Huygens Professional User Guide

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$Medium Refractive index Disolved in 0.25% PPD, 0.0025% DABCO ...$

d2rd 2=πr 2 dand thus r = ------π(EQ 4)For a square pinhole c shape = 1 ⁄ π = 0.564. For circular pinholes c shape = 0.5 to just convertfrom diameter to radius.Airy disk as unit forthe backprojectedradius of a square pinholeThe relation between the edge size of square pinhole in Airy disk units and the backprojectedradius is a combination of (EQ 2) and (EQ 4). Taking into account that (EQ 2) already convertsa diameter into a radius we get:r b=0.69λ ex N----------------------------------------- AirydisksNA(EQ 5)with N Airydisks the number of Airydisks.Computing the backprojected pinhole distance in Nipkow spinning disksAs is the case for the backprojected pinhole diameter, the distances between the pinholes inspinning disks must be divided by the system magnification. For the most used Yokogawa, forexample, the pinhole distance is 2.5 µ. You can check this by stopping the disk. So with an100x lens the backprojected distance is about 2.5 µ.Pinhole radius tablesIn the case of the Leica TCS4d, the Biorad MRC500/600, the Zeiss LSM310 and the ZeissLSM410 type microscopes the pinhole geometry and system magnification is known resultingin the conversion formulas from the following tables:Leica confocal microscopesTCS 4D, SP1, NTThe size of the square pinhole is given as an 8-bit number which maps to the physical pinholeradius given in the following table:TABLE 1. Leica TCS4d pinhole parametersTCS4d Range begin Range endReported parameter (p 8 ) 0 255Size (micron) 20 630 (earlierreported as 500)Pinhole geometrySystem magnification 4.5squareBackprojected pinhole 295 p 8 + 2508radius (nm) -------------------------------- (EQ 6)If the pinhole is specified in Airy disk units, see “Airy disk as unit for the backprojected radiusof a square pinhole” for information on how to convert to a backprojected radius.m obj24 The Huygens Professional User Guide
TCS-SP2The Leica TCS-SP2 has a system magnification of 3.6. However, the size of its square pinholeis usually specified in Airy disk units making it independent of the actual overall magnification.It is dependent though on wavelength and NA of the objective. See “Airy disk as unit forthe backprojected radius of a square pinhole” for information on how to convert to a backprojectedradius.Zeiss confocal microscopesTABLE 2. Zeiss LSM310 (upright) pinhole parametersZeiss LSM310 upright Range begin Range endReported parameter (p 8 ) 0 255Size (micron) 0 500Pinhole geometry squareSystem magnification 1.11Backprojected pinhole996 ⋅ pradius (nm) ------------------ 8(EQ 7)m objTABLE 3. Zeiss LSM410 (inverted) pinhole parametersZeiss LSM410 inverted Range begin Range endReported parameter (p 8 ) 0 255Size (micron) 0 1000Pinhole geometry squareSystem magnification 2.23Backprojected pinhole992 ⋅ pradius (nm) ------------------ 8(EQ 8)TABLE 4. Zeiss LSM510 pinhole parametersm objZeiss LSM510Size (p,diameter,micron)Backprojected pinholep ⋅ 1000radius (nm) ------------------------6.66 ⋅ m(EQ 9)objOlympus confocalmicroscopesTABLE 5. Olympus FluoviewOlympus FluoviewReported parameter 1 2 3 4 5Size (p, diameter, micron) 0 100 150 200 300Pinhole geometry circular?System magnification 3.426Backprojected pinholeradius (nm) --------------------p6.85m obj(EQ 10)The Huygens Professional User Guide 25
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Page 8 and 9: Installation and system requirement
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Page 12 and 13: Deconvolving my first imageFigure 2
Page 14 and 15: Deconvolving my first imageHistogra
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Page 18 and 19: The easy way• Press the Run butto
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Page 30 and 31: Biorad confocal microscopesTABLE 6.
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