1. Reporter: Chien-Chung Tsai
Topic report
Confocal reflective/fluorescence microscope
Reporter: Chien-Chung Tsai
Adviser: Prof. Sheng-Lung Huang
2012/07/26

2. Outline Introduction of confocal microscope
Working principle of confocal microscopes
---Confocal reflective microscope
---Confocal fluorescent microscope
Relative high-end confocal techniques
Lab’s confocal system and its applications
Conclusions

3. Introduction of confocal microscope
Basic concept
In 1957, Marvin Minsky paterned the original confocal model for wide-field fluorescent microscope as shown in left figure. Right figure is the fundamental illustration for confocal point scanning scheme.

4. Introduction of confocal microscope
Point light
Parallel light

5. Introduction of confocal microscope
Spatial resolution

6. Introduction of confocal microscope
3D imaging type
Golvano mirror scanning
PZT oscillation scanning 1
Exit pinhole
Entrance pinhole
Collimator
Focal lens
Beamsplitter
Golvano mirror pair
Close-loop z-axial PZT
Color-corrected telecentric objective lens
Sample
Stage 1
Exit pinhole
Entrance pinhole
Collimator
Focal lens
Beamsplitter
Open-loop z-axial PZT
Objective lens
Sample
2-axes linear stage

7. Working principle of confocal microscope
Confocal reflective microscope
Note: Confocal reflective microscope can detect the surface reflectance of the testing sample. Assume the sample surface is flat, the refractive index profile of the surface on the sample is determined in place of the reflective intensity from designate wavelength coherent light source. Besides, the axial and transversal resolutions can be improved by means of utilizing high NA objective lens and small diameter exit pinhole. Photo diode after the beamsplitter has the function to immediately normalize the power level in order to improve the intensity stability, to push up the refractive index accuracy.
Coherent light source
Collimator
Objective lens
Sample
Photo diode
Exit pinhole
Focal lens
Beamsplitter

8. Working principle of confocal microscope
Confocal fluorescent microscope
Note: The difference between confocal fluorescent and confocal reflective microscopes is the sensing light emerging into the exit pinhole. As the beamsplitter is replaced to the dichroic mirror, the excited fluorescent light from the sample is collected by the objective lens passing through the dichroic mirror, and then is focused on to APD/PMT.
Coherent light source
Collimator
Objective lens
Sample with fluorescence
APD/PMT
Exit pinhole
Focal lens
Dichroic mirror

9. Relative high-end confocal techniques
Spectral confocal reflection microscopy
(a)
(b)
M. J. Booth, R. Juškaitis, and T. Wilson, “Spectral confocal reflection microscopy using a white light source,” J. Eu. Opt. Soc. – Rapid Publication 3, (2008).

10. Relative high-end confocal techniques
Laser scanning confocal fluorescent microscopy
Lodgepole pine (Pinus contorta) pollen grain optical sections. Bulk pollen was mounted in CytoSeal 60 and imaged with a 100x oil immersion objective (no zoom) in 1 micrometer axial steps. Each image in the sequence (1-12) represents the view obtained from steps of 3 micrometers.

11. Lab’s confocal system 2D CCD Eye piece Slit mirror 4 Tube lens
Collimator 3
Collimator 2
Collimator 1
Collimator 4
Mirror 1
Focal lens
objective lens
50X
20X
10X
Sample
3-axial manual stage
Filter set
Tube lens
Power monitor
Turret
Slit mirror 4
2D CCD
Eye piece
Slit mirror 2
Slit mirror 1
Slit mirror 3
Beam splitter
532 nm
635 nm
1024 nm
780 nm
2-axial automatic stage

12. Application of Lab’s confocal system
Doping concentration of crystal fiber
Surface reflectance measurement for refractive index profile
Surface roughness evaluation.
Crystal fiber mode number evaluation.
SHG and THG en-face imaging.

13. Conclusions
Generally speaking, confocal microscope is a functional microscope.
Confocal microscope can eliminate the backgroung noise to push up the image contrast ratio when compared with wide-field optical microscope.
As fluorescent light is strong, PMT/APD can be replaced to photo diode to cost down the price.
Laser scanning confocal microscope is suitable for time-lapse biological image.
Full-field OCT still has high competitive value when compared with confocal microscope

14. Ti:sapphire brightness
A. Dubois, K. Grieve, G. Moneron, R. Lecaque, L. abre, and C. Boccara, “Ultrahigh-resolution full-field optical coherence tomography,” Appl. Opt. 43, 2874 (2004).

15. Ti:sapphire brightness
Halogen Köhler: (nW/ μm2· Ω)
Ti:sapphire: (μW/ μm2· Ω)
A. Dubois, K. Grieve, G. Moneron, R. Lecaque, L. abre, and C. Boccara, “Ultrahigh-resolution full-field optical coherence tomography,” Appl. Opt. 43, 2874 (2004).