Leica’s confocal microscope (Microsystems, Heidelberg) is a no compromise, true point, scanning system with extremely high sensitivity and theoretical maximum x, y and z resolution. This powerful tool for imaging live and fixed specimens consists of a performance microscope, complex software and hardware accessories, a UV fluorescent lamp and a focusing box. Although I generally use this microscope to image fluorescent signals from cells stained with antibodies or Ca2+ sensitive dyes, the microscope can be used for integrating electrophysiological applications, as well.
The operation of the microscope is pretty easy and does not require intensive training; however, the software is very complex and requires time to become fully competent. The Leica program also comes with a tutorial/simulation program and help system that can be used for training, as well as, image analysis. The specific set of acquisition parameters (beam path, objectives, excitation wavelength light, laser scan format and mode) can be easily selected using the program. Various beam splitters are available (such as dichroic, neutral, double dichroic and triple dichroic filters) and excitation/emission wavelengths can be customized allowing the use of a variety of secondary antibodies. The choice of objectives is vast, enabling a broad magnification range in addition to electronic zooming. The scan format determines the spatial and temporal optical levels of the specimen and laser intensity can be set as the percentage value of the laser output. The zoom function enables an enlarged recording of freely selective square frames of a specimen. All aspects are easily controlled and toolbars allow you to insert and customize individual function buttons. Once the parameter configuration is defined, it can be saved and stored as an individual user program. One draw back is the lack of a coarse/fine focus control. Instead, the macro/micro-focusing is done in one step with a focusing knob requiring careful manipulation.
A particularly interesting feature is the ability to image and view multiple images concomitantly. Also, up to five tiled images can be displayed separately or as an overlay (of fluorescent or transmitted light images) and colors can be assigned for the various channels. I found that the optimization of image contrast (the offset and gain value of detection) using color look-up tables glow over and under was especially useful. Optimizing the acquisition parameters is done in continuous scan and filtering can be applied to enhance the image quality.
Data can be acquired using single scan, time series or continuous scan parameters. Scan speed varies from 200 to 2000 image lines per second. Attention must be paid, as sensitive specimens can be bleached by the lower scan speed. I advise restriction of continuous scanning, as subjecting the specimen to continuous light can cause photochemical destruction.
The software needed for image processing and analysis has a broad range of accessories such as instant calibration of images, rotation, tri-dimensional reconstruction, etc. Three-dimensional spatial data set (3D series) can be generated as surface, wireframe or isolines. Arithmetical functions (addition, subtraction, multiplication, division, minimum, maximum, average projection) can be performed and then sent to the experiment. For quantification, measurement functions can be used to measure pixel intensity profiles along a line segment, surface or volumes. To calculate various statistical values (variance, SD, histogram function, number of pixels) within the selected region of interest the mathematical function can also be used. The Leica confocal software allows easy storage in a special Leica data format (lei), but existing individual images can be later on saved as tif or raw files. I recommend saving the files as frequently as possible to prevent loss of data in the event of a system freeze. A drawback is that the system does not save the experiment automatically before shut down.
In conclusion, the great features of the system are excellent resolution, good image capabilities, versatility of data collection and superior quality of images and analysis. The weak points are the focusing methods and software instability during data collection. The technical service is excellent but I suggest purchasing an extended warranty because the system is prone to technical difficulties.
Lorelei Silverman
Faculty of Medicine
Department of Physiology
University of Toronto