TCSPC (Time-Correlated Single-Photon Counting) FLIM data with megapixel resolution can be recorded by using bh TCSPC modules in combination with new 64 bit data acquisition software. The large memory space available in the 64 bit environment allows new FLIM procedures to be used. We demonstrate the performance for applications that require imaging of a large number of cells in a single field of view, for multi-wavelength FLIM, for spatial mosaic imaging, and for recording transient changes in the fluorescence decay after a stimulation of the sample. Image quality was further improved by integrating a parallel counter channel that bypasses the timing electronics of the TCSPC module. Photon numbers from this counter are not affected by dead-time effects. Lifetime images are built up by using intensity data from the parallel counter and fluorescence decay data from the TCSPC electronics.
Lately quite a plethora of concepts have been successfully developed, which take resolution beyond the classical limits
of a light microscope. Among these structured illumination microscopy (SIM) and photo activated localization
microscopy (PALM) hold the promise to provide biologists with unprecedented insights into sub-cellular
organizations. A combination of these methods seems particularly attractive as it allows adapting to the required
resolution and enables to map single molecules or molecule ensembles in the context of highly resolved structures.
SIM achieves two fold resolution enhancements in both lateral and axial directions, so structures can be highly
resolved in 3D. Adapting the structuring to the wavelength opens up the avenue for multi-color staining. Hence the
distribution of one protein and its associated structure can be viewed in the context of others. Since all common
fluorescent dyes can be used sample preparation is straightforward. Besides the classical approach to obtain highly
resolved structures with up to 10 times the classical resolution, the power of PALM lies additionally in its ability to
count and observe single molecules. As such clustering of molecules can be studied as well as many molecules tracked
simultaneously to study their diffusion. New strategies open up the possibility to obtain resolution enhancement in the
axial direction as well. These applications start already to have an impact on our view how a cell is organized and how
different proteins contribute to its make-up.
We present a lifetime imaging technique that simultaneously records fluorescence and phosphorescence lifetime images
in laser scanning systems. It is based on modulating a high-frequency pulsed laser by a signal synchronous with the
pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multi-dimensional TCSPC.
Fluorescence is recorded during the on-phase of the laser, phosphorescence during the off-phase. The technique does not
require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of using excessively high
pulse power for phosphorescence excitation. Laser modulation is achieved either by electrically modulating picosecond
diode lasers, or be controlling the lasers via the AOM of a standard confocal or multiphoton laser scanning microscope.
The diffraction limit in traditional fluorescence microscopy (approximately 200 and 600 nanometers in lateral and axial
directions, respectively) has restricted the applications in
bio-medical research. However, over the last 10 years various
techniques have emerged to overcome this limit. Each of these techniques has its own characteristics that influence its
application in biology. This paper will show how two of the techniques, Structured Illumination Microscopy (SIM) and
PhotoActivated Localization Microscopy (PALM), complement each other in imaging of biological samples beyond the
resolution of classical widefield fluorescence microscopy. As a reference the properties of two well known standard
imaging techniques in this field, confocal Laser Scanning Microscopy (LSM) and Total Internal Reflection (TIRF)
microscopy, are compared to the properties of the two high resolution techniques.
Combined SIM/PALM imaging allows the extremely accurate localization of individual molecules within the context of
various fluorescent structures already resolved in 3D with a resolution of up to 100nm using SIM. Such a combined
system provides the biologist with an unprecedented view of the
sub-cellular organization of life.
The elucidation of diffusion processes and molecular interactions and their relation to compartments and structures will be essential to understand cellular functions in detail. Often it is not the average signal that is of interest but the behaviour of single molecules which behave as individuals. Fluorescence based assays have revolutionized the way we can observe molecules at work in their natural cellular settings and they have now also become available for single molecule studies. These technologies comprise Fluorescence Fluctuation Analysis (FFA) including Fluorescence Correlation Spectroscopy (FCS), Fluorescence Redistribution After Photobleaching (FRAP), Foerster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging (FLIM). Especially for dual colour experiments and when dealing with delicate samples the employment of multiphoton microscopy using the aforementioned technologies can be of great benefit. Ideal instruments to study single molecules would therefore need to accommodate equipment that allow for fast time resolution, adequate detectors and lasers as well as integrated work flows. In this contribution we discuss the newest developments in commercial instrumentation and software at Carl Zeiss towards highly sensitive imaging in combination with spectroscopic analysis.