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\subsection{Backscattered Electrons}  Backscattered electrons 9BSE) (BSE)  are primary electrons that have been elastically scattered by the nuclei of the atoms in the sample. The energy of BSE ranges from $50eV$ to the energy of primary electron beam $I_0$. The backscattering in ESEM is the analogy of Rutherford backscattering spectroscopy (RBS) and the energy of backscattered electrons $E_{BSE}$ is a function of the deflected angle $\alpha$ and the atomic mass M of the target sample. \begin{equation}  \frac{E_{BSE}}{E_0} = \bigg[ \frac{\sqrt{M^2-m^2sin^2\alpha}+mcos\alpha}{M+m} \bigg]^2  \end{equation} 
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A BSE detector surrounds the ESD with light pipes coated with scintillating materials and is placed directly above the sample at the same distance $d$. When BSE reach the scintillator, the emitted light will be directed through the light pipes for signal process. The arrangement makes the detector collect only those BSE with large deflection angle $\alpha$ and it is due to those large angle scattered electrons that the contrast and signal-to-noise ratio (SNR) for light atoms will be improved.   BSE contrast is exploited to determine the different elements in the sample, especially organic samples where imaging of morphology and distribution of internal organelles is of major interest. However, the organic samples are mostly composed of light atoms such as carbon, oxygen and hydrogen for which the BSE contrast is poor. Heavy atoms such as gold, iodine and manganese are introduced to stain the sample. sample as the fixation or indicator.  For example, potassium permanganate ($KMn0_4$)  was applied to \textit{E.gracilis} samples to enhance the BSE images \cite{Collins_1993}. The permanganate binds readily to the biological membranes and the internal structures of a cell can be clearly imaged. Another useful fixation compound, the osmium tetroxide ($OsO_4$), have a huge tendency to interact with cell lipids and proteins, increasing backscattered signals.