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NOTE: THIS FOLLOWS A PIECE ABOUT THE THEORY OF CONFOCAL, IN WHICH THE AIRY DISC ETC IS DISCUSSED.

Choosing pinhole diameter

Why do we have to choose?

In any confocal system, you need to know how to set pinhole diameter, since

contrast and resolution improve as pinhole diameter decreases

but the amount of light getting to the detector decreases. This leads to noisy images due to insufficiency of photons. This can be overcome by averaging more scans or by increasing laser power. Either one increases photo bleaching, or irreversible damage to the sample.

A judicious compromise choice is thus always necessary.

A third reason is specific to the Leica-TCS-NT:

The effect of a given pinhole diameter varies with the wavelength of emitted light. Each detector in the Leica has its own pinhole. To have the same confocality in all three colors, the three pinholes must be set to different diameters according to simple rules given below. For most routine work, this is not a major concern, and a single pinhole size will do.

 

How to relate pinhole diameter to optical parameters

The size of an Airy disc in the specimen plane depends on wavelength and the numerical aperture of the objective lens. It does not depend on the magnification of the lens.

The image of the Airy disc at the pinhole (image plane) is larger than the disc itself due to the magnification of the objective lens plus any subsequent optics.

 

To facilitate calculation, it is best to use optical units (o.u.)

in the specimen plane

for light emitted by FITC (centered on 530 nm, or 0.530 m m) and a lens of N.A. = 1.4, 1 optical unit = 0.060 m m.

the image of 1 optical unit at the pinhole (image plane) is larger than this by the total magnification of the system: In the Leica, Mtotal = Mobjective x Mdetection path = 4.5Mobjective (the factor 4.5 applies only to the Leica)

 

Airy disc size can be expressed in optical units.

 

 

 

 

 

 

 

 

A pinhole centered on an in-focus Airy disc will admit the following %'s of light from the disc:

   

Pinhole Diameter

(Optical Units)

% light admitted

3.8

 

5

 

7.6

 

 

The Leica expresses pinhole diameter in microns. To relate pinhole diameter to the Airy disc for your lens, multiply

 

the number of optical units of disc you want to capture

x

the size of 1 optical unit

x

total magnification

 

i. e.,

 

To capture XX% of the light emitted at 535 nm by an in-focus diffraction-limited source (Airy disc) using a 100x, N.A. 1.4 objective lens, the pinhole diameter should be 208 m m (7.6 optical units).

 

As a general rule, a pinhole diameter of 50-60% of this number (corresponding to the central 3.8 optical units) is recommended, but you should feel free to experiment, most likely toward smaller diameters.

 

Typical pinhole sizes in the Leica according to the recommendation:

 

 

 

Recommended Pinhole Diameter

Lens

530 nm (FITC)

600 nm (T. Red)

700 nm (CY5)

       
10x, N.A. 0.3

48 mm

54 mm

63 mm

40x, N.A. 1.25

46 mm

52 mm

61 mm

100x, N.A. 1.4

103 mm

117 mm

136 mm


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