Method development
- 3D imaging at cryogenic temperatures
During the last year we built up a confocal single-molecule setup that is able to cover the temperature range from 1.4–300 K. At the moment, the setup has the ability to perform high-resolution spectroscopy, polarization analysis, 3D imaging and FLIM (fluorescence life time imaging) over the whole temperature range.
The design of our homemade probe-head in combination with a transfer system allows fast sample exchange even at liquid helium temperatures. This type of transfer system is a nice innovation by our group. A similar system was not reported in the literature before. With help of this transfer system it is possible to prepare samples at room temperature and transfer them into the cold cryostat within several seconds. This fast transfer is a prerequisite to suppress unwanted side effects by slow freezing. The most relevant side effects are drying or degeneration of the sample during long cooling times. This is most relevant for all kind of biological samples; there also cold-degeneration can occur during conventional cooling procedures. Those artifacts are suppressed by our approach completely. In practical terms, the transfer system opens the possibility to investigate numerous samples within one cooling cycle and thus it saves time.
This type of innovation was crucial to increase the applicability and the significance of cryogenic application. Especially single-molecule spectroscopy at low temperature deserved such a simplification. We believe, that the low temperature approach in single-molecule spectroscopy has a huge potential that is not used today just due to some technical limitations. One advantage at low temperatures is the long photostability of chromophores. Thus pigment proteins might become observable, which were not accessible for single-molecule techniques up to now, like blue light photoreceptors. The use of imaging/FLIM will give the possibility to image cells in 3D using the autofluorescence of native pigment proteins. The spatial resolved determination of lifetimes and spectra will allow us to decompose the fluorescence emission at each voxel; thereby e.g. the spatial distribution of pigment proteins within intact cells can be reconstructed that is not accessible until now. Our newly finished setup will give us the possibility to get new insight into a world that is almost unexplored nowadays.
Figure 1: Probehead for 3D imaging in the temperature range between 300 -1.4K.
- Data analysis tools for statistical evaluation of single-molecule data
Data organization, visualization, and analysis of data from single-molecule measurements is done by a software package called SMS-Tool with Graphical User Interface (based on Matlab). This software package and its applicability grow continuously according to the actual demands. Screenshots of the GUI is given in Fig. 2.5.
Actually the program features:
Import, export and organization functionality for single molecule data such as rasterscans, spectra, time-resolved spectra, timetraces and TCSPC data. Additional XML interface and database.
- Convenient data formatting tools such as baseline correction, laser line or cosmic impact removal
- Fast browsing through SMS data.
- Real-time processing features include visualization in 2D and 3D, Zoom, data filtering, data smoothing, simple statistics processing and 2D correlation analysis.
- Analysis tools for removal of spectral diffusion processes
- Automated Zero Phonon-Line (ZPL) detection with various tools for statistical analyses (very useful for large data set).
- Principle Component Analysis (PCA), Independent Component Analysis (ICA) of large data sets.
- Live-mode functionality for use during measurement.
Figure 2 Two screen shots of SMS-TOOL. The original data are displayed within the main window shown on the left side (in both screen shots). On the right side additional windows are shown that illustrate some features of the package. In the upper screen additional windows are for browsing of the individual spectra of a time dependent sequence (upper left), the average spectrum of the whole sequence (upper right) is shown, for the 2D-SCS (synchronous correlation spectrum) (lower left) and 2D-ACS (asynchronous correlation spectrum, lower right) are given. A 3D representation of the original data is given in the window positioned in the middle. In the lower screen shot the ability of the package concerning the evaluation of ZPL positions from a large number of measurement is shown. In the original data (left window) spectral regions can be marked (for e.g positions of ZPLs) for different molecules. In the windows on the right the data from all actual analyzed molecules are displayed (here ~150 data-sets). The upper window shows the collected spectral ranges for all data-sets. In the lower window the statistical analysis based on this data as well as the average spectrum from all analyzed molecules is shown. The parameters of the actual data set are displayed by the window shown in the middle.