CHAPTER 1 - SYSTEM OPERATIONLSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS10/2014 V_01 000000-2071-464 5276.20 Polarization ImagingLinear (plane) polarized light, which is light whose wave goes only one direction, exciting a fluorescentmolecule with a preferred dipole orientation results in polarized emitted light. It provides a contrast-enhancing method that is especially useful in the study of molecules that are fixed in their orientation orare greatly restricted in their rotational diffusion. Anisotropy is directly related to polarization and isdefined as the ratio of the polarized light component intensity to the total light intensity.In polarization microscopy using the LSM 710 and LSM 780 systems the sample is irradiated with verticalpolarized light (in respect to the optical table) from a laser source. The emitted fluorescence is passedsequentially through emission polarizers (analyzers) that are housed in the Notch filter cascade and thattransmit either the vertical (IVV) or horizontal (IVH) polarized emitted light onto the Quasar detector (Lformat fluorescence polarization). Since the vertical component of the emission light is parallel polarizedto the vertical polarized excitation light, it is often also referred to as the parallel component (I p, I||).Likewise, the horizontal polarized emission light is also designated as the perpendicular ("senkrecht" inGerman) component (IS, I|). In the Software the "p" and "s" designations are used.Polarisation P and anisotropy r are defined as:spspIIIIP +−= andspspIIIIr ⋅+−= 2 with 0 ≤ r ≤ 1.Hence they can be interconverted to each other in the following way:rrP +⋅= 23 and PPr +⋅= 32 .In a completely polarized sample (IS=0) the anisotropy r=1. In a completely non-polarized sample (Is=Ip )anisotropy r=0.The formulas for polarization P and anisotropy r as given above are strictly true only, if the opticaltransmission for both emission polarizers are identical. Any differences must be corrected by introducinga correction factor G that is multiplied with I p. Hence the anisotropy r in such a case would be calculatedaccording to:spspIGIIGIr ⋅⋅+⋅−= 2G can be measured using horizontally polarized excitation light and is defined asHHHVIIG = .However, since in the LSM 710 and LSM 780 systems the polarization of the excitation light can not bechanged easily from vertical to horizontal, G has to be determined with an isotropic fluorescent dyesolution as the ratio between the mean intensities Ip and Is , e.g. obtained from the histogram view at theimage container.Please note that the G factor is not the mean intensity of the ratio (R) channel, where every pixelis computed seperately. It has to be calculated from the ratio of the mean intrensities of the Ipand Is images.As the formulas implies:Anisotropy r is the preferential display as anisotropy of single species will be simply additive. Note thatthe ZEN Software provides for a formula to display the anisotropy directly in ratio imaging. Images couldalso be computed in the Processing tool`s Calculator.
