Question

Answer in detail(2 pages)
Describe and compare confocal microscopy and wide-field microscopy in the space below.

Answer 1

Confocal microscopy most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM) is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation.Confocal microscopy technology has contributed greatly to advances in life science research by enabling studies on intact living specimens. The principle of working microscope uses fluorescence optics. Instead of illuminating the whole sample at once, laser light is focused onto a defined spot at a specific depth within the sample. This leads to the emission of fluorescent light at exactly this point. A pinhole inside the optical pathway cuts off signals that are out of focus, thus allowing only the fluorescence signals from the illuminated spot to enter the light detector.

By scanning the specimen in a raster pattern, images of one single optical plane are created. 3D objects can be visualized by scanning several optical planes and stacking them using a suitable microscopy deconvolution software (z-stack). It is also possible to analyze multicolor immunofluorescence stainings using state-of-the-art confocal microscopes that include several lasers and emission/excitation filters.

Most confocal microscopes used in industrial applications are reflection-type. They provide a high-resolution image with all areas in focus throughout the field of view, even for a sample having dents and protrusions on the surface. They enable the non-contact non-destructive measurement of three-dimensional shapes. In this technique we can object point-by-point using a focused laser beam to allow for a 3-D reconstruction. In a conventional microscope you can only see as far as the light can penetrate whereas a confocal microscope images one depth level at a time.

Wide field microscopy

One of the most basic microscopy techniques is known as ‘Widefield Microscopy’. It is fundamentally any technique in which the entire specimen of interest is exposed to the light source with the resulting image being viewed either by the observer or a camera (which can also be attached to a computer monitor).

Principle of Widefield fluorescence microscopy is a variation of light microscopy and the easiest fluorescence imaging mode. The underlying key principle is the use of fluorescent molecules—so-called fluorophores—for the labeling of defined cellular structures. These molecules, such as green fluorescent protein (GFP), absorb light at a specific wavelength (excitation) and emit it at a specific higher wavelength (emission). To visualize the molecule of interest, fluorophore-coupled specific antibodies or proteins, for example, are transferred into the cell. The specimen is then illuminated at the excitation wavelength and viewed through a filter that allows only the emitted wavelength to pass through. Whereas the background is dark, the structures with a bound fluorophore emit light, indicating the presence of the structure of interest.

In contrast to confocal microscopy, the whole specimen is exposed to light in widefield fluorescence microscopy. Fluorescence signals from all focal planes are detected, which leads to lower contrast in thick samples like spheroids and tissue. Therefore, widefield microscopy is best applied with thin specimens with low background autofluorescence, like adherent cells.

Comparison with confocal microscopy

In a widefield microscope the entire focal volume is illuminated but that creates blur from areas out of focus above and below the image plane but a confocal microscope scans a sample with a focused beam of light more than one beam in some platforms.The fluorescence microscope allows to detect the presence and localization of fluorescent molecules in the sample. The confocal microscope is a specific fluorescent microscope that allows obtaining 3D images of the sample with good resolution. This allows to reconstruct a 3D image of the sample.Confocal is better than wide filed because Confocal microscopy offers several distinct advantages over traditional widefield fluorescence microscopy, including the ability to control depth of field, elimination or reduction of background information away from the focal plane (that leads to image degradation), and the capability to collect serial optical. Confocal microscopy offers several distinct advantages over traditional widefield fluorescence microscopy, including the ability to control depth of field, elimination or reduction of background information away from the focal plane (that leads to image degradation), and the capability to collect serial optical sections from thick specimens. The basic key to the confocal approach is the use of spatial filtering techniques to eliminate out-of-focus light or glare in specimens whose thickness exceeds the dimensions of the focal plane. This interactive tutorial explores and compares the differences between specimens when viewed in a confocal versus a widefield fluorescence microscope.