FP CILIA. Customized Intelligent Life-Inspired Arrays. Integrated Project

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1 FP CILIA Customized Intelligent Life-Inspired Arrays Integrated Project Information Society Technologies Future & Emerging Technologies Proactive Initiative BIO-I3 DELIVERABLE: D Public APPLICATION OF REFINED METHOD TO 3D CERCI Actual submission date: October 2, 2008 Start day of project: September 1st, 2005 Duration: 48 months Copyright Members of the CILIA Consortium See for details on the copyright holders. CILIA ( Customized Intelligent Life-Inspired Arrays ) is a project funded by the European Union. For more information on the project, its partners and contributors please see The information contained in this document represents the views of CILIA as of the date they are published. CILIA does not guarantee that any information contained herein is errorfree, or up to date. CILIA MAKES NO WARRANTIES, EXPRESS, IMPLIED, OR STATUTORY, BY PUBLISHING THIS DOCUMENT.

2 CONTENT 1. EXECUTIVE SUMMARY TERMINOLOGY ACQUISITION OF 3D DATA INTRODUCTION METHODOLOGY Confocal microscopy and 3D reconstruction D light microscopy RESULTS Confocal microscopy imaging Mesh generation D light microscopy CONCLUSIONS AND DISCUSSION REFERENCES FP Members of CILIA consortium PUBLIC 2 / 21

3 1. EXECUTIVE SUMMARY Two imaging techniques, confocal microscopy and 3-dimensional (3D) light microscopy, were used in an attempt to acquire the 3D morphology of the cricket cerci mechano-sensory hair array system and its associated features. The advantages and limitations of the techniques are discussed. The results suggest that the techniques are suitable for the characterisation of individual mechanosensors (hair + socket + campaniform) in adult crickets but not suitable for determining the morphology of an array of filiform mechanosensors (distribution, density, directivity). FP Members of CILIA consortium PUBLIC 3 / 21

4 2. TERMINOLOGY Glossary CM FEA IFM µct PMT SEM Confocal Microscopy Finite Element Analysis Infinite Focus Microscope Micro Computed Tomography Photomultiplier tube Scanning Electron Microscopy FP Members of CILIA consortium PUBLIC 4 / 21

5 3. ACQUISITION OF 3D DATA 3.1. INTRODUCTION In two previous deliverable reports (D D reconstructions of hair sensing organs and mechanical/structural properties and D1.1.8 Complete image reference library of crickets using SEM and CM imaging, for interspecies comparison of single hair and array morphology ), we reported on the use of different methods for visualising and modelling the 3D shape characteristics of the individual filiform mechanosensors on the cricket cerci. The methods used were confocal microscopy (CM), Scanning Electron Microscopy (SEM), and Micro Computed Tomography (µct). The main conclusions of these reports were that no single technique would be able to deliver all the relevant information and that data obtained from the various methods would have to be integrated. SEM was identified as an effective and reliable technique for the determination of a range of morphological properties but SEM delivers only 2-dimensional data. Confocal microscopy is a useful supporting tool to SEM in the production of morphological and topological data but also has the potential to aid in the generation of 3-dimensional models and meshes, enabling computational analysis of the mechanical interaction between hairs, sockets and campaniform. It was identified that the reconstruction aspects, particularly for early cricket instars, presented a challenge in terms of resolution and contrast and required further investigation, particularly with respect to confocal microscopy. This report focuses on the use of 3D imaging techniques in order to acquire the 3D morphology of the cricket cerci and in particular, the filiform hair sockets and associated campaniform sensilla. In addition to the use of confocal imaging, we have explored the use of 3D light microscopy. This is a relatively new technique which uses optical 3D imaging for the measurement, analysis and characterisation of surfaces, with a vertical resolution of up to 10 nm. An Infinite Focus Microscope (IFM) was made available by Alicona Imaging at the National Physical Laboratory (NPL), UK METHODOLOGY Confocal microscopy and 3D reconstruction A confocal microscope (Leica Microsystems, GmbH, Germany), as set up and described in deliverable reports D1.1.3 and D1.1.8, was used to capture images of the filiform hair sockets and associated campaniform sensilla, once again relying on the autofluorescence of insect cuticle. 3D images were captured using a light source of wavelength 488 nm at 45% intensity and at 1024 x 1024 pixels resolution. The bespoke micromanipulator, also described in D1.1.8, was used in order to provide four degrees of freedom for micromanipulation of the specimens. For this technique to work accurately it was essential that the specimens be maneuvered so that the sockets could be viewed directly from above. D1.1.3 concluded that confocal microscopy had, at that time, yet to produce any meaningful results in the examination of hatchlings, due to resolution and contrast issues. Only hatchlings were, therefore, used here in order to improve the quality of the images produced. Hatchlings of G. assimilis, G. bimaculatus and G. sigillatus, and the cerci of adult G. FP Members of CILIA consortium PUBLIC 5 / 21

6 sigillatus, were air dried and examined, uncoated in air. The 3D scan data output was combined with image processing tool, ScanIP (Simpleware Ltd, UK) allowing the visualisation and segmentation of the structures. A 3D volumetric mesh was also generated using the mesh generator module, + ScanFE (Simpleware Ltd, UK) and exported for finite element analysis (FEA). FEA has not been carried out at this stage but the files are directly exportable to a number of commonly available analysis tools D light microscopy An optical 3D Infinite Focus Microscope (IFM) (Alicona Imaging GmbH, Austria), with a vertical resolution of up to 10 nm, was used to capture 3D images of cercal sections to include filiform hair sockets and associated campaniform sensilla. The left cerci of young and adult crickets of species G. assimilis, G. bimaculatus and G. sigillatus, were dissected and sputter-coated in gold for 4 minutes. The coated specimens were viewed at 50x and 100x magnification. The estimated vertical resolution was nm at 50x magnification and nm at 100x magnification. FP Members of CILIA consortium PUBLIC 6 / 21

7 3.3. RESULTS Confocal microscopy imaging Figures 1 to 9 illustrate confocal images (left column) generated using the confocal microscope and the segmented 3D reconstructions (right column) of these images generated using the ScanIP software. Figures 1 to 3 show examples of cercal hair arrays from hatchlings and figures 4 to 6 show examples of single sockets from hatchlings. Figure 7 to 9 show examples of single sockets from adults. Species: Gryllus assimilis (1a) Confocal image of a hair array of a young, G. assimilis (Scale bar = µm) (1b) 3D reconstruction of (1a), confocal image of a hair array of a young, G. assimilis Figure 1. Confocal image (left) and subsequent 3D reconstruction (right) of a filiform hair array taken from a cercal section of a G. assimilis cricket hatchling Species: Gryllus bimaculatus (2a) Confocal image of a hair array of a young, G. bimaculatus (Scale bar = µm) (2b) 3D reconstruction of (2a), confocal image of a hair array of a young, G. bimaculatus Figure 2. Confocal image (left) and subsequent 3D reconstruction (right) of a filiform hair array taken from a cercal section of a G. bimaculatus cricket hatchling FP Members of CILIA consortium PUBLIC 7 / 21

8 Species: Gryllodes sigillatus (3a) Confocal image of a hair array of a young, G. sigillatus (Scale bar = µm) (3b) 3D reconstruction of (3a), confocal image of a hair array of a young, G. sigillatus Figure 3. Confocal image (left) and subsequent 3D reconstruction (right) of a filiform hair array taken from a cercal section of a G. sigillatus cricket hatchling Confocal Image 3D reconstruction Species: Gryllus assimilis (4a) Confocal image of a single hair socket of a young, G. assimilis (Scale bar = µm) (4b) 3D reconstruction of (4a), confocal image of a single hair socket of a young, G. assimilis Figure 4. Confocal image (left) and subsequent 3D reconstruction (right) of a filiform hair socket taken from a cercal section of a G. assimilis cricket hatchling FP Members of CILIA consortium PUBLIC 8 / 21

9 Species: Gryllus bimaculatus (5a) Confocal image of a single hair socket of a young, G. bimaculatus (Scale bar = 8.00 µm) (5b) 3D reconstruction of (5a), confocal image of a single hair socket of a young, G. bimaculatus Figure 5. Confocal image (left) and subsequent 3D reconstruction (right) of a filiform hair socket taken from a cercal section of a G. bimaculatus cricket hatchling Species: Gryllodes sigillatus (6a) Confocal image of a single hair socket of a young, G. sigillatus (Scale bar = 2.22 µm) (6b) 3D reconstruction of (6a), confocal image of a single hair socket of a young, G. sigillatus Figure 6. Confocal image (left) and subsequent 3D reconstruction (right) of a filiform hair socket taken from a cercal section of a G. sigillatus cricket hatchling FP Members of CILIA consortium PUBLIC 9 / 21

10 Species: Gryllodes sigillatus (7a) Confocal image of a single hair socket of an adult, G. sigillatus (Scale bar = µm) (7b) 3D reconstruction of (7a), confocal image of a single hair socket of an adult, G. sigillatus Figure 7. Confocal image (left) and subsequent 3D reconstruction (right) of a filiform hair socket taken from a cercal section of an adult G. sigillatus cricket Species: Gryllodes sigillatus (6a) Confocal image of a single hair socket of an adult, G. sigillatus (Scale bar = 8.00 µm) (6b) 3D reconstruction of (6a), confocal image of a single hair socket of an adult, G. sigillatus Figure 8. Confocal image (left) and subsequent 3D reconstruction (right) of a filiform hair socket taken from a cercal section of an adult G. sigillatus cricket FP Members of CILIA consortium PUBLIC 10 / 21

11 Species: Gryllodes sigillatus (6a) Confocal image of a single hair socket of an adult, G. sigillatus (Scale bar = 8.00 µm) (6b) 3D reconstruction of (6a), confocal image of a single hair socket of an adult, G. sigillatus Figure 9. Confocal image (left) and subsequent 3D reconstruction (right) of a filiform hair socket taken from a cercal section of an adult G. sigillatus cricket Figures 1 to 9 illustrate how difficult it is to obtain accurate 3D reconstructions of the hair canopy and its associated features using confocal microscopy. Certain features are identifiable but the morphological characteristics of the mechanosensors are not revealed in their entirety. Problems with contrast and saturation mean that, when converted into greyscale, the confocal images do not present distinguishable features which can be identified by the image processing software. Using Figure 3a as an example, when the confocal image is converted into a greyscale image (Figure 10) the features are much less distinguishable by the image processing software. FP Members of CILIA consortium PUBLIC 11 / 21

12 (a) (b) Figure 10. Greyscale images adapted from a confocal image, illustrating a marginal difference in greyscale values. Figure 10a shows how a pixel close to the bottom left-hand socket of the array, is identified as having a greyscale value of 38. This pixel appears to be located on the cuticular region of FP Members of CILIA consortium PUBLIC 12 / 21

13 the cercus. When the cursor is moved to the left of the image over what appears to be a part of the socket, Figure 10b, the pixel has a greyscale value of 45. This difference in greyscale value of only 7 makes the features of the image virtually indistinguishable. Where sections of the greyscale image appear very dark (i.e. close to black with a greyscale value of 0), these translate during reconstruction as areas of no information i.e. holes. Similarly, where very light areas appeared through saturation from the laser beam, these regions close to the white greyscale value of 255, appear solid (Figure 3b) Mesh generation Figures 11 to 13 illustrate how a 3D reconstructed model can be converted into a mesh for finite element analysis. Figure 11. FE mesh generated from the confocal microscopy data of a hair array of a young, G. sigillatus The model is an un-adapted, smoothed mesh consisting of 819,694 elements (124,501 hexahedral and 695,193 tetrahedral) and 304,426 nodes. The minimum element quality target, measured by the ratio of the shortest to longest sides (where 0.1 is a degenerated or flat triangle and 1.0 is an equilateral tetrahedral element), was set at 0.1 with a mean quality of 0.4 achieved. Extra surface smoothing was not applied. FP Members of CILIA consortium PUBLIC 13 / 21

14 A further example is presented in Figures 12 and 13. The reconstructed model, shown in figure 9b, was previously presented in deliverable report D D reconstructions of hair sensing organs and mechanical/structural properties. The original image (Figure 12a) was taken using confocal microscopy and is of a single socket from an adult G. sigillatus. Figure 12a: Confocal image of a socket and three associated campaniform of an adult G. sigillatus. Figure 12b: 3D reconstruction of the socket and associated campaniform of an adult G. sigillatus Figure 12. A confocal image (a) and a 3D reconstruction (b) of a filiform hair socket and its associated campaniform sensilla The model is an un-adapted, smoothed mesh consisting of 1,392,205 elements (241,131 hexahedral and 1,151,074 tetrahedral) and 527,225 nodes. The minimum element quality target was set at 0.12 and extra surface smoothing was applied. Figure 13a: 3D volumetric finite element mesh of a single hair socket and its associated campaniform sensilla Figure 13b: Close-up of the 3D volumetric finite element mesh showing the combination of mixed tetrahedral and hexahedral elements. Figure 13. A finite element mesh, generated from confocal microscopy data, of a single mechanosensor socket and three associated campaniform. FP Members of CILIA consortium PUBLIC 14 / 21

15 The finite element meshes are generated directly from the scan data. An FE mesh, such as that shown in Figure 13, created by this method is directly exportable to many commercially available FEA and CFD packages with no re-meshing or pre-processing required D light microscopy Figure 14 shows an image typical of that produced by the IFM optical 3D light microscope. The image shows a dorsal section of the left cercus of a young G. sigillatus with four sockets clearly visible, two of which contain filiform hairs. The central socket, highlighted by the box, clearly possesses two associated campaniform organs at approximately 180 to each other (towards the medial and lateral sides of the cercus). Medial Proximal Distal Lateral Figure 14. 2D image showing four filiform hair sockets of the cercus, as obtained using 3D light microscopy. The boxed area is the region of interest. FP Members of CILIA consortium PUBLIC 15 / 21

16 The topology of the cercal surface can be obtained using the IFM analysis software from which a screenshot is shown (Figure 15). Figure 15. Screenshot of the IFM analysis software illustrating how the topology of the cercus can be analysed. The locations of the red and green crosses in the image, refer to the red and green cursors, providing information with regards to the location of the reference lines. Figure 15 shows a profile analysis of a socket and one of its associated campaniform sensilla. Measurement tools permit a profile analysis of the z direction elevation. A line has been drawn across the socket (top image) and the graph underneath shows the z direction elevation of the selection. In this example, the height of the socket was measured as 4.80 µm in relation to the cuticle on the lateral side of the socket. FP Members of CILIA consortium PUBLIC 16 / 21

17 The conical shape of the cercus can also be characterised by the radius of curvature along a specified direction which can be obtained from the elevation profile obtained (figure 16). Figure 16. Screenshot of the IFM analysis software illustrating how the radius of curvature of the cercus can be measured. A profile analysis line was drawn perpendicular to the long axis of the cercus and the radius of curvature was measured to be 55.0 µm. FP Members of CILIA consortium PUBLIC 17 / 21

18 Figure 17 is an IFM optical image of part of the left cercus of a young (hatchling) Gryllus assimilis with two clear images of campaniform sensilla, highlighted by the boxes. #2 #1 Figure 17. IFM optical image of part of the left cercus of a young Gryllus assimilis. The image in figure 17 clearly shows the cap and collar arrangements, as described by many authors including Grünert and Gnatzy (1987), of two campaniform sensilla (highlighted by the boxes). Using the same topology analysis tool as described above (Figure 15) a surface analysis of one of the sensilla (# 2) is presented in figure 18. FP Members of CILIA consortium PUBLIC 18 / 21

19 Figure 18. IFM screenshot illustrating the results of a topological analysis of a single campaniform sensillum in relation to the surrounding cuticle. The topological analysis of the campaniform sensillum (Figure 18) does not reveal the domeshaped features known of this structure. From the distal to the proximal ends of the cercus, right to left of the image, the region between the two cursors slopes at a height of 970 nm and an angle of 10. The diameter of the mechanosensor, measured as the distance between the two cursors, is measured as 3.0 µm which is typical of the diameter of this type of sensillum. However, the dome-shaped cap and the rounded collar can not be observed CONCLUSIONS AND DISCUSSION Confocal and optical 3D-microscopy are useful tools for a great many applications but as has been observed previously using microct (D1.1.3), the complex morphology of the cricket mechanosensory system stretches the capabilities of these techniques to their limits. Both techniques have provided some promising results with regards to the 3D morphological acquisition of mechanosenors but they need to be optimised. In order to detect the features of the campaniform sensilla, for example, the vertical resolution needs to be much higher than that achieved. FP Members of CILIA consortium PUBLIC 19 / 21

20 The segmentation and 3D reconstruction of confocal images works well for single mechanosensors of adult crickets and for an array of mechanosensors in hatchlings. However, this technique is unsuitable for the imaging and reconstruction of mechanosensor arrays in adult crickets due to the small focal point used in confocal microscopy. The pinhole focal point of the confocal microscope means that only small areas of the cercus are in focus at any one time so structures outside the focal point are not detected by the photomultiplier tube (PMT) of the microscope. It is also less successful for the 3D acquisition of single mechanosensors of hatchlings due to the necessity for high magnification, high resolution images. Additionally, the complex geometry of the mechanosensory units means that the FE models produced require high numbers of hexahedral and tetrahedral elements resulting in large file sizes (up to 400 MB). These are computationally expensive to run using FEA. In addition, the amount of post-processing needed on the generated meshes to "repair" the missing patches is likely to be considerable. Many commercial Finite Elements packages do have some "mesh repair" capability (often used with automeshing functions). However, they work reasonably well on models (2D or 3D) of far less surface-geometry complexity than the cerci-sockets-campaniform system. In the case of the cerci, the "repair" will have to be done largely "by hand" using additional visual information from the microscopy. The results suggest that the techniques discussed here are suitable for the characterisation, in some detail, of individual mechanosensors (hair + socket + campaniform) but not suitable for determining the morphology of an array of filiform mechanosensors (distribution, density, directivity), especially in adults. The technique used by John Miller (pers. comm.), which consists of splitting the cercus longitudinally and flattening it out as a planar surface, does eliminate the problem associated with the conical shape of the cerci. This simplifies considerably the acquisition of images from which the array parameters can be obtained, converting a 3D reconstruction problem into a 2D one. However, this approach is also very time-consuming and, at this stage, not really suitable for rapid reconstruction of array morphology. In principle all the techniques mentioned can be used on the various species of crickets (phylogeny), and for various developmental stages (ontogeny), bearing in mind the respective advantages and limitations. FP Members of CILIA consortium PUBLIC 20 / 21

21 4. REFERENCES Grünert U., & Gnatzy, W., (1987) Campaniform sensilla of Caliphora vicina (Insecta, Diptera). II. Typology. Zoomorphology, 106, FP Members of CILIA consortium PUBLIC 21 / 21