上海交通大学：《Structural and Chemical Characterization of Materi》教学资源_OM4 Confocal Microscopy

Confocal Microscopy http://v.youku.com/v_show/id_XMjMyNzE3NjQ0.html

Confocal Microscopy http://v.youku.com/v_show/id_XMjMyNzE3NjQ0.html

Optical Sectioning and 3D reconstruction V

Optical Sectioning and 3D reconstruction x y z

Optical Sectioning and 3D reconstruction 口

Optical Sectioning and 3D reconstruction x y z

Optical Sectioning and 3D reconstruction 0 一·

Optical Sectioning and 3D reconstruction x y z =

Fluorescence Illumination of a single point Camera Tube lens Excitation light Emission light Objective lens Sample Problem-fluorescence is emitted along entire illuminated cone,not just at focus

Fluorescence Illumination of a single point Sample Objective lens Excitation light Tube lens Emission light Problem – fluorescence is emitted along entire illuminated cone, not just at focus Camera

The confocal microscope Scan excitation spot point- Detector by-point to build up image Pinhole Tube lens Excitation light Emission light Objective lens Sample

The confocal microscope Sample Objective lens Excitation light Tube lens Emission light Pinhole Detector Scan excitation spot point- by-point to build up image

How big should your pinhole be? Want pinhole to pass entire Airy disk X Airy disk diameter ≈1.22元/NA Width of point spread function at pinhole: Airy disk diameter x magnification of lens

How big should your pinhole be? Want pinhole to pass entire Airy disk Airy disk diameter ≈ 1.22   /NA Y X Width of point spread function at pinhole: Airy disk diameter × magnification of lens

How big should your pinhole be? Width of point spread function at pinhole Airy disk diameter x magnification of lens 1 Airy unit =resolution of lens×magnification of lens×2 -100x 1.4 NA:resolution 220nm,so 1 Airy unit 44 um -40x/1.3 NA:resolution 235nm,so 1 Airy unit 19 um -20x/0.75 NA:resolution 407nm,so 1 Airy unit 16 um 10x/0.45 NA:resolution 678nm,so 1 Airy unit 14 um

How big should your pinhole be? • Width of point spread function at pinhole = Airy disk diameter × magnification of lens = 1 Airy unit = resolution of lens × magnification of lens × 2 – 100x / 1.4 NA: resolution = 220nm, so 1 Airy unit = 44  m – 40x / 1.3 NA: resolution = 235nm, so 1 Airy unit = 19  m – 20x / 0.75 NA: resolution = 407nm, so 1 Airy unit = 16  m – 10x / 0.45 NA: resolution = 678nm, so 1 Airy unit = 14  m

Pinhole size ·C1si:30,60,100,150um ·Spinning Disk:50um All are substantially larger than Airy Disk for low magnification lenses. -On spinning disk,can use 1.5x magnification changer

Pinhole size • C1si: 30, 60, 100, 150 m • Spinning Disk: 50 m • All are substantially larger than Airy Disk for low magnification lenses. – On spinning disk, can use 1.5x magnification changer

Light sources Excitation light must be focused to a diffraction limited spot Excitation light Could be done with an arc lamp and pinhole-but very inefficient Enter the laser: Perfectly collimated and Objective lens high power Sample

Light sources Sample Objective lens Excitation light Excitation light must be focused to a diffraction limited spot Could be done with an arc lamp and pinhole – but very inefficient Enter the laser: Perfectly collimated and high power