\subsection{Temperature control}
\label{sec.temp_control}

A temperature control apparatus for the AFM should be capable of maintaining biologically relevant temperatures (0{\dg}C to 50{\dg}C).
For most proteins, this range is sufficient to measure the energy landscape roughness.
The controller must not introduce much mechanical noise or thermal drift into the system.
With our commercial AFM, we have the added constraint that the
temperature module must have a small size to fit inside the
MultiMode head.

A temperature module satisfying these conditions was constructed around a Peltier thermoelectric heater/cooler (see Figure~\ref{fig.temp_control}).
\begin{figure}[bp]
 \centering
 \hspace{\stretch{1}}
 \sbox{\tempbox}{\includegraphics{mp/temp_control.1}}
 \subfloat[Control module.]{\label{fig.temp_control}\usebox{\tempbox}}
 \hspace{\stretch{1}}
 \subfloat[Peltier.]{\label{fig.peltier}\raisebox{0.5\ht\tempbox-0.5\height}[0pt][0pt]{\includegraphics{mp/temp_control.2}}}
 \hspace{\stretch{1}}
 \caption{(a) Our temperature control module.
    The surface $a$ is cooled or heated by one side of the Peltier thermoelectric device $b$.
    The other side of the Peltier is kept near room temperature using the  brass heat sink $c$.
    The heat sink itself can be anchored to room temperature by gravity-fed water flowing through a U-shaped channel $d$ to $e$.
    The module is mounted directly on our piezo scanner $f$.
    (b) A Peltier operating in heating mode.  Regions of P- and N-type semiconductor connect hot and cold conducting plates.
    %The working principle is explained in the text.
    }
\end{figure}
A Peltier functions by applying a voltage to regions of P- and N-type semiconductor in series (Fig.~\ref{fig.peltier}).
%Conduction in N-type semiconductors is mainly through thermally excited electrons and in P-type semiconductors is mainly through thermally excited holes.
Applying a positive voltage as shown in Figure~\ref{fig.peltier} heats the sample.
% by constantly pumping hot conductors in both semiconductors towards surface, which heats the surface.
Reversing the applied voltage cools the surface.
% by pumping hot conductors away.
A commercial thermoelectric controller (model MTCA 6040, from Melcor, Trenton, NJ) monitors the sample temperature with a thermocouple and powers the Peltier as required to maintain a set temperature.

The characteristics of the completed device are very encouraging\cite{yang06}.
Constant sample temperatures can be maintained over the entire cooling range as shown in Figure~\ref{fig.temp_stability}.
The system also responds relatively quickly to even large changes in temperature setpoint as demonstrated in Figure~\ref{fig.temp_response}.
% (The timescale of a single bond-and-unfold sequence is $\sim$10 seconds).
In order to set an upper limit on the vibrational noise due to the addition of the temperature control module, we imaged sputtered gold at our cold temperature extreme of 5{\dg}C and also under normal conditions at 25{\dg}C.
As shown in Figure~\ref{fig.resolution}, we see nanometer sized features in both images, which puts an acceptable upper bound on noise in the entire apparatus.
% there is no noticable change in the resolution of the images, indicating that the system does not introduce significant temperature dependent noise.

\begin{figure}[btp]
 \centering
 \hspace{\stretch{1}}
 \sbox{\tempbox}{\includegraphics[width=0.3\textwidth]{yang_temp_stability}}
 \subfloat[Temperature stability.]{\label{fig.temp_stability}\usebox{\tempbox}}
 \hspace{\stretch{1}}
 \subfloat[Temperature response.]{\label{fig.temp_response}\includegraphics[height=\ht\tempbox]{yang_temp_response}}
 \hspace{\stretch{1}}
 \subfloat[Resolution.]{\label{fig.resolution}\raisebox{0.5\ht\tempbox-0.5\height}[0pt][0pt]{\includegraphics[width=0.3\textwidth]{yang_resolution}}}
 \hspace{\stretch{1}}
 \caption{Performance of the temperature control system\cite{yang06}.
          (a) The stability of the sample temperature for setpoints of 5, 15, and 25{\dg}C.
	  The temperature stabilized at 5.1, 14.9, and 25.1{\dg}C respectively, with fluctuations of less than 0.1{\dg}C.
	  (b) The rate of temperature response to a setpoint change from 25 to 5{\dg}C.
	  (c) Images of sputtered gold at (c.a) 5{\dg}C and (c.b) 25{\dg}C.
	  The images are from different regions of the sample and were acquired in pure water in the tapping mode.  Both images have a size of 500 x 500 nm$^2$.
}
 \label{fig.temp_perform}
\end{figure}

In conclusion, the temperature control system satisfies all the requirements necessary to allow temperature dependent unfolding experiments.
In Section~\ref{sec.ubiquitin} we present some Ubiquitin unfolding results taken with this system.
