Wide Field (WF) Fluorescent Microscopy

WF microscopy is the most basic form of fluorescence microscopy and is commonly used in traditional cell biology. In a WF microscope, a parallel beam of light illuminates an entire specimen at once to excite the fluorophore using a filter block. The resulting fluorescence (including that for a multi-probed sample) of the specimen can be viewed at once, as a whole. Such images can be viewed by the naked eye or captured with a camera. Though traditionally, excitation light is provided by a mercury or xenon high-pressure bulb, where the required wavelengths are selected with optical filters, longer-lasting fiber-coupled metal halide lamp systems may be preferred.

Laser scanning microscopes can alternatively illuminate a single small point of the specimen, and then continue to scan across the specimen to create an image. More recently, bright, single-wavelength light-emitting diodes (LEDs) have become popular excitation light sources as they offer a long lifespan, and a tighter wavelength control. Though simple in nature, the key to successful WF microscopy lies with the specimen selection. Thinner specimens will more readily generate informative images, which may limit imaging to single cells or thin organelles that often closely adhere to glass surfaces.

Advantages of WF microscopy lie in that the entire image is illuminated simultaneously, allowing for fast imaging. WF microscopy is inexpensive and offers simplicity as well as flexibility compared to other techniques. WF microscopy allows a researcher to observe specimens in real-time by the naked eye; a great benefit for the definitive selection of fluorescent cells.

Some disadvantages of the technique however lie in that the rate of image acquisition is determined by the capabilities of the secondary camera or photomultiplier system. Care must be taken to minimize observation times and photobleaching, especially if the sample is to be imaged after manual observation. WF microscopy, inherently, may generate low image resolution and low contrast as lower excitation intensities must be used.

Output images have a high potential for shading artifacts due to uneven illumination, though issues may be partially addressed with the addition of specialized beam-splitting devices or cameras that use particular detectors. If closely associated fluorophores are used the image may be depicted as a bright blur lacking structural orientation, rather than a discrete object. Spatial resolution issues are also inherent with this particular technique, and WF microscopy lacks 3D imaging capabilities.

Tools:

Fluorescence Spectrum Viewer