Laser scanning confocal fluorescence microscopy: an overview Liu Jie B

Introduction Principle Application Conclusion

live cell fluorescence microscopy How do proteins interact with ligands, other proteins, DNA inside or on the membrane of cells? People have determined from biochemical and genetic experiments spanning diverse approaches from in vitro reconstitution of cellular processes to atomic resolution structure determination. These techniques only provide a static, snapshot view of cells. The commonest approach for studying dynamic cellular events is live cell fluorescence microscopy

Types of fluorescence microscope systems Widefield microscopes Scanning confocal microscopes Spinning disk confocal microscopes

Sensitivity of detection Confocal Objective Detector Light source Speed of acquisition Viability of the specimen

1. Confocal principle The excitation laser beam is focused through advanced optics into the sample solution. The fluorescence light emitted by the dye molecules is collected by the same objective and focused on the small pinhole. The focal point in the sample and the pinhole lie in conjugate planes. The optical arrangement of the focal points is called ‘confocal’. Thus, light only from the focal point (plane) can pass the pinhole and be measured by the detectors.

2.Objective 数值孔径： numerical aperture （ NA) 物镜的主要技术参数，是判断物镜而言性能高 低的重要标志。 物镜前透镜与被检物体之间介质的折射率（ h ） 和孔径角（ u ）半数的正弦之乘积。用公式表示如 下： NA=hsinu/2 It is normally preferred to use an objective with the highest numerical aperture to maximize the brightness of the image.

3. Detector The more sensitive the detector, the lower the illumination intensity needed. Using an intensified camera is one way of increasing sensitivity, at the expense of increasing noise in the image. Sensitive back-illuminated charge- coupled device (CCD) cameras with thinned chips are available.

4. Laser excitation power The signal-to-noise ratio of the fluorescence image data increases with the laser power. Therefore, the laser power should be as high as possible. the laser excitation power should be low enough to prevent photo-induced reactions in the specimens thereby affecting the data.

Speed of acquisition Filter wheel configurations of the scan head are slower in switching. CCD cameras acquire a whole field of view at once. Resonant galvanometers(which are optional on many commercial systems).

Viability of the specimen Illumination causes photobleaching and therefore cell damage,everything possible should be done to limit the duration and intensity of illumination. Shut off illumination light when it is not needed; this is inherent in confocal systems

Zeiss LSM510– ConfoCor2 Combi system

Laser scanning confocal fluorescence microscope image of 138 nM TMR- tetheredγ-venin incubated with human blood cells.

Laser scanning confocal fluorescence microscope image of a leukocyte showing clustered binding of 138 nM TMR- tetheredγ-venin

Applications of LSCFM imaging are very diverse,and they are constantly developing. For example,in the world of therapeutic peptides, hormones,cytokines, regulatory factors,growth factors,or monoclonal antibodies used in medicine,we have move from relatively basic studies to more quantitative analysis of distances between structures and detection in various cellular compartments.

Other Imaging Modes Bright-field imaging Total internal reflection(TIRFM) Fluorescence correlation spectroscopy Photobleaching and photoactivation approaches Fluorescence resonance energy transfer (FRET) Fluorescence lifetime imaging (FLIM)