Confocal microscopy provides many advantages over conventional widefield microscopy for life sciences applications. It allows control of depth-of-field and the ability to collect serial optical sections from thick specimens. Confocal microscopy can be used to create 3D images of the structures within cells. Examining these structures can help researchers observe the internal workings of cellular processes.
In conventional widefield optical microscopes, secondary fluorescence emitted from a specimen often occurs through the excited volume and obscures resolution of features that lie in the objective focal plane. Thicker specimens exhibit such a high degree of fluorescence that much of the detail is lost. Spatial filtering techniques eliminate out-of-focus light in specimens whose thickness exceeds the immediate plane of focus.
Advances in Confocal Microscopy
Confocal laser scanning and spinning disk confocal microscopy let researchers generate 3D images of organelles within living cells. High-quality images can be obtained from specimens prepared for conventional fluorescence microscopy. There is also a growing number of cell biology applications that require imaging of both fixed and living cells and tissues.
Recent advances in confocal microscopy have made possible multi-dimensional views of living cells and tissues that include image information in 3D over time. This information is presented in multiple colors. Having temporal data collected from time-lapse experiments or through real-time image acquisition is a powerful tool for cellular biology. The capabilities of confocal microscopy increase as new laser systems are being developed to limit cell damage and as computer processing speeds and storage capacities increase.
Laser Scanning Confocal Microscopy
The primary advantage of laser scanning confocal microscopy is to produce thin optical sections through fluorescent specimens that have a thickness beyond 50 micrometers. Images are collected by coordinating incremental changes in the microscope fine focus mechanism (using a stepper motor) with sequential image acquisition at each step.
Contrast and definition are greatly improved over other techniques due to reduction of background fluorescence and improved signal-to-noise. Artifacts are also removed that would occur during physical sectioning or fluorescent staining by optical sectioning. Living specimens can be examined under a variety of conditions with enhanced clarity.
Spinning Disk Confocal Microscopy
The spinning disk confocal microscope lets researches use lower light levels and obtain more accurate cell physiology with lower fluorophore concentrations. The benefits of delivering higher-efficiency imaging at lower laser powers include less photobleaching, phototoxicity, and is less expensive than confocal laser scanning microscopes.
The many advantages confocal microscopy provides over conventional widefield microscopy for life sciences applications helps researchers observe the internal workings of cellular processes.
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