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

CONFOCAL MICROSCOPY IN A NEW LIGHT

Title: Introduction to Confocal MicroscopyPresented by: Dr. Andrew Dixon Date: May 2009

Introduction to Confocal Microscopy

3

An Introduction to Confocal An Introduction to Confocal MicroscopyMicroscopyAn Introduction to Confocal An Introduction to Confocal MicroscopyMicroscopy

• 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?What is the Problem?What is the Problem?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-FocusAll-in-FocusAll-in-FocusAll-in-Focus

True color information in 3D topographyBump dimension: 13.8 µm height and 79 µm width

6

Marvin Minsky’s IdeaMarvin Minsky’s Idea Marvin Minsky’s IdeaMarvin 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 PrincipleThe Confocal PrincipleThe Confocal PrincipleThe 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 SectionThe Optical SectionThe Optical SectionThe Optical Section

Optical section ‘thickness’ depends on objective lens NA. Lateral and axial resolution are related.

0

2

4

6

8

10

12

14

16

18

0 0.5 1 1.5

lateral resolution - micron

op

tica

l sec

tio

n -

mic

ron

9

The Power of Confocal ImagingThe Power of Confocal ImagingThe Power of Confocal ImagingThe Power of Confocal Imaging

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.

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

Increasing Imaging SpeedIncreasing Imaging SpeedIncreasing Imaging SpeedIncreasing 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

From Optical Section to 3-D imageFrom Optical Section to 3-D imageFrom Optical Section to 3-D imageFrom 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 PossibilitiesA World of Possibilities……A World of PossibilitiesA 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 Research

Material Sciences

13

In Conclusion…In Conclusion…In Conclusion…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

EndEndEndEnd

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

Advanced Confocal Microscopy: Axio CSM 700

16

Axio CSM 700 – System Axio CSM 700 – System OverviewOverviewAxio CSM 700 – System Axio CSM 700 – System OverviewOverview

Xe illuminator, conf. microscope, controller, user PC

17

Innovative Confocal MethodInnovative Confocal MethodInnovative Confocal MethodInnovative Confocal Method

1 Xe illuminator

2 Multi slit grid for scanning

instead of scan mirrors

3 Beam splitter

4 Sample / focal plane

5 Digital detector which also

provides digital confocal

apertures

1

2

3

4

5

18

3D Image Acquisition3D Image Acquisition3D Image Acquisition3D 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

AdvantagesAdvantagesAdvantagesAdvantages

• High acquisition speed (up to > 100 fps)

• True colour confocal microscopy

• High resolution

Optical 3D profilometer

20

Optical resolution: XY Optical resolution: XY Optical resolution: XY Optical resolution: XY

No resolutionNo contrast

x,y

I

21


Cut-off distance reached,but contrast is equal to zero


x,y

I

22



Maximum resolutionRayleigh criterium

d(x,y) ~ f * / NA

f= 0.37 … 0.61


x,y

I

Strictlyconfocal

Classical

23



ResolutionMaximum contrast

Maximum resolutionRayleigh criterium

d(x,y) ~ f * / NA

f= 0.37 … 0.61


x,y

I

Strictlyconfocal

Classical

24

Lateral Resolution Limit: Grid Lateral Resolution Limit: Grid Lateral Resolution Limit: Grid Lateral Resolution Limit: Grid Sample: Nanoscale critical dimension standards (supracon AG Jena) Colour channel: blueObjective: Epiplan-APOCHROMAT 150x/0.95

200 nm L&S 150 nm L&SResolution limit

25

Test Sample:Validated depth measurement sample with 80 nm steps

Objective: EC Epiplan-APOCHROMAT 100x/0.95

True height: 80 nmMeasured height: 87 nmDifference: 7 nm

Axial Detection LimitAxial Detection Limit Axial Detection LimitAxial Detection Limit

26

High Range of SamplesHigh Range of SamplesHigh Range of SamplesHigh 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 Comparison of confocal microscopymicroscopyComparison of confocal Comparison of confocal microscopymicroscopy

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

Axio CSM 700 …Axio CSM 700 …Axio CSM 700 …Axio CSM 700 …

… opening up

new worlds of microanalysis.

Title: Applications for Topographic Measurements in Materials EngineeringPresented by: Ralf Loeffler Date: May 2009

Applications for Topographic Measurements in Materials Engineering

30

Application ExamplesApplication ExamplesApplication ExamplesApplication Examples

• Geometry inspection on cutting plate.• Failure analysis on turbine blade.• Tribology on high performance steel.

31

Geometry Inspection – Cutting PlateGeometry Inspection – Cutting PlateGeometry Inspection – Cutting PlateGeometry 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

top view


Functional parameters influencing performance: angle and radius of cutting edge

side view

3D-µCT surface rendering

resolution: 10 µm/vx

33

rake

honing 1 (r1)

honing 2 (r2)

toolflank

chamfer


Definition of functional parameters

rake

honing 1 (r1)

honing 2 (r2)

chamfer

tool flank

34


rakechamf

er

cutting

edge

r1

r2

focus image

rake angle

35


only one cutting edge radius (honing) approx 200 µm

rake

cutting

edge

wear groove

height: approx. 11 µm

width approx. 49 µm

36


Quantitative Measurement: new plate

Roughness - along cutting edgeRz = 2.2 µm Ra=0.3 µm

37

Roughness - along cutting edge


Quantitative Measurement: worn plate

Rz = 10.9 µm Ra=1.0 µm

38

New cutting plate Worn cutting plate

Roughness (Ra) (along cutting edge)

• 0.3 µm • 1.0 µm

Roughness (RZ)(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 Geometry Inspection – Cutting PlatePlate

39


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 BladeFailure Analysis – Turbine BladeFailure Analysis – Turbine BladeFailure 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 inhot vapour atmosphere

41


Top view: sections with distinct surface wear

no wear intermediate wear

high wear

section 1 section 2 section 3

section 1 section 2 section 3

42


Ra = 0.7 µm Rz = 27.3 µm

Note milling marks

no wear

Roughness Measurement

topography profile

area measurement

43


Note wear and milling marks

Ra = 1.4 µm Rz = 21.7µm

intermediate wear


topography profile

area measurement

44


area measurement

Ra = 2.7 µm Rz = 109.4 µm

Note deep wear marks

high wear


R1= 630 µmDepth = 65 µm

45


R1 = 550 µm

Depth = 65 µm

R1 = 530 µm

Depth = 65 µm

Note aligned wear marks

high wear

area measurement

Ra = 4.0 µm Rz = 45.4 µm


12 3 4 5

2 3 4 5

1 2 3 4 5

1 3 4 5

46

Failure Analysis Failure Analysis –– Turbine Blade Turbine BladeFailure Analysis Failure Analysis –– Turbine Blade Turbine Blade

R1 = 450 µm

Depth = 70 µm

Image acquisition: 50x

high wear

2D topography profile

47

Section 1 No wear

Section 2High 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

Failure Analysis Failure Analysis –– Turbine Blade Turbine Blade

48


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 CompositesTribology – Maraging Steel CompositesTribology – Maraging Steel CompositesTribology – 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


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


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


Axio CSM 700 analysis with all-focus image

• Excellent visualization

• Wear mechanism: only by leveling ceramic particles

• Axio CSM 700 data in accordancewith SEM micrograph observation

• Scanning of entire wear markat high magnification

width = 300 µm

height ≤ 1 µm

53


Axio CSM 700 vs. White Light InterferometerIdentical 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

EndEndEndEnd

Title: Exotic Applications in Confocal MicroscopyPresented by: Dr. Steve MetcalfeDate: May 2009

Exotic Applications in Confocal Microscopy

56

Example ApplicationsExample ApplicationsExample ApplicationsExample 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

FoamFoamFoamFoam

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 & FabricsPaper & FabricsPaper & FabricsPaper & 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

Ink on PaperInk on PaperInk on PaperInk 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

CCD with Bayer Mask SensorCCD with Bayer Mask SensorCCD with Bayer Mask SensorCCD 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

Polymer Film on Metal Polymer Film on Metal SubstrateSubstratePolymer Film on Metal Polymer Film on Metal SubstrateSubstrate

•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 stripPCB Track - Conductive stripPCB Track - Conductive stripPCB Track - Conductive strip

It is easy to see the 3D topography of this sample.

63

Rendered PCB TrackRendered PCB TrackRendered PCB TrackRendered 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

MeasurementsMeasurementsMeasurementsMeasurements

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 BumpsSolder BumpsSolder BumpsSolder Bumps

No noise but true colour information in 3D topography

Bump dimension: 13.8 µm height and 79 µm width

66

TFT Spacer – Touch Panel SpacerTFT Spacer – Touch Panel SpacerTFT Spacer – Touch Panel SpacerTFT 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

Photo VoltaicPhoto VoltaicPhoto VoltaicPhoto 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

Laser Scribes on Thin Film Solar CellsLaser Scribes on Thin Film Solar CellsLaser Scribes on Thin Film Solar CellsLaser 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

Photo Voltaic at 100 x ObjectivePhoto Voltaic at 100 x ObjectivePhoto Voltaic at 100 x ObjectivePhoto Voltaic at 100 x Objective

Full colour data is available at high magnifications

70

Pattern on SiC waferPattern on SiC waferPattern on SiC waferPattern 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.

71

Metal Mold for Light Guide PanelMetal Mold for Light Guide PanelMetal Mold for Light Guide PanelMetal Mold for Light Guide Panel

These complex surfaces can be visualised readily with confocal techniques.

72

Micro Lens ArrayMicro Lens ArrayMicro Lens ArrayMicro 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

Fresnel lensFresnel lensFresnel lensFresnel 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

Topography of cosmetics materialsTopography of cosmetics materialsTopography of cosmetics materialsTopography 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

Precision Assembly & Precision Assembly & Manufacture Manufacture Precision Assembly & Precision Assembly & Manufacture 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

Precision Assembly & Precision Assembly & ManufactureManufacturePrecision Assembly & Precision Assembly & ManufactureManufacture

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

Acknowledgements Acknowledgements Acknowledgements Acknowledgements • Wikipedia for historic information.

• Chris Spindloe STFC Rutherford for his help with the Laser Target images.Last 2 slides

78

End End End End

Questions and Answers

Documents

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