Editorial Feature

What is Confocal Microscopy?

Confocal microscopy is an optical imaging technique that relies on spatial filtering methods to remove contributions to the image from regions of the sample that are not immediately in focus.

confocal microscopy

Image Credit: Elizaveta Galitckaia/Shutterstock.com

Unlike conventional microscopy methods that simultaneously illuminate the entire sample area, confocal microscopy uses an excitation beam focused on the sample and then relies on detecting the resulting fluorescence to recreate the image of the sample.1

Most modern confocal microscopes use a laser beam as the excitation source for the sample.

The incident light is reflected from a mirror assembly and focused onto a sample.

The resulting fluorescence is then passed through spatial pinhole filters to remove any contributions of fluorescence from regions that are out of focus in the sample.

The mirror assembly can then be translated to scan the beam across the sample and build up a more extensive spatial or depth profile. This is why one of the most common variations of confocal microscopy is known as laser scanning confocal microscopy.

The main advantage of confocal microscopy is the ability to reconstruct 3D fluorescence images from samples prepared in the same way as standard fluorescence microscopy.

As well as for 3D reconstructions of the cell structure, the image information can also be recorded as a time-lapse to watch the temporal evolution of the cell’s behavior.2

However, one of the main disadvantages of confocal microscopy is that image acquisition can be slow due to the need to raster scan the beam over the sample.

Many focal spot sizes for confocal microscopy will be intentionally small to achieve the best spatial resolution, which will further increase scan times. For taking 3D profiles, the problem is further enhanced as 2D images at multiple focal depths will need to be acquired.

Confocal microscopy can image thicker samples than strictly transmission-based methods and samples. A sample thickness of around 50 µm is usually ideal, though using the method with thicker samples is possible.3

Typical species imaged with confocal microscopy include cells and tissues for morphological and dynamic analysis.4

The highest spatial resolutions achievable in confocal microscopy are around 180 nm lateral by 500 nm axial - without additional steps to correct image aberrations.5 This is sufficient for many biological applications and some applications in nanomaterials and materials development more generally.6

What is Confocal Microscopy Used For?

A significant application of confocal microscopy is in the field of histology.

Histology and histopathology involve looking at abnormalities and mutations in biological tissue to identify cellular changes related to disease.7

Histology is beneficial not just as a medical diagnosis tool but identifying a treatment target for diseases and understanding the cellular mechanisms involved in disease progression.

The wealth of information achievable through histology research can be used to grade the severity of tumors and to identify whether a new pharmaceutical is interacting with the correct target region or not.

Confocal microscopy is a particularly powerful tool for histological studies looking at skin samples.8 Typically used in reflectance mode for such studies with a near-infrared laser source for excitation, species in the skin such as melanin and keratin give strong reflectance signals. This information can then distinguish between benign and malignant skin lesions for a rapid and accurate diagnosis.9

Other applications of confocal microscopy in cell biology include ophthalmology.10 From evaluation of corneal thickness to nerve densities and keratocyte density, confocal microscopy is an ideal method for use on ophthalmological samples as it does not require additional thinning.

With the correct focal length settings, full-thickness corneal samples can be used. It is, therefore, possible to build up depth and density profiles of the distributions of various species in the sample of interest.

Another advantage of confocal microscopy with slit-lamps for biological samples is that these can be used to control the amount of light scatter for the sample.10 Excess scattering and unwanted fluorescence from samples can lead to blurring of achievable spatial resolution of the method.

Confocal Microscopy Developments

Several methods are being developed through hardware implementations or post-processing approaches to improve the spatial resolution achievable in confocal microscopy measurements.5

Brighter light sources are also making a greater array of multiphoton imaging methods possible, which can overcome some issues with the penetration depths of specific wavelengths of light into biological samples.

A highly active area of development for confocal microscopy is moving toward super-resolution methods that enable smaller spatial resolutions than the diffraction limit of the wavelength of light used.11

Particularly for medical applications, the use of machine learning algorithms and automated scanning and focusing procedures are being used to overcome the limitations of the slow scan speeds, either by full automation of the image acquisition procedure or minimizing the number of images required for a full 3D reconstruction.

References and Further Reading

  1. Nwaneshiudu, A., Kuschal, C., Sakamoto, F. H., Anderson, R. R., Schwarzenberger, K., & Young, R. C. (2012). Introduction to Confocal Microscopy. Journal of Investigative Dermatology, 132(12), 1–5. https://doi.org/10.1038/jid.2012.429
  2. Lee, J. E., Liang, K. J., Fariss, R. N., & Wong, W. T. (2008). Ex vivo dynamic imaging of retinal microglia using time-lapse confocal microscopy. Investigative Ophthalmology and Visual Science, 49(9), 4169–4176. https://doi.org/10.1167/iovs.08-2076
  3. Smith, C. L. (2011). Basic confocal microscopy. Current Protocols in Molecular Biology, 56(1), 2-2. https://doi.org/10.1002/0471142727.mb1411s81
  4. Bayguinov, P. O., Oakley, D. M., Shih, C. C., Geanon, D. J., Joens, M. S., & Fitzpatrick, J. A. (2018). Modern laser scanning confocal microscopy. Current Protocols in Cytometry, 85(1), e39. https://doi.org/10.1002/cpcy.39
  5. Fouquet, C., Gilles, J. F., Heck, N., Santos, M. Dos, Schwartzmann, R., Cannaya, V., Morel, M. P., Davidson, R. S., Trembleau, A., & Bolte, S. (2015). Improving axial resolution in confocal microscopy with new high refractive index mounting media. PLoS ONE, 10(3), 1–17. https://doi.org/10.1371/journal.pone.0121096
  6. Senthil Kumar, P., Grace Pavithra, K., & Naushad, M. (2019). Characterization techniques for nanomaterials. In Nanomaterials for Solar Cell Applications. Elsevier Inc. https://doi.org/10.1016/B978-0-12-813337-8.00004-7
  7. Musumeci, G. (2014). Past, present and future: overview on histology and histopathology. Journal of Histology and Histopathology, 1(1), 5. https://doi.org/10.7243/2055-091x-1-5
  8. Rajadhyaksha, M., González, S., Zavislan, J. M., Anderson, R. R., & Webb, R. H. (1999). In vivo confocal scanning laser microscopy of human skin II: Advances in instrumentation and comparison with histology. Journal of Investigative Dermatology, 113(3), 293–303. https://doi.org/10.1046/j.1523-1747.1999.00690.x
  9. Filoni, A., & Alaibac, M. (2019). Reflectance Confocal Microscopy in Evaluating Skin Cancer: A Clinicians's Perspective. Frontiers in Oncology, 9, 1457. https://doi.org/10.3389/fonc.2019.01457
  10. Erie, J. C., McLaren, J. W., & Patel, S. V. (2009). Confocal Microscopy in Ophthalmology. American Journal of Ophthalmology, 148(5), 639–646. https://doi.org/10.1016/j.ajo.2009.06.022
  11. Sheppard, C. J. R. (2021). The development of microscopy for super-resolution: Confocal microscopy, and image scanning microscopy. Applied Sciences, 11(19). https://doi.org/10.3390/app11198981

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Rebecca Ingle, Ph.D

Written by

Rebecca Ingle, Ph.D

Dr. Rebecca Ingle is a researcher in the field of ultrafast spectroscopy, where she specializes in using X-ray and optical spectroscopies to track precisely what happens during light-triggered chemical reactions.


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