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\rhead{Lewis Guignard EE230  Page \thepage}
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\date{\today}
\title{EE230 }
\author{Lewis Guignard}

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{\fontsize{1cm}{1 em}\selectfont EE230 }

\section{Introduction}

The objective of this lab exercise is to gain proficiency using and understanding of fluorescence microscopy, and the usage of both excitation and emission filters therein.  We set up a fluorescence microscope apparatus and view different slides implementing different fluorophores, through two different filters to gain insight into the usage and benefits of this technique.

\section{Methods}

Viewing was performed with an HBO Mercury lamp source.  This source is 10-100x brighter than a standard incandescent source, giving powers needed for fluorescent microscopy.  The lamp is bright enough to eventually bleach the sample, so when not in use, lamp was blocked from lighting the sample.  For samples, we used a test slide from FocalCheck, containing several samples of 2.5 $\mu$m beads, which emitted light in several wavelength bands.  We also observed a sample of cells, different parts of which had applied different fluorophore types.
\newline

{\fontsize{0.5cm}{1.2em}\selectfont Procedure}
\begin{enumerate}
	\item Kohler illumination calibration.  This was performed in a slightly different manner from bright-field microscopy, as we were working in reflection mode at this point, and with a different source.  The fine Kohler tuning is as follows:
	\begin{enumerate}
		\item Align light path: remove an objective plug using gloves, and rotate this opening so light will travel to sample.
		\item Put a piece of paper on the sample stage, so the light can be seen at that point.  At this point the HBO source can be turned on.
		\item Bulb is on, so maximum brightness doesn't have to be waited on.  Elsewise, turn bulb on and allow to warm up.
		\item  Use adjustment screws on the bulb houseing for $x,y$ positioning, as well as focusing screw.  Bulb to the paper sample orientation is optimized by adjusting these three parameters, until the bulb is centered on its brightest point.
		\item at this point the aperture and filter stops are brought back down to proper levels: aperture such that the most light is allowed in, and filter such that the light cone is just larger than the aperture viewed at the piece of paper.
		\item We then replace the cap on the objective opening, and the microscope is ready for use
	\end{enumerate}
	\item At this point, observation of samples began.  We first looked at each filter cube without a sample in place, and recorded what color we was observed. (see Table~\ref{tab:block_color})
	\item Next was to observe the fluorescent microsphere sample, which contained five sections, each having beads of the same size, but with a different fluorophore attached.  We then recorded how 'bright' we thought each slide was under each block's filtration (See Table ~\ref{tab:block_intensity} and Figure ~\ref{fig:Spectrum}).
	\item Immediately after taking the data of Tables ~\ref{tab:block_color} and ~\ref{tab:block_intensity}, the samples were returned to their light proof container, so that they would not become photo-bleached over time.
	\item Lastly, we loaded a biological sample of cells onto the stage, and looked under different filters.  The sample contained three different fluorophores attached to three different parts of the cell.  The objective was to match the proper fluorophore to the cell part it had been attached to by how bright it was in each filter block, see Table ~\ref{tab:Cell_parts}.  We further took photos of the cell under both blocks of light, and then superimposed the images, to gain as much information as possible at once (see Figure ~\ref{fig:CameraPhone})
	\item before returning the cell sample to its light-proof container, we attempted to view it through brightfield, and noted any distinction between parts was nearly impossible, proving the value of fluorescence microscopy.
		
\end{enumerate}

\newpage

\section{Results}

	Part II. Observations of Fluorescence:
	We noted block color (excitation filter) as per Table ~\ref{tab:block_color}. As there is only one light source, the the color of the emitted light is the \textbf{pass band of the block filter}. 
	 
	\begin{table}[h]
	  \centering
	  \begin{tabular}{ c  c  c }\\ \hline
	                   & \multicolumn{1}{c}{Cube 1} & \multicolumn{1}{c}{Cube 2}\\
	            Color  & Green & Red\\    
	        \hline
	  \end{tabular}
	  \caption{Color of each filter block (excitation filter)
	     \label{tab:block_color}}
	\end{table}
	
	Visibility of each set of beads was defined in one of three options: Not visible (N), visible (Y), or faintly visible (faint).  Further definition was decided ambiguous between observers.
	
	\begin{table}[h]
		\centering
		\begin{tabular}{ c  c  c c c c}\\ \hline
		   Visible?    & 1 & 2 & 3 & 4 & 5\\
		   Block 1     & N & faint & Y & Y & Y \\
		   Block 2     & faint & Y & Y & faint & faint \\    
		   \hline
		\end{tabular}
		\caption{visibility of beads under different blocks.  Color is defined in Table ~\ref{tab:block_color}  )
		\label{tab:block_intensity}}
	\end{table}
	
	\begin{figure}[h]
	  \centering
	  \includegraphics[width=10cm]{Spectrum}
	  \caption{Subjective intensity measurements of each fluorophore sample under each filter block - giving a spectral response.}
	  \label{fig:Spectrum}
	\end{figure}

	\begin{table}[h]
	  \centering
	  \begin{tabular}{ c  c  c c}\\ \hline
  		\textbf{Fluorophore} & \textbf{excitation wavelength ($\lambda$)} & \textbf{emission wavelength ($\lambda$)} & \textbf{Cell Part} \\
  		Alexa Fluor\textregistered 488 phalloidin & 495 & 518 & F-actin \\
  		DAPI & 358 & 461 & Nuclei \\
  		MitoTracker\textregistered Red CMXRos & 579 & 599 & Mitochondria \\
	        \hline
	  \end{tabular}
	  \caption{Mapping cell part to fluorophore}
	     \label{tab:Cell_parts}
	\end{table}	

	\begin{figure}[h]
	  \centering
	  \includegraphics[width=10cm]{BiFluorescentImage}
	  \caption{Two superimposed photos of the red-block and green-block images of the biological cell sample.}
	  \label{fig:CameraPhone}
	\end{figure}	
	
	
\newpage

\section{Conclusion}

In conclusion, we note the validity of the fluorescence technique in creating enough contrast to see objects that cannot normally be seen under bright-field microscopy.  The excitation filters are helpful to only allow the light of interest onto the sample, limiting photo-bleaching of the rest of the high-intensity light, and bringing out more contrast from the fluorophore.  Post - or emission filtering gives even higher contrast.  A careful choosing of which excitation, and emission filters to use will give a good contrast for a given fluorophore, as can be seen in Figure ~\ref{fig:CameraPhone}.  


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