Temperature controlled FCS measurements with a thermally decoupled sample holder using a Solid Immersion Lens
Rao R., Serov A., Sidler T., Lopez A., Gösch M., Rigler R., Lasser T.
We present a temperature controlled sample holder, which is thermally decoupled from the microscope body and objective. The sample holder is specifically designed for measurements performed at the single molecule level, e.g. Fluorescent Correlation Spectroscopy (FCS) experiments. The thermal decoupling is achieved by using a containing the Solid Immersion Lens (SIL) in combination with a low NA air objective. This system allows us a high NA (>1.2) optical performance as needed for FCS experiments. The SIL and the temperature control is conveniently integrated into the sample plate which fits into the conventional sample stage of an existing Zeiss ConfoCor microscope. This sample holder was used with Rhodamine Green molecules in a concentration of 10nM, excited with an Ar+ laser at 488nm. Essential experimental parameters such as count rate per molecule (CPM), diffusion time and molecule concentration are investigated. The SIL-system performance is compared to the performance of a high NA water immersion objective.
With the SIL based detection system, measurements on temperature controlled biological and chemical samples become feasible. Measurements on small sample volumes are not affected by the heat gradient, which is present when heating is applied to samples that are directly in contact with enormous heat reservoirs like the microscope body. The presented SIL based set-up is intended to be used for biological and chemical studies, where accurate control and variation of the sample-temperature is critical. Serov A., Rao R., Gösch M., Anhut T., Martin D., Brunner R., Rigler R., Lasser T., Sub-wavelength resolution for fluorescent correlation spectroscopy using a Solid Immersion Lens, Proc. SPIE 5486 (2004)