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Writer's pictureSubham

The Microscopy Series: Confocal Microscope

The #ConfocalMicroscope is a sophisticated modification of the #FluorescenceMicroscope with specially placed #PinholeApertures. The apertures enhance the #OpticalResolution and #SpatialResolution producing stunningly crisp pictures with a lot of information. Lets understand the #optics involved here.

Image courtesy: ibidi.com

Principle of Construction

  1. Light Source: Primarily #laser rays emitted from a specific machine fitted with a #BeamExpander that broadens the scope of the #laser beam.

  2. Illuminating Aperture: This small, pinhole aperture is placed between the light source and #DichroicMirror. It acts as a filter to allow only chosen laser rays to illuminate the sample.

  3. Dichroic Mirror: Also known as #BeamSplitter this is placed beween the illuminating aperture and the Objective lens in an inclined state.

  4. Objective Lens: This is placed between the dichroic mirror and the sample. This is characterized by high #NumericalAperture. It focuses a finely focused laser beam on a #diffraction-limited spot in the sample and rapidly scan across the sample at a single depth, thereby illuminating a thin plane. The sample absorbs the short wavelength rays and emit longer wavelength rays. The objective lens forms an image of that #diffraction-limited spot on the plane of the #ConfocalPinholeAperture.

  5. Confocal Pinhole Aperture: This is a small pinhole aperture placed on the #ConjugatePlane of the #FocalPlane of the #ObjectiveLens. As objective lens forms the image at the same plane as this, the #IlluminatingAperture and the #ConfocalPinholeAperture are "confocal" in situation. This acts as a #SpatialFilter and allows only the in-focus emitted rays to be imaged. Out-of-focus rays or #glare from above and below the plane of focus of the object is eliminated.

  6. Emission Filter: The wavelength range of #fluorescence spectrum is selected by an #EmissionFilter which also acts as a barrier by blocking the excitation laser rays.

  7. Photomultiplier Detector: It detects the incoming rays and send the signal electronically to a #Computer which then constructs the final image.

Merits

  1. Image's optical resolution particularly in the sample depth direction is much #better.

  2. Its particularly good in #3DImaging and surface profiling of samples.

  3. It has the ability to serially produce thin (0.5-1.5uM) optical sections.

  4. #Contrast and #Definition are drastically improved over widefield techniques.

  5. It has the ability to adjust #magnification electronically i.e; zoom filter.

Demerits

  1. Because of pinholes, increased resolution is at the cost of #decreased signal intensity, but it can be corrected with long exposures.

  2. High intensity laser beams are #harmful to living cells.

  3. Expensive.

Applications

  1. Imaging #thicker specimens like multicellular embryos.

  2. Optical sectioning and 3D reconstruction of biological samples.

  3. Studying location of #organelles and #macromolecules within cells.

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