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Useful information, guides, and how-tos on imaging.

Light Microscopy

One of the major advantages provided by confocal microscopy is “optical sectioning” of thick sample, which is achieved by eliminating fluorescence from out-of-focal plane. The apparent thickness of optical sections (depth of focus) in confocal microscopy is a function of both the numerical aperture of the objective and the confocal pinhole aperture size. Smaller aperture, which eliminates more out-of-focal fluorescence, will result in thinner optical sections but dimmer images, which can be compensated by increasing photomultiplier (detector) sensitivity and gain or laser power level. Larger pinhole apertures provide thicker optical sections but less crisp images. This thickness information will be useful to estimate the number of optical sections so that there is no gap between sections upon projection of all the sections. The table below shows the apparent optical section thickness of the objectives in the Leica SP2 systems, which are calculated empirically at given different pinhole aperture sizes.

 

Representative Optical Sections of selected objectives

Objective

Section Thickness (µm) at given pin-hole (AE)

M

NA

0.5 AE

1 AE

2 AE

4 AE

10x

0.4

6.9

7.5

15.5

21.7

20x

0.7

5.6

6.3

7.2

8.2

40x

1.3

1.6

2.0

2.8

5.3

63x

1.4

1.2

1.5

2.6

4.3

Electron Microscopy

Laser Capture Microdissection

Imanging manipulation and analysis

Web links

Fundamentals, advanced knowledge, and applications of light microscopy and digital imaging: Molecular Expressions, Olympus Microscopy Resource Center,  Nikon MicroscopyU