the two sets of myofilaments (actin and myosin) arranged in a hexagonal lattice. Like DNA molecules, the Themonomers basic structure of the actin of and the myosin sarcomere filaments of are a striatedhelically arranged muscle, and either they give vertebrate rise to very or similar insect, is the diffraction patterns. The theory of diffraction from a helix was established in 1950s by F. Crick and two sets of myofilaments (actin and myosin) arranged in a hexagonal lattice. Like DNA molecules, other researchers [21]. They demonstrated that the Fourier transform of a helix is described as a series the monomersof Bessel functions of the actin of the and first myosinkind. In a filamentstwo-dimensional are helically diagram, arrangedthe Bessel function and theys take give the form rise to very similarof diffraction a series of patterns.layer-like features The theory arranged of diffraction in an X-shape from. Therefore, a helix waseach establishedof the layer-like in reflection 1950s bys F. Crick and otheris called researchers a layer-line [21 ].reflection. They demonstrated The helical pitch that of thethe Fourieractin filament transform is different of a helixfrom that is described of the as a series ofmyosin Bessel filament functions so that of the two first filaments kind. In give a two-dimensionalrise to layer-line reflections diagram, at different the Bessel positions functions in take the formthe of diffrac a seriestion pattern of layer-like (although features a few of arrangedthem overlap in with an X-shape. each other). Therefore, each of the layer-like In the usual configuration of diffraction recording from muscle specimens, the long axis of the reflections is called a layer-line reflection. The helical pitch of the actin filament is different from that muscle fiber is made perpendicular to the axis of the incident X-ray beam. In the diffraction pattern of the myosinrecorded filament in this way, so thattwo axes the can two be filaments defined. One give is riseperpendicular to layer-line to the reflections fiber axis and at differentis called the positions in the diffractionequator. The pattern reflections (although that arise a fewalong of the them equator overlap are withcalled eachequatorial other). reflections. Equatorial In the usual configuration of diffraction recording from muscle specimens, the long axis of the muscle fiber is made perpendicular to the axis of the incident X-ray beam. In the diffraction pattern recorded in this way, two axes can be defined. One is perpendicular to the fiber axis and is called the equator. The reflections that arise along the equator are called equatorial reflections. Equatorial reflections are the strongest of all reflections from muscles and are dominated by the contribution from the hexagonal lattice of the filaments. Int. J. Mol.Int. J. Sci.Mol.2018 Sci. ,201819,, 1748 19, x FOR PEER REVIEW 5 of 195 of 19

reflections are the strongest of all reflections from muscles and are dominated by the contribution Thefrom axisthe hexagonal along the lattice fiber of isthe called filaments. the meridian. There are periodical structures along the myofilamentsThe axis such along as troponin, the fiber and is theircalled periodicity the meridian. is reflected There inare reflections periodical onstructures the meridian. along Theythe are calledmyofilaments meridional reflections.such as troponin The, helices and their of periodicity the myosin is filaments reflected i andn reflections actin filaments on the meridian. are discontinuous, They i.e., theyare alsocalled exhibit meridional periodicities reflections. along The the helices filament of the axis. myosin Therefore, filaments there and are aactin number filaments of meridional are discontinuous, i.e., they also exhibit periodicities along the filament axis. Therefore, there are a reflections arising from the monomers of actin and myosin. The reflections not on either the meridian or number of meridional reflections arising from the monomers of actin and myosin. The reflections not the equator are the layer-line reflections, which arise from the helical arrangement of actin and myosin on either the meridian or the equator are the layer-line reflections, which arise from the helical filaments.arrangement Two typical of actin 2-dimensional and myosin filaments. diffraction Two patterns typical from 2-dimensional rabbit skeletal diffraction muscle patterns fibers arefrom shown in Figurerabbit3. skeletal One is takenmuscle in fibers the relaxed are shown state in (seeFigure Figure 3. One3A) is and,taken except in the forrelaxed the first,state sixth,(see Fig andure seventh3A) actinand layer-line, except reflections, for the first the, sixth, myosin and layer-lineseventh actin reflections layer-line dominate, reflections, which the reflectsmyosin alayer good-line helical orderreflections of the helix dominate, of myosin which heads. reflect Thes a other good ishelical taken order in rigor of the (see helix Figure of myosin3B) and heads. the myosin The other layer-line is reflectionstaken in are rigor replaced (see Fig byure numerous 3B) and the actin myosin layer-line layer-line reflections. reflections This are indicates replaced by that numerous the myosin actin heads attachlayer to actin-line reflections. in a stereospecific This indicates manner, thatfollowing the myosin the heads helical attach symmetry to actin in of a actinstereospecific so that themanner, mass that contributesfollowing to thethe actinhelical layer-line symmetry reflections of actin so is that increased. the mass At that the contributes same time, to the the intensities actin layer of-line the two reflections is increased. At the same time, the intensities of the two major equatorial reflections (1,0 major equatorial reflections (1,0 and 1,1) are reversed, which reflects the movement of mass from the and 1,1) are reversed, which reflects the movement of mass from the myosin filaments to the actin myosin filaments to the actin filaments. In this way, one can obtain information about the molecular filaments. In this way, one can obtain information about the molecular behavior within muscle fiber behaviorby directly within interpreting muscle fiber the bydiffraction directly pattern. interpreting the diffraction pattern.

Figure 3. Diffraction pattern recorded from rabbit skeletal muscle fibers. (A) relaxed; (B) rigor. The Figure 3. Diffraction pattern recorded from rabbit skeletal muscle fibers. (A) relaxed; (B) rigor. patterns were recorded from an array of 30 skinned single fibers of psoas muscle. Note that a series The patternsof myosin were layer recorded-line reflections from an (MLL, array green of 30 letters) skinned in the single relaxed fibers pattern of psoas are replaced muscle. Noteby a serie thats aof series of myosinactin layer layer-line-line reflections reflections (ALL, (MLL, purple green letters). letters) in the relaxed pattern are replaced by a series of actin layer-line reflections (ALL, purple letters). 2.3. What is Made Possible by the Advent of Synchrotron Radiation 2.3. WhatThe is Made diffraction Possible patterns by the Adventseen in ofFig Synchrotronure 3 are called Radiation static diffraction patterns recorded under Theconstant diffraction conditions patterns of the seenspecimen in Figure with 3detectors are called without static using diffraction the capacity patterns of time recorded-resolved under recordings (traditionally, X-ray films). Before the days of synchrotron radiation facilities, laboratory constant conditions of the specimen with detectors without using the capacity of time-resolved X-ray sources were used in combination with low-sensitivity (by today’s standard) X-ray films. The recordings (traditionally, X-ray films). Before the days of synchrotron radiation facilities, laboratory strongest laboratory X-ray source was a rotating-anode X-ray generator but, even with this, it took a X-raywhole sources day wereto record used a diffraction in combination pattern withequivalent low-sensitivity to the ones (byshown today’s in Figure standard) 3 from a X-ray whole films. The strongestsartorius muscle laboratory from a X-rayfrog. source was a rotating-anode X-ray generator but, even with this, it took a wholeThe advantage day to recordof sync ahrotron diffraction radiation pattern X-ray equivalent beams, especially to the ones those shown from so in-called Figure third3 from- a wholegeneration sartorius synchrotron muscle from radiation a frog. facilities, is their overwhelming intensity, and in terms of brilliance The(a unit advantage expressing ofthe synchrotronbrightness of beams), radiation the X X-ray-ray beams beams, from especially a beamline thoseequipped from with so-called an third-generation synchrotron radiation facilities, is their overwhelming intensity, and in terms of brilliance (a unit expressing the brightness of beams), the X-ray beams from a beamline equipped with an undulator and a monochromator is 108 times brighter than those emitted from a rotating-anode generator (in the case of SPring-8). An undulator is a device inserted in the orbit of electrons in Int. J. Mol. Sci. 2018, 19, 1748 6 of 19 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 19 the synchrotronundulator and ring a monochromator and can generate is 108 strong times brighter and highly than those oriented emitted X-ray from beams. a rotating An-anode undulator consistsgenerator of a series (in the of case magnets of SPring with-8). alternatingAn undulator polarities, is a device whichinserted makes in the orbit the electronof electrons orbit in the literally synchrotron ring and can generate strong and highly oriented X-ray beams. An undulator consists of undulate. Each time the electron orbit is bent, strong radiation is generated. The diffraction patterns a series of magnets with alternating polarities, which makes the electron orbit literally undulate. Each in Figuretime3 the were electron recorded orbit is in bent, one strong of such radiation beamlines, is generated. BL45XU The diffraction of SPring-8 patterns in Japan in Fig [ ure22], 3 whichwere can 12 deliverrecorded 10 photons/s. in one of such The beamlines, detectors BL45XU available of todaySPring are-8 in much Japan more[22], which sensitive can thandeliver X-ray 1012 films and theyphotons/s. include The Imaging detectors Plates,available CCD today (charge-coupled are much more sensitive device) than or X CMOS-ray films (complementary and they include metal oxideImaging silicon)-type Plates, semiconductorCCD (charge-coupled detectors device) that or CMOS either (compl detectementary X-ray photons metal oxide directly silicon) or-type are used in combinationsemiconductor with detectors image intensifiersthat either detect or phosphor X-ray photons plates directly followed or are by used fiber in optics.combination By using with such detectors,image one intensifiers can record or phosphor diffraction plates patterns followed as seenby fiber in optics. Figure By4 in using a matter such detectors, of a few one seconds can record from a set diffraction patterns as seen in Figure 4 in a matter of a few seconds from a set of ~30 single muscle of ~30 single muscle fibers in which the mass of sample in the beam is much smaller than in the case of fibers in which the mass of sample in the beam is much smaller than in the case of the laboratory X- the laboratoryray source X-ray, using source, a sartorius using muscle a sartorius as a specimen muscle. as a specimen.

Figure 4. Comparison of diffraction patterns from vertebrate skeletal muscle fibers (left, identical to Figurethe 4. oneComparison in Figure 3A) of and diffraction insect flight patterns muscle from fibers vertebrate (right, from skeletal giant waterbug muscle Lethocerus fibers (left). , identical to the one in Figure3A) and insect flight muscle fibers ( right, from giant waterbug Lethocerus). By utilizing these highly bright and well-oriented synchrotron radiation X-ray beams, one can perform time-resolved diffraction recordings at a high time resolution. In addition, diffraction By utilizing these highly bright and well-oriented synchrotron radiation X-ray beams, one can recording from a very small area of the specimen can be achieved using X-ray microbeams (X-ray perform time-resolved diffraction recordings at a high time resolution. In addition, diffraction recording beams with a micrometer-order diameter) as well as a combination of both. In the following sections, from awe very outline small some area examples of the specimen of the application can be achieved of synchrotron using radiation X-ray microbeams X-ray beams (X-ray to insect beams flight with a micrometer-ordermuscle. diameter) as well as a combination of both. In the following sections, we outline some examples of the application of synchrotron radiation X-ray beams to insect flight muscle. 3. Brief Introduction to Insect Flight Muscle 3. Brief IntroductionThe flight muscles to Insect of insects Flight are Muscle the muscles responsible for the wing-beat. They consist of Thevoluminous flight muscles power muscles of insects that areprovide the musclesthe driving responsible force of the for wing the-beat wing-beat., and smaller They steering consist of voluminousmuscles powerthat adjust muscles the angle that of providethe wings theso that driving the insects force can of steer the themselves wing-beat, during and smallerflight. Most steering of the winged insects have two antagonistic pairs of power muscles called dorsal longitudinal muscle muscles that adjust the angle of the wings so that the insects can steer themselves during flight. (DLM) and dorso-ventral muscle (DVM). They do not drive the wings directly, but they do it Most of the winged insects have two antagonistic pairs of power muscles called dorsal longitudinal muscle (DLM) and dorso-ventral muscle (DVM). They do not drive the wings directly, but they do it indirectly by deforming the thoracic exoskeleton. For this reason, they are called indirect flight muscles. They occupy most of the space of the thoracic exoskeleton and they have been the major target for X-ray diffraction studies as well as other physiological and structural studies. Int. J. Mol. Sci. 2018, 19, 1748 7 of 19

Some small insects can beat their wings at very high frequencies, e.g., ~500 Hz in mosquitoes (e.g., [23–25]) and up to 1000 Hz in smaller midges [26]. These frequencies are not realized by the repetition of usual contraction-relaxation cycles in which each is triggered by a motor nerve impulse. These insects have asynchronous flight muscles in which the muscle undergoes autonomous oscillation while the intracellular calcium concentration is held constant by low-frequency motor nerve impulses [27–32]). The molecular mechanism for this high-frequency autonomous oscillation has been the main interest for insect muscle researchers. The key function is stretch activation, which refers to the delayed rise of active force after an externally applied stretch [27]. Its molecular mechanism is still under investigation. Structurally, asynchronous flight muscle is also special. The arrangement of contractile proteins is so regular that a sarcomere of asynchronous flight muscle can be regarded as a kind of protein crystal. Because of this high regularity, the lower-order layer line reflections consist of a series of discrete spots, which would appear in the diffraction pattern from a protein crystal (e.g., [33]. See also Figure4). It is speculated that this high regularity of structure is related to the function of stretch activation [34,35]. The flight muscle fibers isolated from a giant waterbug, Lethocerus have been the standard material for flight muscle research and they give rise to very high-quality diffraction patterns. Its muscle fibers are long (~10 mm) and easy to mount and are also suitable for mechanical measurements. Today, however, diffraction patterns can be recorded from the flight muscles of much smaller insects, which are so small that diffraction recording from their flight muscle would have been unimaginable before the days of synchrotron radiation.

4. Diffraction Recordings from Drosophila Flight Muscle The advent of third-generation synchrotron radiation facilities has made it possible to perform time-resolved X-ray diffraction studies on the flight muscles of Lethocerus and other relatively large insects at millisecond time resolutions. During the recordings, either sinusoidal oscillations [36] or step length changes [37] were applied to isolated flight muscle fibers. An important study was done by George et al. [38] who performed time-resolved X-ray diffraction recording from the flight muscle of a hawkmoth, Manduca, while applying sinusoidal length changes. The muscle was not isolated, but left in situ and mechanically connected between a servomotor and a force transducer. Since the muscle fibers were intact, they were activated by electrical stimulation. The milestone achievement is Irving and Maughan’s work on the tiny fruitfly, Drosophila melanogaster [39]. Their work is novel not only because the diffraction was recorded from the flight muscle of such a tiny insect, but also because it was from living and beating flies (tethered flight) and the time resolution was as high as 1 ms, which was required to take snapshots of diffraction patterns during the 200-Hz wing-beat of Drosophila. This high time resolution was achieved by the use of fast-rotating X-ray shutters synchronized with the wing-beat so that diffraction patterns for a certain phase of the wing-beat were excised. Later their work was further refined and more reflection spots were analyzed with better accuracy [40]. In their work, the X-ray beams were passed along the dorso-ventral axis on the midline, and diffraction patterns were recorded from DLM. Another study of recording diffraction patterns from Drosophila flight muscle was done by Iwamoto et al. [41]. In this study, the time course of development of the myofibrillar structure was followed in the pupal stage. In this study, the X-ray beams were directed through the side of the thorax so that diffraction patterns were recorded from both DLM and DVM. Additionally, diffraction patterns from what was thought to be the jump muscle were also recorded. It is not a flight muscle, and from the intensity ratio of the equatorial reflection, it is considered to have a myofilament lattice structure different from that of flight muscle. The number ratio of actin to myosin filaments is 3:1 in flight muscle, but the diffraction pattern from the jump muscle is best explained if the ratio is 5:1. In this study, diffraction recordings from single myofibrils by using X-ray microbeams were also included (see below). Int. J. Mol. Sci. 2018, 19, 1748 8 of 19

5. Sub-Millisecond Time-Resolved Diffraction Recordings from Bumblebee Flight Muscle Each of the beamlines in the third-generation synchrotron radiation facilities, such as BL45XU of SPring-8, is usually equipped with an undulator and a monochromator. A monochromator generates highly monochromatized X-ray beams (∆λ/λ = 0.01% where λ is the wavelength) but the flux outside the bandwidth is not utilized. The high-flux BL40XU beamline of SPring-8 is special in that it has a helical undulator, which generates a helically rotating magnetic field [42]. The X-rays produced by this undulator are already substantially monochromatic and, by omitting a monochromator, the beamline achieves an X-ray flux of 1015 photons/s, i.e., 1000 times brighter than in BL45XU. This is because of the wider X-ray bandwidth (∆λ/λ = 2%) so that there is a tradeoff between the bandwidth and the flux. By utilizing this exceptionally high X-ray flux, one can perform very fast time-resolved X-ray diffraction recording when fast recording detectors are used. During the beginning of the study, a 3-CCD (charge-coupled device) camera was used in BL40XU in combination with an image intensifier. The 3-CCD camera had three CCD sensor chips that were alternately used to increase the frame rate. With a full-frame size (640 × 480 pixels), a frame rate of ~290 frames/s was achieved. By using this system, a number of fast time-resolved diffraction recordings were made on muscle specimens, including the structural changes of skeletal muscle fibers after photolysis of caged ATP [43] or caged calcium [44] and an early study on the flight muscles from bumblebee and giant waterbug [37]. The problems with this detector were that its sensitivity was not very high because the light path was split to three by prisms, and there were some frame-to-frame variations because the sensitivities of the three CCD chips were not quite identical. It also required a complex timing circuit to synchronize with external events such as the UV flash for photolysis of caged compounds or mechanical stretch. Today, the 3-CCD detector has been replaced by faster CMOS (complementary metal oxide silicon) detectors. Each of them has a single CMOS sensor chip so that there is no frame-to-frame variation. A frame rate of up to 6400–7000 frames/s is achieved with a mega-pixel frame size (1024 × 1024 pixels). Later models have even higher sensitivities. The timing circuit can also be simpler. By using one of these fast CMOS cameras, the responses of the diffraction patterns a step stretch (complete in 1 ms) of skinned bumblebee flight muscle fibers, which were recorded at a frame rate of 2000 frames/s [45]. It is shown that the earliest changes in the diffraction pattern after stretch are the reciprocal intensification of the 1,1,1 reflection and the diminution of the 2,0,1 reflection (the numbers are the crystallographic Miller indices, which indicates which crystallographic plane the reflection comes from). By varying the experimental conditions, these intensity changes were demonstrated to occur conspicuously only when the flight muscle fibers were calcium-activated and it was suggested that the intensity changes report the structural changes that trigger stretch activation. Three-dimensional model calculations showed that the intensity changes can be explained by the deformation of myosin heads while they are weakly attached to actin and these calculations have led to a proposal that such a deformation promotes the conversion of the myosin heads from the weakly attached form to the strongly attached, force-producing form. This mechanism would explain stretch activation essential for the action of asynchronous flight muscle. Fast diffraction recordings were also performed by using live bumblebees during tethered flight [46] (see Figure5). Unlike the case of Drosophila [39,40], the X-ray beams were incident from the side of the bumblebee thorax so that the diffraction patterns of both DLM and DVM were recorded at the same time. It was shown that the two muscles act almost exactly in anti-phase with a phase shift of 180 degrees. The frame rate was 5000 frames/s and, because the wing-beat frequencies of the bees were ~120 Hz, ~40 frames were taken for each wing beat. This frame rate made it possible to follow the time course of intensity changes during the wing-beat in detail. It was shown that the reciprocal intensity changes of the 1,1,1 and 2,0,1 reflections occur conspicuously in live bees and they occur at the right timing when the flight muscle is being stretched by its antagonistic pair. This result shows that the stretch-induced structural changes responsible for these intensity changes occur under physiological conditions in live flying bees. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 19 stretchInt. J. Mol.-induced Sci. 2018, structural19, 1748 changes responsible for these intensity changes occur under physiological9 of 19 conditions in live flying bees.

Figure 5. A single frame of X X-ray-ray diffraction movie recorded from the flight flight muscles of live, beating bumblebees (1 frame = 0.2 ms). The The regions regions of of interest interest (blue (blue rectangles) rectangles) are are shown shown in in a greater magnificationmagnification on the left. Taken from Iwamoto & Yagi [[4646].].

6. Radiation Damage Issues 6. Radiation Damage Issues Although, in principle, X-ray diffraction recording is a non-invasive method to study the Although, in principle, X-ray diffraction recording is a non-invasive method to study the structure structure of biological specimens, irradiation caused by intense X-ray beams leads to radiation of biological specimens, irradiation caused by intense X-ray beams leads to radiation damage. This is damage. This is mainly because, in an aqueous environment, free radicals are generated and they mainly because, in an aqueous environment, free radicals are generated and they diffuse around diffuse around to cause damage to biomolecules (e.g., [47]). Due to this, hydrated biological to cause damage to biomolecules (e.g., [47]). Due to this, hydrated biological specimens are more specimens are more vulnerable to radiation damage than non-hydrated specimens. In the case of the vulnerable to radiation damage than non-hydrated specimens. In the case of the BL45XU beamline BL45XU beamline (flux = 1012 photons/s), radiation damage becomes evident after a few seconds of (flux = 1012 photons/s), radiation damage becomes evident after a few seconds of exposure. Radiation exposure. Radiation damage can be alleviated to some extent by inclusion of a reducing agent such damage can be alleviated to some extent by inclusion of a reducing agent such as glutathione or as glutathione or dithiothreitol and catalase (e.g., ref. [48]). dithiothreitol and catalase (e.g., ref. [48]). Another method to reduce radiation damage is to move the specimen during exposure so that Another method to reduce radiation damage is to move the specimen during exposure so the X-ray beams do not keep hitting the same part of the specimen. This can be applied to uniform that the X-ray beams do not keep hitting the same part of the specimen. This can be applied to specimens. Isolated muscle fibers can be regarded as uniform along their long axis, so that they can uniform specimens. Isolated muscle fibers can be regarded as uniform along their long axis, so that be shifted by the distance of the beam diameter after each short exposure or they can be moved they can be shifted by the distance of the beam diameter after each short exposure or they can continuously during a longer exposure. The latter method is suitable for time resolved be moved continuously during a longer exposure. The latter method is suitable for time resolved measurements. In the case of experiments using caged compounds, the full flux of the BL40XU measurements. In the case of experiments using caged compounds, the full flux of the BL40XU beamline (1015 photons/s) was given and the fibers were moved along their long axis at the speed of beamline (1015 photons/s) was given and the fibers were moved along their long axis at the speed of 100 mm/s by using a motorized mechanical stage [37,43]. 100 mm/s by using a motorized mechanical stage [37,43]. For recordings of static diffraction patterns, a very effective way to reduce radiation damage is For recordings of static diffraction patterns, a very effective way to reduce radiation damage is to to quick-freeze the specimen and keep it at a very low temperature until the exposure is finished (e.g., quick-freeze the specimen and keep it at a very low temperature until the exposure is finished (e.g., [49]). [49]). By doing so, biological specimens can be at least ~500 times more resistant to radiation damage By doing so, biological specimens can be at least ~500 times more resistant to radiation damage than than unfrozen specimens [50]. This is a very common technique for protein crystallography. If a unfrozen specimens [50]. This is a very common technique for protein crystallography. If a hydrated hydrated specimen is frozen slowly, crystalline ice (hexagonal ice) is formed and this destroys the specimen is frozen slowly, crystalline ice (hexagonal ice) is formed and this destroys the specimen specimen because of volume expansion. To avoid this, the water in the specimen must be frozen very because of volume expansion. To avoid this, the water in the specimen must be frozen very quickly to quickly to form amorphous (vitrified) ice. For electron microscopy, muscle fibers are often plunged form amorphous (vitrified) ice. For electron microscopy, muscle fibers are often plunged onto a cooled onto a cooled copper block (e.g., [51]), but the vitrified layer is restricted to 10 µm or less from the copper block (e.g., [51]), but the vitrified layer is restricted to 10 µm or less from the plunged surface. plunged surface. To vitrify a greater volume of the specimen, the use of so-called cryoprotectants is To vitrify a greater volume of the specimen, the use of so-called cryoprotectants is very effective (e.g., [52]). very effective (e.g., [52]). These include glycerol, dimethyl sulfoxide, and 2-methyl pentanediol. These These include glycerol, dimethyl sulfoxide, and 2-methyl pentanediol. These chemicals yield effective chemicals yield effective freezing results for specimens of the size of a single muscle fiber if they are freezing results for specimens of the size of a single muscle fiber if they are used at a ~20% concentration. For comparison of various cryoprotectants and their concentrations, see Iwamoto [53].

Int. J. Mol. Sci. 2018, 19, 1748 10 of 19

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 10 of 19

7. Diffractionused at a Recordings ~20% concentration. Using X-rayFor comparison Microbeams of various cryoprotectants and their concentrations, see Iwamoto [53]. X-ray microbeams refer to X-ray beams with a diameter of the order of micrometers. These can be created7. Diffraction by focusing Recordings optics suchUsing as X- aray KB Microbeams mirror and a Fresnel zone plate, which was mentioned in the section onX-ray the microbeams X-ray microscope. refer to X-ray However, beams with these a diameter optics usuallyof the order have of micrometers short focus. These and maycan not be suitablebe for created small-angle by focusing scattering optics such experiments as a KB mirror such and a as Fresnel diffraction zone plate, recording which was from mentioned muscle. in A more suitablethe and section simple on opticalthe X-ray system microscope. is to useHowever, pinholes. these Usuallyoptics usually two orhave more short pinholes focus and are may used not inbe tandem. The firstsuitable (upstream) for small pinhole-angle definesscattering the experiments diameter such of the as microbeamdiffraction recording (defining from pinhole) muscle. andA more the second (downstream)suitable pinholeand simple cuts optic offal the system parasitic is to scatteringuse pinholes. generated Usually two by theor more edge pinholes of the first are pinholeused in (guard tandem. The first (upstream) pinhole defines the diameter of the microbeam (defining pinhole) and pinhole). The practical lower limit of the microbeam diameter created this way is ~2 µm. If one wishes the second (downstream) pinhole cuts off the parasitic scattering generated by the edge of the first to use anpinhole aperture (guard smaller pinhole). than The this, practical one lower will belimit forced of the to microbeam bring the diameter sample created very close this way to theis ~2 defining pinholeµm. because If one thewishes beam to use rapidly an aperture expands smaller to a than larger this diameter, one will be because forced to of bring Fraunhofer the sample diffraction. very Byclose using to X-raythe defining microbeams, pinhole onebecause may the obtain beam diffractionrapidly expands patterns to a fromlarger adiameter very confined becausearea of of the specimen.Fraunhofer The microbeam diffraction. diameter of ~2 µm is comparable to that of a single myofibril so that one is By using X-ray microbeams, one may obtain diffraction patterns from a very confined area of expected to be able to record a diffraction pattern from a single myofibril. the specimen. The microbeam diameter of ~2 µm is comparable to that of a single myofibril so that Theone first is expected example to tobe recordable to record diffraction a diffraction patterns pattern from from single a single myofibrils myofibril. was done in the BL45XU beamline usingThe first bumblebee example flightto record muscle diffraction fibers. patterns The patterns from single were myofibrils not recorded was done from in isolatedthe BL45XU myofibrils. Unlike thebeamline usual using configuration bumblebee in flight fiber muscle diffraction, fibers. the The microbeams patterns were were not directed recorded along from theisolated fiber axis in an end-onmyofibrils. configuration Unlike the (see usual Figure configuration6). If the inmyofibrils fiber diffraction, are completelythe microbeams straight were directed and the along beams are completelythe fiber parallel axis in to an the end myofibrillar-on configuration axis, ( thensee Fig oneure should6). If the reasonably myofibrils are expect completely to record straight a pattern and from the beams are completely parallel to the myofibrillar axis, then one should reasonably expect to a single myofibril. The results were that the recorded patterns were expected from a single hexagonal record a pattern from a single myofibril. The results were that the recorded patterns were expected lattice offrom myofilaments a single hexagonal and lattice the reflections of myofilaments were and arranged the reflections in a hexagonalwere arranged array in a [hexagonal54] (the Fourier transformarray of [54 a] hexagonal(the Fourier transform lattice is of a a hexagonal hexagonal lattice lattice). is a hexagonal Therefore, lattice). the Therefore, geometry the of geometry the beam and the specimenof the beam was and considered the specimen to was have considered been nearly to hav undere been nearly the ideal under straight-parallel the ideal straight-para conditionsllel as mentionedconditions above. as mentioned The myofibrils above. The ofbumblebee myofibrils of flightbumblebee muscle flight are muscle thicker are thicker than thosethan those of vertebrate of skeletalvertebrate muscle (diameter,skeletal muscle ~3 µ(diameter,m) and are~3 µm) isolated and are from isolated each from other each because other ofbecause the abundanceof the of abundance of mitochondria. These factors make it easier to record diffraction patterns from single mitochondria.myofibrils. These factors make it easier to record diffraction patterns from single myofibrils.

Figure 6. Geometry of diffraction recording (end-on configuration) from a single myofibril within a Figure 6. Geometry of diffraction recording (end-on configuration) from a single myofibril within a flight muscle fiber of a bumblebee by using an X-ray microbeam. The beam diameter was 2 µm and flight muscle fiber of a bumblebee by using an X-ray microbeam. The beam diameter was 2 µm and one of the first diffraction patterns recorded from a single myofibril is shown on the right. The pattern shown here is identical to the one shown in Reference [54].

Importantly, a diffraction pattern from a single hexagonal lattice was recorded despite the fact that there were ~1000 sarcomeres in series within the beam path (the length of the specimen was ~3 mm). Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 11 of 19 Int. J. Mol. Sci. 2018, 19, 1748 11 of 19 one of the first diffraction patterns recorded from a single myofibril is shown on the right. The pattern shown here is identical to the one shown in Reference [54]. The length of 3 mm is close to the full length of myofibrils in the bumblebee thorax. This means that the orientationsImportantly, of the hexagonala diffraction latticespattern from of successive a single hexagonal sarcomeres lattice are was identical recorded throughout despite the fact the entire that there were ~1000 sarcomeres in series within the beam path (the length of the specimen was ~3 length of the myofibril. A myofibril of bumblebee flight muscle can be regarded as a single giant mm). The length of 3 mm is close to the full length of myofibrils in the bumblebee thorax. This means proteinthat crystal. the orientations of the hexagonal lattices of successive sarcomeres are identical throughout the entire length of the myofibril. A myofibril of bumblebee flight muscle can be regarded as a single 8. Quickgiant Freezing protein ofcrystal. Flight Muscle Specimens The experiment described above was done in the BL45XU beamline by using hydrated specimens 8. Quick Freezing of Flight Muscle Specimens that were not quite stable or resistant to radiation damage. The quality of the diffraction patterns was not satisfactory.The Toexperiment improve described the quality above of diffraction was done patterns,in the BL45XU we started beamline to quick-freeze by using hydrated the specimens. specimens that were not quite stable or resistant to radiation damage. The quality of the diffraction Thepatterns first successful was not satisfactory. recordings To from improve quick-frozen the quality muscleof diffraction specimens patterns, were we publishedstarted to quick in 2005- [50]. Diffractionfreeze patterns the specimens. were recorded from both a whole muscle fiber (of rabbit skeletal muscle) and an isolated myofibrilThe first ofsuccessful bumblebee recordings in the from normal quick-frozen configuration muscle specimens of recording were published in which in the2005 X-ray [50]. beam axis wasDiffraction perpendicular patterns towere the recorded fiber axis. from From both a thewhole single muscle rabbit fiber muscle(of rabbit fiber, skeletal a numbermuscle) and of an layer-line reflectionsisolated were myofibril clearly recordedof bumblebee (this in is the not normal a microbeam configuration experiment). of recording It isin unusual which the that X-ray these beam layer-line axis was perpendicular to the fiber axis. From the single rabbit muscle fiber, a number of layer-line reflections are recorded from a single muscle fiber and this is because a much longer exposure time is reflections were clearly recorded (this is not a microbeam experiment). It is unusual that these layer- possibleline for reflections quick-frozen are recorded specimens. from a single muscle fiber and this is because a much longer exposure Thetime myofibrils is possible from for quick bumblebee-frozen specimens. flight muscle were affixed onto a thin plastic film before freezing. Because theThe beam myofibrils diameter from was bumblebee ~2 µm, it flight should muscle illuminate were affixed the area onto equivalent a thin plastic to a film single before sarcomere, which wasfreezing. ~3 µ Becausem long. the The beam 1,0 diameter and 2,0 equatorialwas ~2 µm, reflectionsit should illuminate were clearly the area recorded equivalent (see to a Figure single 7). It is again unusualsarcomere, that which a diffraction was ~3 µm pattern long. The is 1,0 recorded and 2,0 fromequatorial a single reflections sarcomere were clearly of a single recorded myofibril (see and Figure 7). It is again unusual that a diffraction pattern is recorded from a single sarcomere of a single again it owes to a long exposure time allowed for frozen specimens. myofibril and again it owes to a long exposure time allowed for frozen specimens.

Figure 7. Equatorial reflections recorded from a single sarcomere within a frozen myofibril of Figurebumblebee 7. Equatorial attached reflections on a thin plastic recorded film. The from myofibrillar a single axis sarcomere is in the horizontal within adirection. frozen This myofibril is of bumblebeethe color attached version on of athe thin figure plastic that appeared film. The in myofibrillarReference [50]. axis is in the horizontal direction. This is the color version of the figure that appeared in Reference [50]. 9. Evolution of the Myofilament Structure of Winged Insects 9. EvolutionAs of described the Myofilament in Section 7, Structure the myofibril of of Winged bumblebee Insects flight muscle has been shown to have a structure of a giant single protein crystal. To know whether it is true for other winged insects, we Asexamined described the in lattice Section structure7, the of myofibril myofibrils offrom bumblebee a wide variety flight of muscle winged hasinsects been by shownusing the to have a structuremicrobeam of a giant end-on single diffraction protein method crystal. as described To know above. whether This time it is, the true specimens for other were winged quick- insects, we examinedfrozen to the improve lattice their structure stabilityof [55 myofibrils]. from a wide variety of winged insects by using the microbeam end-onFigure 8 diffractionshows one of method the end- ason describeddiffraction above.patterns Thisrecorded time, from the the specimens myofibril were in a frozen quick-frozen to improveflight their muscle stability fiber from [55]. a beetle, Aulacophora femoralis. When compared with the patterns from Figure 8 shows one of the end-on diffraction patterns recorded from the myofibril in a frozen flight muscle fiber from a beetle, Aulacophora femoralis. When compared with the patterns from unfrozen specimens, the improvement of the quality of the diffraction pattern is evident, and reflections of up to 5,0 are observed. The results of end-on diffraction recording from 50 insect species showed that insect species with asynchronous flight muscles have the aforementioned giant single-crystal-like myofibrils without any exception. These insects include Diptera (flies, mosquitoes, etc.), Hymenoptera (bees, wasps, etc.), Coleoptera (beetles), and a part of Hemiptera (true bugs including giant waterbugs). Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 12 of 19

unfrozen specimens, the improvement of the quality of the diffraction pattern is evident, and Int. J. Mol.reflections Sci. 2018, of19 ,up 1748 to 5,0 are observed. The results of end-on diffraction recording from 50 insect species 12 of 19 showed that insect species with asynchronous flight muscles have the aforementioned giant single- crystal-like myofibrils without any exception. These insects include Diptera (flies, mosquitoes, etc.), Yet, insectsHymenoptera with synchronous (bees, wasps, flight etc.), musclesColeoptera generally (beetles), and do nota part have of Hemiptera giant-single-crystal-like (true bugs including myofibrils. These insectsgiant waterbug includes). LepidopteraYet, insects with (butterflies synchronous and flight moths), muscles Orthoptera generally do (grasshoppers not have giant-single and- locusts), and manycrystal other-like orders.myofibrils. In addition,These insects myofibrils include fromLepidoptera the flight (butterflies muscle and of Odonatamoths), Orthoptera (dragonflies and (grasshoppers and locusts), and many other orders. In addition, myofibrils from the flight muscle of damselflies) show some single-crystal-like features. Odonata (dragonflies and damselflies) show some single-crystal-like features.

Figure 8. End-on diffraction pattern from a single myofibril of the flight muscle of a beetle, Aulacophora Figurefemoralis 8. End-on. The diffraction specimen patternwas quick from-frozen a single and myofibrilthe pattern of was the recorded flight muscle at the of liquid a beetle,-nitrogenAulacophora femoralistemperature.. The specimen The left image was quick-frozenis the schematic anddiagram the showing pattern the was positions recorded of the at reflection the liquid-nitrogen spots temperature.expected The from left the imagehexagonal is thelattice schematic of myofilaments. diagram Taken showing from Reference the positions [45]. of the reflection spots expected from the hexagonal lattice of myofilaments. Taken from Reference [45]. It is surprising that the myofibrils of asynchronous flight muscles have unanimously highly ordered structure regardless of the orders to which the insects belong. This is because asynchronous Itflight is surprising muscles are that believed the myofibrils to have occurred of asynchronous many times flightindependently muscles inhave the course unanimously of insect highly orderedevolution. structure The regardless universal occurrence of the orders of the to giant which-single the-protein insects-crystal belong.-like This myofibr is becauseils suggests asynchronous that flight musclesthis structure are plays believed an essential to have role occurred in the function many of timesasynchronous independently flight muscle, in thewhich course ensures of insect evolution.uniform The transmission universal occurrence of force or oflength the giant-single-protein-crystal-likechanges required for the proper generation myofibrils of the suggests stretch- that this activated force. structure plays an essential role in the function of asynchronous flight muscle, which ensures uniform transmission10. Future of force Directions or length changes required for the proper generation of the stretch-activated force.

10. Future DirectionsBecause the phase problem is inevitable in X-ray diffraction recordings, the right interpretation of diffraction patterns requires a proper understanding of diffraction theory. But diffraction theory is Becausenot easy the to understand phase problem and this is makes inevitable researcher in X-rays prefer diffraction more direct recordings, methods that the can right directly interpretation show of diffractionobjects patterns in real space. requires a proper understanding of diffraction theory. But diffraction theory is Recently, however, we have increasing knowledge about how to preserve or recover the phase not easy to understand and this makes researchers prefer more direct methods that can directly show information in X-ray diffraction experiments, which is owed to advanced algorithms and improved objectscomputer in real space. performance. The techniques to restore the real-space images of the object by using this Recently,knowledge however, are collectively we have called increasing coherent diffractive knowledge imaging about (CDI) how or tolens preserve less imaging or recover[56–58]. To the phase informationdo this, in X-raycoherent diffraction illumination experiments, of objects whichis requir ised. owed Coherence to advanced refers algorithmsto the property and improvedof computerelectromagnetic performance. waves The in techniqueswhich the phase to restoreof the wave the is real-space the same for images all the ofphotons the object like in by laser using this knowledgebeams. are X collectively-ray beams with called laser coherent-like coherence diffractive are typically imaging generated (CDI) or in lens X-ray less free imaging electron [56 laser–58 ]. To do (XFEL) facilities, but partly coherent X-ray beams are available in the conventional synchrotron this, coherent illumination of objects is required. Coherence refers to the property of electromagnetic radiation facilities as well. waves in which the phase of the wave is the same for all the photons like in laser beams. X-ray beams with laser-like coherence are typically generated in X-ray free electron laser (XFEL) facilities, but partly coherent X-ray beams are available in the conventional synchrotron radiation facilities as well. One of the CDI techniques is to restore lost phase information by iterative error-reducing calculations and is often called the oversampling method [59–62]. For details of the method, readers are encouraged to refer to the literature and here only an outline is described. The calculation, illustrated in Figure9, starts with the observed diffraction pattern to which random phases are assigned. By performing a reverse Fourier transformation, one obtains a real-space image of the object, but the obtained image is incorrect. Although one does not know the exact shape Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 13 of 19

One of the CDI techniques is to restore lost phase information by iterative error-reducing calculations and is often called the oversampling method [59–62]. For details of the method, readers are encouraged to refer to the literature and here only an outline is described. The calculation, illustrated in Figure 9, starts with the observed diffraction pattern to which randomInt. J. Mol. Sci.phases2018, 19are, 1748 assigned. By performing a reverse Fourier transformation, one obtains a13 real of 19- space image of the object, but the obtained image is incorrect. Although one does not know the exact shape of the object (the final goal of the calculations is to know it), one usually has some crude knowledgeof the object about (the finalits morphology, goal of the calculations e.g., its size. isThen to know one notices it), one that usually unlikely has somedensities crude are knowledge generated inabout the itsimage morphology, and these e.g., unlikely its size. densities Then one are notices either thatreduced unlikely or deleted. densities The are Fourier generated transform in the imageation ofand this these image unlikely (diffraction densities pattern) are either has a reduced new set orof deleted.phases that The are Fourier expected transformation to be more accurate of this image than (diffractionthe initial set pattern) of random has a phases. new set The of phases amplitudes that are of expectedthe calculated to be morediffraction accurate pattern than theare initialadjusted set to of therandom observed phases. ones. The amplitudes of the calculated diffraction pattern are adjusted to the observed ones.

Figure 9. Iterative phase-retrieval algorithm (oversampling method). The recorded diffraction pattern Figure 9. Iterative phase-retrieval algorithm (oversampling method). The recorded diffraction pattern from a specimen (in reciprocal space, its phase information is missing) is given a random set of phases from a specimen (in reciprocal space, its phase information is missing) is given a random set of phases (Top left). Then the pattern is subjected to inverse Fourier transformation (FFT−1) to obtain the real- (Top left). Then the pattern is subjected to inverse Fourier transformation (FFT−1) to obtain the space image (electron density distribution) of the specimen (Top right). This is incorrect because of real-space image (electron density distribution) of the specimen (Top right). This is incorrect because the incorrect set of phases and it has densities in unlikely positions. Usually the approximate size of of the incorrect set of phases and it has densities in unlikely positions. Usually the approximate size of the specimen is known and we assume this area is likely to contain the specimen. This area is called the specimen is known and we assume this area is likely to contain the specimen. This area is called a support (the smaller square) and is assumed to be somewhat greater than the expected size of the specimen. To successfully retrieve the correct phase informati information,on, the entire area of data collection (the larger square) square) must must be be at at least least 2 2×× greatergreater than than the the actual actual size size of ofthe the sample. sample. This This is to is ensure to ensure that thatthe samplingthe sampling frequency frequency of data of data is at isleast at least 2× finer 2× finerthan the than Nyquist the Nyquist frequency, frequency, which which is defined is defined as f = as1/s, f w=here 1/s, wheres is the s issize the of size the ofspecimen the specimen (This (Thisis why is whyit is called it is called the oversampling the oversampling method). method). Next, Next, the densitiesthe densities that that fall falloutside outside the the support support are are reduced reduced either either to to zero zero (error (error-reduction-reduction algorithm) algorithm) or or to to a small fraction of the obtained densities (hybrid input-outputinput-output algorithm or HIO algorithm) ( Bottom right). After this, a Fourier transformation is applied to this corrected image to obtain a new diffraction pattern (Bottom left). This has a set of phases that is expected to be more correct than the initial set. By retaining this set of phases, the amplitudes are adjusted to the recorded values (Middle left) and the calculations are repeated in a clockwise direction. If all the conditions are right, the image of the sample converges to the true one after hundreds to thousands of iterations. Based on Reference [62]. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 14 of 19

right). After this, a Fourier transformation is applied to this corrected image to obtain a new diffraction pattern (Bottom left). This has a set of phases that is expected to be more correct than the initial set. By retaining this set of phases, the amplitudes are adjusted to the recorded values (Middle left) and the calculations are repeated in a clockwise direction. If all the conditions are right, the image Int. J. Mol.of the Sci. sample2018, 19 converges, 1748 to the true one after hundreds to thousands of iterations. Based on Reference14 of 19 [62].

ByBy repeatingrepeating thesethese cyclicalcyclical calculations,calculations, oneone maymay finallyfinally restorerestore thethe correctcorrect setset ofof phasesphases andand restorerestore thethe truetrue imageimage ofof the the object. object. AmongAmong suchsuch iterativeiterative algorithms,algorithms, thethe mostmost widelywidely usedused oneone isis calledcalled thethe hybridhybrid input-outputinput-output (HIO)(HIO) algorithmalgorithm [[5959]] in in whichwhich unlikelyunlikely densitiesdensities areare notnot forcedforced toto zerozero butbut toto smallsmall positivepositivevalues. values. ThisThis typetype ofof algorithmalgorithm isis regardedregarded asas aa kindkind ofof modelmodel fittingfitting thatthat isis employedemployed inin manymany fieldsfields ofof sciencescience includingincluding proteinprotein crystallography.crystallography. OneOne comparescompares thethe observedobserved diffractiondiffraction patternpattern andand thethe patternpattern calculatedcalculated fromfrom thethe determineddetermined structurestructure andand infersinfers thatthat thethe determineddetermined structurestructure isis correctcorrect whenwhen thethe error error between between the the two two is is reasonably reasonably small. small. TheThe problemsproblems commoncommon toto thisthis kindkind ofof iterativeiterative modelmodel fittingfitting algorithm algorithm include:include: (1)(1) thethe successsuccess ofof fittingfitting dependsdepends heavilyheavily on the goodness of of the the starting starting model model,, (2) (2) a agood good signal signal-to-noise-to-noise ratio ratio is isrequired required for for the the diffraction diffraction pattern pattern,, and and (3) (3)the the calculation calculationss tend tend to be to trapped be trapped by local by local minima. minima. The TheHIO HIO algorithm algorithm is known is known to be efficiently to be efficiently trapped trapped by local by minima, local minima, so that this so algorithm that this algorithmis often used is oftenin combination used in combination with other algorithms, with other such algorithms, as a genetic such algorithm, as a genetic to find algorithm, the global to findminimum the global [63]. minimumAnother [63]. CDI technique is a holographic method and the representative type is called Fourier- transformAnother holography CDI technique (FTH) [ is64– a70 holographic]. Unlike the methoditerative andphase the-retrieval representative algorithms type as described is called Fourier-transformabove, phase information holography is recorded (FTH) in [64 the–70 diffraction]. Unlike thepattern iterative itself. phase-retrieval In the case of FTH, algorithms a small asX- describedray-scattering above, object phase (reference information point) is is recorded placed near in the the diffraction sample. The pattern X-rays itself. scattered In the by case the of sample FTH, aand small those X-ray-scattering scattered by object the reference (reference point point) interfere is placed nearwith theeach sample. other, The which X-rays creat scatteredes Moir byé- thelike sampleinterference and those waves. scattered The phase by the infor referencemation point is preserved interfere in with these each waves. other, When which an creates inverse Moir Fourieré-like interferencetransformation waves. is performed The phase on information the diffraction is preserved pattern in after these assuming waves. When that anthe inverse phase Fourieris zero transformationeverywhere, the is performedimage of the on sample the diffraction is restored pattern (see after Figure assuming 10). The that trick the is phase that isthe zero inverse everywhere, Fourier thetransform image ofation the of sample a diffraction is restored pattern (see with Figure zero 10). phases The trick (the is Patterson that the inverse function) Fourier is an transformationautocorrelation offunction a diffraction of the patterndensities with of the zero sample. phases If (the a reference Patterson point function) exists isat an a distance autocorrelation farther than function the largest of the densitiesdiameter of of the the sample. sample, If the a reference correlation point func existstionat between a distance the farther sample than and the the largest reference diameter point ofis thethe sample,density thedistribution correlation of function the sample between itself theand sample it does and not the overlap reference with point the rest is the of density the autocorrelation distribution offunction. the sample itself and it does not overlap with the rest of the autocorrelation function.

Figure 10. Principle of Fourier transform holography (FTH). (A) Diffraction from a sample (SPring-8 Figure 10. Principle of Fourier transform holography (FTH). (A) Diffraction from a sample (SPring-8 logo) and its inverse Fourier transformation (FFT−1) calculated by assuming that the phases are all logo) and its inverse Fourier transformation (FFT−1) calculated by assuming that the phases are all zero. This results in what is called the Patterson function, which is an autocorrelation function of the zero. This results in what is called the Patterson function, which is an autocorrelation function of the sample. This is a real-space image but it is very difficult to imagine what the sample looks like by sample. This is a real-space image but it is very difficult to imagine what the sample looks like by watching this function. (B) Diffraction from a sample with a reference point. The phase information is preserved in the Moiré pattern (caused by the interference between the sample and the reference) and, as a result, the real-space image of the sample is restored by performing inverse Fourier transformation. This is an experiment on a computer. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 15 of 19

watching this function. (B) Diffraction from a sample with a reference point. The phase information is preserved in the Moiré pattern (caused by the interference between the sample and the reference)

Int. J. Mol.and Sci., as2018 a result,, 19, 1748 the real-space image of the sample is restored by performing inverse Fourier15 of 19 transformation. This is an experiment on a computer.

TheThe meritmerit of of the the FTH FTH method method is is that that the the principle principle is simpleis simple and, and in,theory, in theory it does, it does not requirenot require any trickyany tricky algorithms algorithms but only but only one inverseone inverse Fourier Fourier transformation transformation operation operation to restore to restore the imagethe image of the of sample.the sample. Thedemerit The demerit of this of methodthis method is that is one that has one to has place to place a reference a reference point atpoint a certain at a certain distance distance away fromaway the from sample. the sample. The spatial The resolutionspatial resolution is determined is determined by the size by of the the size reference of the point.reference The point. larger The the referencelarger the point, reference the stronger point, the the stronger interference the isinterference so that it is is easier so that to retrieveit is easier the to image retrieve at an the expense image ofat spatialan expense resolution. of spatial resolution. BothBoth thethe iterative phase phase retrieval retrieval method method and and the the FTH FTH work work fairly fairly well well with with samples samples with withhigh highcontrast contrast such such as metal as metal nanoparticles, nanoparticles, but but it itis ismore more difficult difficult to to retrieve retrieve images fromfrom biologicalbiological samples.samples. InIn bothboth methods,methods, thethe achievedachieved spatialspatial resolutionresolution isis currentlycurrently ofof thethe order order ofof 1010 nm,nm, whichwhich isis unsatisfactoryunsatisfactory for forbiological biologicalspecimens. specimens. TheThe applicationapplication ofof thesethese lens-lesslens-less imagingimaging techniquestechniques toto conventionalconventional fiberfiber diffractiondiffraction patternspatterns hashas notnot beenbeen triedtried extensively,extensively, butbut consideringconsidering thethe rapidrapid progressprogress ofof algorithms algorithms andand thethe improvedimproved performanceperformance ofof computers, we we are are expected expected to to be be able able to toretrieve retrieve real real-space-space images images of the of themuscle muscle fine finestructure structure from from the theconventional conventional diffraction diffraction patterns patterns in inthe the near near future future instead instead of analyzing analyzing thethe diffractiondiffraction patterns patterns in in the the reciprocal reciprocal space space we we are are currently currently completing. completing. AnAn attemptattempt toto retrieveretrieve thethe 3-dimensional3-dimensional real-spacereal-space structurestructure ofof thethe actomyosinactomyosin complexcomplex waswas donedone byby iterativeiterative errorerror reductionreduction as as earlyearly asas 20072007 beforebefore thethe daysdays ofof XFELXFEL [71[71].]. AlthoughAlthough atat aa lowlow resolution,resolution, the the 3-dimensional3-dimensional structurestructure ofof thethe rigor rigor complexcomplex betweenbetween actinactin andand myosinmyosin subfragment-1subfragment-1 fromfrom variousvarious sources and and its its structural structural changes changes upon upon binding binding of ofADP ADP were were successfully successfully restored restored (see (seeFigure Figure 11). 11Today). Today we have we havemuch much better better knowledge knowledge and calculation and calculation power power and we and should we should be able be to ablerestore to restore the real the-space real-space 3-dimensional 3-dimensional image image of the of themolecular molecular machinery machinery of of muscle muscle in in a shortershorter calculationcalculation time time and and at at aa higherhigher spatialspatial resolutionresolution ifif wewe workwork hardhard toto applyapply thesethese emergingemerging techniques techniques toto musclemuscle research.research.

FigureFigure11. 11.StructureStructure ofof thethe complexcomplex betweenbetween myosinmyosin S1S1 (subfragment-1)(subfragment-1) fragmentfragment andand actinactin restoredrestored fromfrom diffraction diffraction patterns. patterns. The The sources sources of of S1 S1 are: are: ( A(A)) rabbit rabbit fast fast skeletal, skeletal, ( B(B)) rabbit rabbit cardiac/slow cardiac/slow skeletal,skeletal, (C) chicken gizzard smooth, (D) human nonmuscle IIA, and (E) human nonmuscle IIB. The green and purple amino-acid backbones are the best-fit structures for the electron-density envelope of the rigor and ADP-bound complexes, respectively. Taken from Reference [71]. Int. J. Mol. Sci. 2018, 19, 1748 16 of 19

11. Conclusions This review has explained how the bright and well-oriented synchrotron radiation X-rays can be utilized for muscle research especially for research of insect flight muscle. The techniques involved here include fast time-resolved diffraction recording and the recording from a single myofibril by using X-ray microbeams and rapid-freezing techniques. The interpretation of diffraction patterns still requires a proper understanding of diffraction theory and this can be an obstacle for general researchers to employ this powerful non-invasive technique. However, this situation will soon be changed when effective algorithms are available for restoring real-space images of specimens with ease.

Conflicts of Interest: The author declares no conflict of interest.

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