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.
We demonstrated that the dispersion of scanning microscope optics (including a Zeiss 40x/1.2 Apochromat objective)
can be compensated by means of chirped mirrors over a bandwidth of 170 nm at 800 nm. The interferometric
autocorrelation trace recorded at the focus of the microscope objective with a two-photon diode indicated a pulse
duration of < 12 fs. The propagation time difference of the system can be minimized by proper choice of the
components, enabling sub-12-fs pulse delivery with a completely filled 40x/0.8 Zeiss Achroplan water immersion
Research in the life sciences increasingly involves the investigation of fast dynamic processes at the cellular and subcellular level. It requires tools to image complex systems with high temporal resolution in three-dimensional space. For this task, we introduce the concept of a fast fluorescence line scanner providing image acquisition speeds in excess of 100 frames per second at 512×512 pixels. Because the system preserves the capability for optical sectioning of confocal systems, it allows us to observe processes with three-dimensional resolution. We describe the principle of operation, the optical characteristics of the microscope, and cover several applications in particular from the field of cell and developmental biology. A commercial system based on the line scanning concept has been realized by Carl Zeiss (LSM 5 LIVE).
Research in the Life Sciences increasingly involves the investigation of fast dynamic processes at the cellular and sub-cellular level. It requires tools to image complex systems with high temporal resolution in three-dimensional space. For this task we introduce a fast fluorescence line scanner with image acquisition speeds in excess of 100 frames per second at 512x512 pixels and with a more than 10- fold increased sensitivity compared to point scanning confocal systems. Since the system preserves the capability for optical sectioning of confocal systems it allows to observe processes in three dimensions. We describe the principle of operation, the optical characteristics of the microscope and cover several applications in particular from the field of developmental biology.
If laser scanning microscopy with enhanced spatial or temporal resolution is performed on sensible biological samples it is essential to prevent damage or substantial alteration of the object due to intense laser illumination. Considering a simplified model for temperature rise and photochemical reactions conclusions concerning scanning and measuring conditions are drawn. A method for improving the spatial resolution of the laser scanning microscope by image processing is described. This method is based on a local deconvolution procedure and yields an improvement of the resolution of about 1. 7 in a discussed example. It is applied to images of chromosomes. For time-resolved fluorescence microscopy on the basis of time-correlated single photon counting methods for obtaining images of fluorescence decay parameters under the condition of weak fluorescence intensity are discussed. As an example intensity and relaxation time images of a tumour cell incubated with HpD are presented. 1.