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CONFOCAL MICROSCOPY IN A NEW LIGHT. Title: Introduction to Confocal Microscopy Presented by: Dr. Andrew Dixon Date: May 2009 Introduction to Confocal.

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Presentation on theme: "CONFOCAL MICROSCOPY IN A NEW LIGHT. Title: Introduction to Confocal Microscopy Presented by: Dr. Andrew Dixon Date: May 2009 Introduction to Confocal."— Presentation transcript:

1 CONFOCAL MICROSCOPY IN A NEW LIGHT

2 Title: Introduction to Confocal Microscopy Presented by: Dr. Andrew Dixon Date: May 2009 Introduction to Confocal Microscopy

3 3 An Introduction to Confocal Microscopy What is the problem? Marvin Minskys idea The confocal principle The power of confocal imaging Increasing imaging speed Imaging in 3-D Summary of key points

4 4 What is the Problem? Optical microscope images contain both in-focus and out-of-focus detail How can one produce an image which only includes the in-focus detail?

5 5 All-in-FocusAll-in-Focus True color information in 3D topography Bump dimension: 13.8 µm height and 79 µm width

6 6 Marvin Minskys Idea Marvin Minskys Idea Instead of collecting the complete image at one time, Minsky proposed to build up the image point by point. In this way one can introduce additional optical components in the light collection path to block the out-of-focus light from contributing to the image. Marvin Minsky Inventor of the confocal microscope Harvard (1955) US Patent 3,013,467

7 7 The Confocal Principle The sample is illuminated with a focused spot of light. Light from the sample is re-focused at the confocal aperture. Only in-focus signal reaches the detector illumination Confocal aperture detector sample Focus Cone Specimen X/Y Image X Y

8 8 The Optical Section Optical section thickness depends on objective lens NA. Lateral and axial resolution are related.

9 9 The Power of Confocal Imaging In the mid 80s mirror scanning systems were developed that adapted a conventional microscope for confocal imaging. Scientists became very excited by the images they could obtain, without having to prepare very thin section samples. Conventional image Confocal image Bio-Rad MRC-500 (Dr. W.B. Amos, MRC Cambridge) Example images show tubulin structure in fertilized sea urchin egg immuno-labelled for fluorescence contrast. (scale bar 50 micron)

10 10 Increasing Imaging Speed Scanning a focused illumination spot, point by point is relatively slow. Several alternative schemes have been developed to increase imaging speed. Another approach is to illuminate the sample with a focused line of light. This is the system used in the Axio CSM 700 from Carl Zeiss. One approach is to illuminate the sample simultaneously with multiple spots of light.

11 11 From Optical Section to 3-D image A series of optical section images can be combined into a single all in focus image, or manipulated to provide quantitative information about surface profile, surface roughness etc.

12 12 …A World of Possibilities True color information in 3D topography Surface profiling. Surface roughness Neurons in a Brainbow transgenic mouse, labeled with multiple hues of fluorescent proteins. Extended focus image (Dr. J. Livet Harvard University) Biological ResearchMaterial Sciences

13 13 In Conclusion… Exceptional contrast optical section images Non-contact probing and profiling Not restricted to single color imaging Imaging at high speed Qualitative and quantitative 3-D characterization High resolution surface profiling Confocal microscopy delivers…

14 14 EndEnd

15 Title: Advanced Confocal Microscopy: Axio CSM 700 Presented by: Dr. Franz Reischer Date: May 2009 Advanced Confocal Microscopy: Axio CSM 700

16 16 Axio CSM 700 – System Overview Xe illuminator, conf. microscope, controller, user PC

17 17 Innovative Confocal Method 1Xe illuminator 2Multi slit grid for scanning instead of scan mirrors 3Beam splitter 4Sample / focal plane 5Digital detector which also provides digital confocal apertures 1 2 3 4 5

18 18 3D Image Acquisition 3D topographies, height maps, profilometry, and roughness analysis are all based on the acquisition of Z stacks. Axio CSM 700 always measures the current position of the stage using a laser linear scale with 10 nm increments and 24 bit.

19 19 AdvantagesAdvantages High acquisition speed (up to > 100 fps) True colour confocal microscopy High resolution Optical 3D profilometer

20 20 Optical resolution: XY No resolution No contrast x,y I

21 21 Optical resolution: XY Cut-off distance reached, but contrast is equal to zero No resolution No contrast x,y I

22 22 Optical resolution: XY Cut-off distance reached, but contrast is equal to zero Maximum resolution Rayleigh criterium d(x,y) ~ f * / NA f= 0.37 … 0.61 No resolution No contrast x,y I Strictly confocal Classical

23 23 Optical resolution: XY Cut-off distance reached, but contrast is equal to zero Resolution Maximum contrast Maximum resolution Rayleigh criterium d(x,y) ~ f * / NA f= 0.37 … 0.61 No resolution No contrast x,y I Strictly confocal Classical

24 24 Lateral Resolution Limit: Grid Sample:Nanoscale critical dimension standards (supracon AG Jena) Colour channel:blue Objective:Epiplan-APOCHROMAT 150x/0.95 200 nm L&S150 nm L&S Resolution limit

25 25 Test Sample: Validated depth measurement sample with 80 nm steps Objective: EC Epiplan-APOCHROMAT 100x/0.95 True height: 80 nm Measured height: 87 nm Difference: 7 nm Axial Detection Limit Axial Detection Limit

26 26 High Range of Samples Surfaces with low as well as high reflectivity, incl. polished metals & totally smooth glass. Top surface of coatings and substrates under transparent layers. Film thickness measurement of transparent layers starting at ~ 1 µm

27 27 Comparison of confocal microscopy Typical light microscope Scanning electron microscope Tactile instruments for roughness measurement True colour confocal microscope Without preparation High resolved viewing with large depth of field Display in true colour 3D measurements in sub- micrometer range

28 28 Axio CSM 700 … … opening up new worlds of microanalysis.

29 Title: Applications for Topographic Measurements in Materials Engineering Presented by: Ralf Loeffler Date: May 2009 Applications for Topographic Measurements in Materials Engineering

30 30 Application Examples Geometry inspection on cutting plate. Failure analysis on turbine blade. Tribology on high performance steel.

31 31 Geometry Inspection – Cutting Plate Turning and milling are the most important machining steps in metal processing / machining Cutting plates consist of coated (TiCN) hard-metal (WC) Important factors on wear behaviour: plate material and geometry of cutting edge, but also material of workpiece Empirical approach to improve wear properties of cutting plates mostly qualitative characterization of tool wear Quantitative characterization enables accurate measurement of important parameters influencing tool performance (roughness and geometry of cutting edge, e.g. honing and erosion) Goal: high tool life / endurance at high feed rates

32 32 top view Geometry Inspection – Cutting Plate Functional parameters influencing performance: angle and radius of cutting edge side view 3D-µCT surface rendering resolution: 10 µm/vx

33 33 rake honing 1 (r 1 ) honing 2 (r 2 ) tool flank chamfer Geometry Inspection – Cutting Plate Definition of functional parameters rake honing 1 (r 1 ) honing 2 (r 2 ) chamfer tool flank

34 34 Geometry Inspection – Cutting Plate rake chamfer cutting edge r1r1 r2r2 focus image rake angle

35 35 Geometry Inspection – Cutting Plate only one cutting edge radius (honing) approx 200 µm rake cutting edge wear groove height: approx. 11 µm width approx. 49 µm

36 36 Geometry Inspection – Cutting Plate Quantitative Measurement: new plate Roughness - along cutting edge R z = 2.2 µm R a =0.3 µm

37 37 Roughness - along cutting edge Geometry Inspection – Cutting Plate Quantitative Measurement: worn plate R z = 10.9 µm R a =1.0 µm

38 38 New cutting plateWorn cutting plate Roughness (R a ) (along cutting edge) 0.3 µm 1.0 µm Roughness (R Z ) (along cutting edge) 2.2 µm 10.9 µm rake angle 18 deg 19 deg cutting edge radius 47 µm 100 µm not determined Features no wear mechanism: adhesive wear Geometry Inspection – Cutting Plate

39 39 Geometry Inspection – Cutting Plate Conclusion Complex sample geometry limits accessibility positioning of sample essential Standard methods limited to qualitative evaluation Confocal Axio CSM 700 allows qualitative and quantitative analysis Wear can be quantified by means of roughness, flattening (erosion) and angle widening

40 40 Failure Analysis – Turbine Blade Sample: blade of compressor unit (turbine) Status: failed, surface wear detected Material: austenitic steel Manufacturing: milling in one piece, blades not welded on ring Environment: rotation speed approx. 300 m/s in hot vapour atmosphere

41 41 Failure Analysis – Turbine Blade Top view: sections with distinct surface wear no wearintermediate wearhigh wear section 1section 2section 3 section 1 section 2section 3

42 42 Failure Analysis – Turbine Blade R a = 0.7 µm R z = 27.3 µm Note milling marks no wear Roughness Measurement topography profile area measurement

43 43 Failure Analysis – Turbine Blade Note wear and milling marks R a = 1.4 µm R z = 21.7µm intermediate wear Roughness Measurement topography profile area measurement

44 44 Failure Analysis – Turbine Blade area measurement R a = 2.7 µm R z = 109.4 µm Note deep wear marks high wear Roughness Measurement R 1 = 630 µm Depth = 65 µm

45 45 Failure Analysis – Turbine Blade R 1 = 550 µm Depth = 65 µm R 1 = 530 µm Depth = 65 µm Note aligned wear marks high wear area measurement R a = 4.0 µm R z = 45.4 µm Roughness Measurement 1 2 3 45 2 3 4 5 12345 1345

46 46 Failure Analysis – Turbine Blade R 1 = 450 µm Depth = 70 µm Image acquisition: 50x high wear 2D topography profile

47 47 Section 1 No wear Section 2 High wear Section 3 Intermediate wear Roughness (R a ) 0.7 µm 4.0 µm 1.4 µm Roughness (R Z ) 27.3 µm 45.4 µm 21.7 µm Features Milling marks dominate No wear marks Deep, round wear marks Milling marks barley visible Small, rather round wear marks Milling marks clearly visible Failure Analysis – Turbine Blade

48 48 Failure Analysis – Turbine Blade Conclusion Wear can be quantified by means of R a -value Due to large spherical defects R z -value increases in areas with coarse defect structure Shape of defect may be linked to prevailing mechanism, either erosion or cavitation

49 49 Tribology – Maraging Steel Composites Tribology testing of new, exceptionally hard Metal-Matrix-Composites fuel injection systems Wear depth < 2 µm white light interferometer Need: reliable, accurate and fast measurement system with high precision and visual presentation of the data Pin on disc testing by 1500 MPa need for materials with excellent wear properties

50 50 Tribology – Maraging Steel Composites Wear mark virtually absent (depth < 2 µm) limited analytical methods available SEM micrograph of a steel-ceramic composite before testing SEM micrograph of a steel-ceramic composite after testing Note: only ceramic exhibits signs of wear and tear outs

51 51 Tribology – Maraging Steel Composites Wear mark on a steel-ceramic composite excellent graphical representation LOM (bright field) micrograph of a wear mark on a steel-ceramic composite 3D visualization of a wear mark on a steel-ceramic composite using the Axio CSM 700

52 52 Tribology – Maraging Steel Composites Axio CSM 700 analysis with all-focus image Excellent visualization Wear mechanism: only by leveling ceramic particles Axio CSM 700 data in accordance with SEM micrograph observation Scanning of entire wear mark at high magnification width = 300 µm height 1 µm

53 53 Tribology – Maraging Steel Composites Axio CSM 700 vs. White Light Interferometer Identical results to the WLI analysis method Advantages of the Axio CSM 700 are its speed and visual representation of data as all-in-one snapshots with height, focus and true color images Axio CSM 700 (20x) WLI 2D-profile

54 54 EndEnd

55 Title: Exotic Applications in Confocal Microscopy Presented by: Dr. Steve Metcalfe Date: May 2009 Exotic Applications in Confocal Microscopy

56 56 Example Applications Foam Paper CCD array Polymer Film on Metal Substrate Electronics PCB Electronics sub assemblies Solar Cell (Photo Voltaic Materials) SiC Wafers Light Guide MicroLens Arraay Fresnel lens Precision Assembly STFC

57 57 FoamFoam Dynamic processes like foam can be accessed, (as long as they stay still long enough to collect the images) The high speed frame mode can help here. The structures of foam are very important for a number of disciplines.

58 58 Paper & Fabrics For example, ink penetration is used to examine ink quality as well as counterfeit material compared to original writings. Fabrics can also be examined for penetration of spray on coatings and surface contamination Examination of filters for particles and particle volume are also examples These days all materials come under scrutiny and fibrous materials like Paper and Fabrics cannot escape the quest for knowledge in the development of new materials.

59 59 Ink on Paper Pigment ink keeps on the surface and dye ink penetrates into the paper. SEM also can detect the same phenomenon but the SEM could not visualize colour information.

60 60 CCD with Bayer Mask Sensor Objects which are arrays can be inspected. Image analysis measurements can be made by thresholding out based on colour. It is then possible to measure individual features, Counting sizing and volume data are all available. Out of interest, note that there are more green pixels than read or blue. This is due to the colour response of the human eye

61 61 Polymer Film on Metal Substrate Laminate. Using advanced techniques both surfaces or a laminate can be inspected. 2 individual scans are completed one for the top surface and one for the lower. Both of these scans can then be superimposed and viewed on the 3D display. Layer thickness can then be ascertained.

62 62 PCB Track - Conductive strip It is easy to see the 3D topography of this sample.

63 63 Rendered PCB Track The surface image can be rendered with a true colour image as above or colour coded in height. A combination of these display techniques help to reveal the surface structures in their true form.

64 64 MeasurementsMeasurements Data relating to the angle and radius can be obtained. Also distance and height at the same time. Compared to tactile methods we can see both the small and large surfaces.

65 65 Solder Bumps No noise but true colour information in 3D topography Bump dimension: 13.8 µm height and 79 µm width

66 66 TFT Spacer – Touch Panel Spacer Measurements and images are presented for these common electronic spacers. A touchscreen is a display which can detect the presence and location of a touch within the display area. The term generally refers to touch or contact to the display of the device by a finger or hand. (Wikipedia) Now a common place technology used on games, mobile phones and many other electronic devices.

67 67 Photo Voltaic Advanced thin-film photovoltaic cells are multi layer structures. Surface topography, roughness and form are all important to the performance of the material

68 68 Laser Scribes on Thin Film Solar Cells Reports and data are assembled from all the data sources. Namely topographical data from the Z scan. Rendered data in full colour. A horizontal scan across the laser scribe is overlaid and the corresponding with measurements presented. The entire length of the scribe can be measured and resulting data for average, min, max and Standard Deviation can be found.

69 69 Photo Voltaic at 100 x Objective Full colour data is available at high magnifications

70 70 Pattern on SiC wafer Grooved pattern on SiC coated with SiO 2. In normal reflection mode distances are measured too short because of the SiO 2 coating. New first peak method and knowledge of refraction index gives correct distances between SiC top surface and grooves.

71 71 Metal Mold for Light Guide Panel These complex surfaces can be visualised readily with confocal techniques.

72 72 Micro Lens Array Microlenses are small lenses, generally with diameters less than a millimetre (mm) and often as small as 10 micrometres (µm). The small sizes of the lenses means that a simple design can give good optical quality but sometimes unwanted effects arise due to optical diffraction at the small features. Microoptics in nature. Examples of microoptics are to be found in nature ranging from simple structures to gather light for photosynthesis in leaves to compound eyes in insects. As methods of forming microlenses and detector arrays are further developed then the ability to mimic optical designs found in nature will lead to new compact optical systems

73 73 Fresnel lens A Fresnel lens is a type of lens invented by French physicist Augustin-Jean Fresnel. Originally developed for lighthouses. Measurements of surface properties are easily achieved.

74 74 Topography of cosmetics materials Collected images can be used to measure skin replica blending condition of lipstick, manicure and foundation. 3D image of human skin, silicone replica 3D image of human hair Lipstick

75 75 Precision Assembly & Manufacture STFC have extensive expertise in the process of micro-fabrication at the sub-mm level and an understanding of the problems that this poses

76 76 Precision Assembly & Manufacture Laser targets assembled under a microscope. Some items are conical in shape. Roughness of curved surfaces will be measured. A difficult task for other instruments due to the conical shape of the part.

77 77 AcknowledgementsAcknowledgements Wikipedia for historic information. Chris Spindloe STFC Rutherford for his help with the Laser Target images. Last 2 slides

78 78 EndEnd Questions and Answers


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