Confocal microscopy was invented by Marvin Minsky in 1957 and aims to improve resolution over traditional microscopy. It uses point illumination and a pinhole to exclude out-of-focus light and produce thin optical sections and high-contrast images. The key components are a laser light source, dichromatic mirror, pinholes, and photodetector. Confocal microscopy finds applications in cell biology and materials science by allowing optical sectioning and 3D reconstruction. It provides advantages like non-invasiveness, live cell imaging, and depth analysis, but has disadvantages such as photobleaching and loss of intensity.

1. CONFOCAL MICROSCOPY
By
Prabhat Kumar
M.S. Brain & Cognition sciences
Submitted
to
Dr. Sarfraj Ahmad Siddiqui

2. CONFOCAL MICROSCOPY
 Introduction
 Principle
 Components
 Applications
 Advantage
 Disadvantage
 References

3. Introduction
 Marvin Minsky made the first confocal microscope in 1957, he was
postdoc at Harvard University.
 Also known as Confocal laser microscopy.
 This microscopy basically aim to solve the problem that is in different
microscopy techniques that is the lack of resolution.
 Confocal microscopy making balance between optical microscopy and Marvin Minsky
electron microscopy techniques in term of resolution. By using this
technique we get better resolution when we comparison other
optical microscopy.
 Uses pinhole to produce high resolution images because its exclude out of the focus light.
 So images have better contrast and are less hazy.

4.  Z-stack: A set of confocal images taken from the specimen so that the image area along the x- and
y-axis remains the same but the distance from the objective (z-axis) is different for each image. As
the distance between adjacent images is precisely controlled in the z-stack, it can be used to form
a 3-dimensional image.
 A series of thin slices of the specimen are assembled to generate a 3-dimensional image.(Optical
Sectioning )
 Fluorescence- emission of light by substance that has absorbed light & remission of light stop as
soon as incident light is cutoff. It occurs when electrons move
from their ground state to an excited state.
 These electrons keep the same spin as in the ground state,
but when they return to the ground state they emit energy.
 This energy has a longer wavelength than the originally
absorbed energy. If this longer wavelength is within the visible
spectrum, then we can see a glowing light.
 Example of fluorescence are :- DAPI, ALEXA , FITC , GFP etc.

5. Principle
 Similar to the wide field microscope, the confocal 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 cut off signals that are
out of focus, thus allowing only the fluorescence signals from
the illuminated spot to enter the light detector.
 3D objects can be visualized by scanning several optical planes
and stacking them using a suitable microscopy software (z-
stack).

6. Components
 Light Source Laser :- laser light can be chosen via
selection device and are matched with fluorophore
used in experiment.
 Pinhole :- Apertures placed near the light source and
detector enable the microscope to produce thin optical
sections of focal planes in the specimen. Its exclude the
out of focus light rays.
 Dichromic Mirror :- This is a filter which separates the
excitation from the emitted light in fluorescence beam
path of microscope, also called as beam splitter.
• Photomultiplier Detector :- Highly sensitive detectors
tube used in confocal microscopes to gain an amplified
electrical signal from a weak light source.

7. Applications
 Confocal microscopy has been adapted into numerous fields such as Cell biology, Life
sciences, Semiconductor inspection, Materials science, Neuroanatomy, and
Neurophysiology.
 Confocal Microscopy is a powerful tool for studying signaling mechanisms.
 Key applications include:
oDetail structure of specific part of cell
oLocalizations of proteins.
oImaging two or more fluorescence stains.
oAcquisition of high quality images for presentation.

8. Advantage
 It is non-invasive(no requirement of much effort ) microscopy techniques.
 We can do Qualitative as well as quantitative studies .
 We can do function and dynamic analysis.
 No requirement of vacuum .
 Examine live or fixed cell.
 We get 3-D image in confocal microscopy.
 Setup cost and maintenance cost are comparatively very low when we comparison with
electron microscopy.
 Precise and focus.
 Provides deep analysis of any part of specimen .

9. Disadvantage
 Damage to specimen in case of live cell.
 Decrees in Intensity of the incident light.
 The fluorophore should tagged properly to part of the specimen.
 Fluorophore should be sensitive enough for the given excitation wave length, so that they can
emit the light.
 Photo bleaching: photochemical alteration of a dye or a fluorophore molecule such that it
permanently is unable to fluorescence.

10. Image by Confocal Microscope
Fig : An Intestine Section Fig: Kidney cells (fluorescence vs Confocal microscope)

11. References
 Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology.
 Confocal Microscopy Denis Semwogerere Eric R. Weeks, Emory University, Atlanta, Georgia, U.S.A.
 https://www.olympuslifescience.com/en/microscoperesource/primer/techniques/confocal/gloss
ary/
 http://www.physics.emory.edu/faculty/weeks//confocal/
 https://ibidi.com/content/216-confocal-microscopy
 http://microscopy.berkeley.edu/courses/tlm/clsm/index.html