OUTLINE
7.1 Optical Microscopy 7.2 Confocal Microscopy 7.3 Applications of Confocal Microscopy in Biomedicine
7.1 Optical Microscopy
Three goals:
produce a magnified image of the specimen, separate the details in the image, render the details visible to the human eye or camera.
第七章 共焦显微光学
Chapter 7 Confocal Microscopy Optics
东南大学先进光子学中心 Advanced Photonics Center Southeast University
崔一平 CUI Yiping Cyp@
355
532
3.0
1.0 Ti:Sapphire
2535
188
790 395 790
2.0 2.0 0.8
506 1012 81
Pinhole
Scanning System
Detectors
Resolution and Contrast
Response of A Optical System Point Spread Function (PSF) The properties of the intensity point spread function in the image plane as well as in the axial direction are major factors in determining the resolution of a microscope. Intensity point spread function extends in all three dimensions Lateral components of the intensity distribution: Airy disk.
Image appears on same side of lens as object, cannot be projected onto a screen: virtual image (upright, not inverted).
Light reflected from the rose enters the lens in straight lines, refracted and focused by the lens to produce a virtual image on the retina. Image of the rose magnified: perceive actual size of object to be at infinity; eyes trace light rays back in straight lines to virtual image
Resolution and Contrast
Resolution and Contrast
Order Zero Crossings 3.8 7.0 10.2 Peaks I 100 1.7 0.4
1 2 3
4
13.3
0.2
Resolution and Contrast
Resolution and Contrast
Simple single lens devices that are often hand-held, such as a magnifying glass. Multiple-lens designs with objectives and condensers (compound)
Microscopy: History
Resolution and Contrast
Axial Resolution Widefield Microscope: No optical sectioning capability (a) Confocal Microscope: Optical sectioning capability (b)
advances in fluorochrome stains and monoclonal antibody techniques: explosive growth in the use of fluorescence microscopy in both biomedical analysis and cell biology. optical microscope most important biomedical optic
f< So < 2f
Image formation on Retina
do~25cm
Combination of Lens and Eye
Simple microscope: bi-convex lens, image perceived by eye as if it were at a distance of 10 inches or 25 centimeters (near point)
Human Medulla
Rabbit Muscle Fibers
sunflower pollen grain
Lasers
The far field begins at a distance, z, defined by
(A(0) is the beam diameter at the exit aperture and is the laser wavelength)
Simple Compound
Microscopy: History
Microscopy: History
Microscopy: Importance
Biomedical sciences: overall morphological features of specimens; quantitative tool
Combination of Lens and Eye
MP = doD at l = f; Decrease l or L, increase MP: MP = doD + 1 at l = 0; L= do Increase l or L, decrease MP (D = 1/f); do = near pt~25 cm
In Widefield Microscope Rayleigh criterion for resolution states that two points are resolved when the first minimum (zero crossing) of one Airy disk is aligned with the central maximum of the second Airy disk. The contrast value is 26.4 percent rlateral = 0.6 / NA
Forensic scientists: hairs, fibers, clothing, blood stains, bullets, and other items associated with crimes
Simple Magnification
So >>>2f
So > 2f
So = 2f
Benefits of Confocal Microscopy
Reduced blurring of the image from light scattering Increased effective resolution Improved signal to noise ratio Z-axis scanning, Depth perception in Zsectioned images Magnification can be adjusted electronically
Resolution and Contrast
In confocal configurations Pointwise Illumination Scanning + Pointwise Detection PSFconf.=PSFillum.*PSFdetc. ~70%*PSFwidefield Therefore, rlateral = 0.4 / NA

Explosive growth in physical and materials sciences; semiconductor industry,

observe surface features of high-tech materials and integrated circuits

Control depth of field Elimination or reduction of background information away from the focal plane Capability to collect serial optical sections from thick specimens
z = A02/
Wavelength(nm)
Beam Diameter(mm) Argon-Ion
Far Field Distance(cm)
488 514
0.6 1.0 Helium-Neon
74 195
543
594 612 632
0.4
0.7 0.7 0.7 Nd:YAG
30
Confocal Microscopy