1. CONFOCAL MICROSCOPY IN A NEW LIGHT

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

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

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

6. Marvin Minsky’s 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. 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. The Optical Section
Optical section ‘thickness’ depends on objective lens NA. Lateral and axial resolution are related.

9. The Power of Confocal Imaging
Conventional image
In the mid 80’s 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.
(Dr. W.B. Amos, MRC Cambridge)
Confocal image
Bio-Rad MRC-500
Example images show tubulin structure in fertilized sea urchin egg immuno-labelled for fluorescence contrast. (scale bar 50 micron)

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.
One approach is to illuminate the sample simultaneously with multiple spots of light.
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.

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

13. In Conclusion… Confocal microscopy delivers…
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. End

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

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

17. Innovative Confocal Method1 2 3 4 5
Xe illuminator
Multi slit grid for scanning instead of scan mirrors
Beam splitter
Sample / focal plane
Digital detector which also provides digital confocal apertures

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. Advantages High acquisition speed (up to > 100 fps)
True colour confocal microscopy
High resolution
 Optical 3D profilometer

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

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

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

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

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&S
Resolution limit
150 nm L&S

25. Axial Detection Limit 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: nm

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

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

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

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

33. Geometry Inspection – Cutting Plate
Definition of functional parameters
rake
honing 1 (r1)
chamfer
rake
chamfer
honing 1 (r1)
tool flank
honing 2 (r2)
honing 2 (r2)
tool flank

34. Geometry Inspection – Cutting Plate
chamfer r1
rake r2
cutting edge
focus image
rake angle

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

36. Geometry Inspection – Cutting Plate
Quantitative Measurement: new plate
Roughness - along cutting edge
Rz = 2.2 µm Ra=0.3 µm

37. Geometry Inspection – Cutting Plate
Quantitative Measurement: worn plate
Roughness - along cutting edge
Rz = 10.9 µm Ra=1.0 µm

38. Geometry Inspection – Cutting Plate
New cutting plate
Worn cutting plate
Roughness (Ra)
(along cutting edge)
0.3 µm
1.0 µm
Roughness (RZ)
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

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. 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. Failure Analysis – Turbine Blade
Top view: sections with distinct surface wear
no wear
high wear
intermediate wear
section 1
section 2
section 3
section 1
section 2
section 3

42. Failure Analysis – Turbine Blade
no wear
Roughness Measurement
Note milling marks
area measurement
Ra = 0.7 µm Rz = 27.3 µm
topography profile

43. Failure Analysis – Turbine Blade
intermediate wear
Roughness Measurement
Note wear and milling marks
area measurement
Ra = 1.4 µm Rz = 21.7µm
topography profile

44. Failure Analysis – Turbine Blade
high wear
R1= 630 µm
Depth = 65 µm
Note deep wear marks
Roughness Measurement
area measurement
Ra = 2.7 µm Rz = µm

45. Failure Analysis – Turbine Blade1 2 3 4 5
high wear
R1 = 530 µm
Depth = 65 µm
R1 = 550 µm
Depth = 65 µm 1 4 5 3 2 1 3 4 5
Note aligned wear marks
Roughness Measurement 5 3 4 2
area measurement
Ra = 4.0 µm Rz = 45.4 µm

46. Failure Analysis – Turbine Blade
high wear
R1 = 450 µm
Depth = 70 µm
2D topography profile
Image acquisition: 50x

47. Failure Analysis – Turbine Blade
Section 1
No wear
Section 2
High wear
Section 3
Intermediate wear
Roughness (Ra)
0.7 µm
4.0 µm
1.4 µm
Roughness (RZ)
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

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

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

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. 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. 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. End

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

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
Varity of applications
The ability to measure and characterise a wide range of surfaces is one on the strength of the technique
Along with the mainstream materials applications of roughness and topography come other exotic applications.
Due to the nature of research you can never be sure what's coming next but it will almost certainly be interesting.
Any surface that gives some reflected data can be characterised.
Here are some examples.

57. Foam
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.
Foaming on Coffee for example is one of the goals of coffee makers, more foam and longer lasting is perceived as very important quality factors.
Also food stuffs, chocolate texture and taste come from the surface quality and foam structures.
Heat protecting tumescent paints that are used to protect buildings. They swell up in the event of a fire and give the metal frames a longer resistance to the effects of the heat.
Foams are also widely used in heat exchangers, catalytic convertors and other chemical exchange systems. Also relevant to building and construction materials.
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. Paper & Fabrics
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.
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.
For example ink penetration is used to examine ink quality as well as counterfeited 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

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 SEM could not catch color information.
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. 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
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. 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.
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. PCB Track - Conductive strip
It is easy to see the 3D topography of the sample here.
Data from PCB
It is easy to see the 3D topography of this sample.

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.
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. Measurements
Data relating to the angle and radius can be obtained.
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. Solder Bumps No noise but true colour information in 3D topography
No noise, but true color information in 3D topography
Bump dimension: 13.8 um height and 79 um width
No noise but true colour information in 3D topography
Bump dimension: 13.8 µm height and 79 µm width

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

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

70. Pattern on SiC wafer Grooved pattern on SiC coated with SiO2.
In normal reflection mode distances are measured too short because of the SiO2 coating.
New first peak method and knowledge of refraction index gives correct distances between SiC top surface and grooves.
The data from a scan is converted with knowledge of the Refractive index.

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

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

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
In the Central Laser Facility at Rutherford, part of the Science and Technology Facilities Council.
Components assembled under a microscope

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.
In the Central Laser Facility at Rutherford, part of the Science and Technology Facilities Council.
Components assembled under a microscope

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

78. End
Questions and Answers .