Checking the Biorad system magnification

The Biorad MRC500/600/1024 microscopes have a very high magnification in the detection sys­tem. The fixed system magnification is, according to <a href="#Pawley_95">Pawley 95</a>, 53 &times; m_tube, with m_tube between 1.0 and 1.56 (the fluorescence attachment multiplies with a factor of 1.25 and another factor of 1.25 would be introduced by the 'DIC' attach­ment).

The factor of 53 includes the 8&times; "eyepiece" just below the scan head, but does not include that variability in magnification due to the variations in tube-length that are result from align­ing the system. The total magnification should be confirmed experimentally.

The high system magnification allows you to view the diffraction pattern (Airy Disk) at the pin­hole plane directly by eye. To enable you to verify the correctness of the factor 53 used in the Backprojected Pinhole Calculator for your instrument we outline the way you can derive the system magnification:

In a Biorad MRC600 with a 1.3 Numerical Aperture 60&times; objective, the Airy disk has a diameter of around 2 - 2.5 mm at the pinhole plane. The diameter of the first Airy zero ring is 7.6 lateral Optical Units (o.u.), us­ing the following transformation to express a distance x in dimensionless o.u.,

v = x 2&pi; NA / &lambda;.

In the system above an o.u. is 0.3 ± 0.033 mm. At the specimen plane (Back Projected) a lateral o.u. is in this case around 61 nm. The total magnification appears thus to be 4918, the system magnification 4918 / 60 = 82 ± 9. This value corresponds well with the largest possible system magnification for the MRC600, including the two abovementioned attachments (53 &times; 1.25 &times; 1.25 = 83).

Pawley 95: Pawley, J. B. Handbook of biological confocal microscopy, 2nd ed. 1995. Plenum Press, New York and London. ISBN 0-306-448262. Page 30.