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

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

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.

Introduction of confocal microscope Point light Parallel light

Introduction of confocal microscope Spatial resolution

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

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

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

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, 08026 (2008).

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. http://www.olympus.com

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

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.

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

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).

Ti:sapphire brightness Halogen Köhler: 63.66 (nW/ μm2· Ω) Ti:sapphire: 6.333 (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).