Confocal Microscopy

Confocal microscopy is an optical imaging technique for fluorescent samples that increases the resolution as compared to traditional light microscopy. Confocal microscopy also allows for optical sectioning of the sample.

How it works – A laser is scanned across a sample surface and the resulting emitted light is passed through a pinhole, rejecting out of focus light.  The intensity and position of the emitted light is recorded to make an image of the sample surface.  A 3D image can be obtained by moving through z with optical sections.

There are several types of confocal microscopes, including laser scanning and spinning disk.  In laser scanning confocal, the laser is scanned across the sample surface using mirrors. With spinning disk confocal, multiple apertures are contained in a disk with can spin rapidly and illuminate the sample, allowing for faster scanning rates.

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Instrumentation Available

Key Attributes

  • Reduces out of focus light as compared to widefield optical imaging
  • Spinning disk confocal allows for higher scanning speeds and reduces phototoxicity and photobleaching
  • Multiple laser lines (405, 488, 561, 640) and objectives (10x, 20x, 40x. 60x) available for imaging

 

Strengths

  • Optical sectioning reduces the contribution of out of focus light to the image
  • Can control depth of field
  • Can optically section through sample and create a 3D image

Limitations

  • The sample must have autofluorescence or be labeled with a fluorescent probe
  • Laser scanning can cause photobleaching or damage to the sample
  • Imaging limited to wavelengths of available laser lines

Applications

  • Imaging and recording the spatial locations of labeled molecules within a sample
  • Live imaging the motion of labeled molecules within a cell
  • Observing colocalization of molecules

Additional Reading

Video of confocal microscopy – 3 minute video explaining confocal microscopy

Introduction to Confocal Microscopy