Confocal microscopy

CONFOCAL MICROSCOPY
1. History – Vikram Aditya (17/06014)
2. Introduction – Vikram Aditya (17/06014)
3. Instrumentation – Vikram Aditya (17/06014)
4. Principle – Damini Pandita (17/06022)
5. Sample preparation – Deepanshu(17/06020)
6. Advantages and applications – Mansi (17/06016), Ujjwal (17/06018)
7. Limitations and Disadvantages – Shruti Khandelwal (17/06019)
8. Developments & variants – Damini (17/06022), Deepanshu(17/06020)
Guided by
Dr. Sunita Singh
Shivaji College, DU

HISTORY
The basic concept of confocal microscopy was originally developed by Marvin Minsky in the mid-
1950s (patented in 1957) when he was a postdoctoral student at Harvard University. Minsky wanted to
image neural networks in unstained preparations of brain tissue and was driven by the desire to image
biological events at they occur in living systems. Minsky's invention remained largely unnoticed, due
most probably to the lack of intense light sources necessary for imaging and the computer horsepower
required to handle large amounts of data.
Following Minsky's work, M. David Egger and Mojmir Petran fabricated a multiple-beam confocal
microscope in the late 1960s that utilized a spinning (Nipkow) disk for examining unstained brain
sections and ganglion cells.
Continuing in this arena, Egger went on to develop the first mechanically scanned confocal laser
microscope, and published the first recognizable images of cells in 1973.
During the late 1970s and the 1980s, advances in computer and laser technology, coupled to new
algorithms for digital manipulation of images, led to a growing interest in confocal microscopy.
Source: https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/confocal/confocalintro/

INTRODUCTION
Confocal microscopy offers several advantages over conventional widefield optical
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 immediate plane of focus.

INSTRUMENTATION
Fig. Ray diagram of a confocal microscope

INSTRUMENTATION
Fig. Image of a confocal microscope

INSTRUMENTATION

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 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.
Source: https://ibidi.com/content/216-confocal-microscopy
PRINCIPLE
Excitation and emission light
pathways in a basic confocal
microscope configuration.

SAMPLE PREPARATION
Specimens that have three-
dimensional structure that is
to be studied with the confocal
microscope, have to be
mounted in such a way as to
preserve the structure. Some
sort of spacer, such as fishing
line or a piece of coverslip, is
commonly placed between the
slide and the coverslip to avoid
deforming the specimen.
When living samples are to be
studied, it is usually necessary
to mount them in a chamber
that provides all of the
necessary requirements for
life, and that will also allow
sufficient access by the
objective lens to image the
desired area.

Specimen properties that affect light transmission, such as opacity and turbidity,
can greatly influence the depth of penetration of the laser beam into the specimen,
and consequently the structures that can be imaged.
If sufficient laser penetration cannot be achieved with a whole mount specimen,
thick sections can be cut using a microtome.
The protocols described in this section address the specimen preparation
techniques using synthetic fluorophores coupled to immunofluorescence that are
necessary to investigate fixed adherent cells and tissue cryosections using widefield
and confocal fluorescence microscopy.
SAMPLE PREPARATION

STAINING PROTOCOLS
Triple-Staining Adherent Cells with MitoTracker, Phalloidin (or Phallacidin), and Nuclear Dyes.
Staining Adherent Cells with Intermediate Filament Primary Antibodies and Synthetic Fluorophores.
Staining Cells and Tissue Cryosections with Tubulin Primary Antibodies, Phallotoxins, and Synthetic Fluorophores.
Staining Adherent Cells with Cytokeratin Primary Antibodies and Synthetic Fluorophores.
Triple-Staining Tissue Cryosections with Wheat Germ Agglutinin, Phalloidin, and Nuclear Dyes.
Immunofluorescence with Brain Tissue Cryosections.
Immunofluorescence with Brain Tissue Floating Cryosections.
https://www.microscopyu.com/techniques/confocal/specimen-preparation-and-imaging
https://www.olympus-lifescience.com/pt/microscope-resource/primer/techniques/confocal/applications/protocols/
https://www.slideshare.net/mobile/indiandentalacademy/microscopy-dental-applications-endodontic-courses-by-indian-dental-academy

 Images are with resolutions of up to 1.4 times greater than that
of conventional microscopy as it eliminates out-of-focus light, using
spatial filtering eliminate flare in samples that are thicker.
 Higher level of sensitivity, due to the in-built highly sensitive light
detectors.
 Enhanced signal-to-noise ratio, as slower scans and highly
sensitive photomultipliers provide better contrast.
 Controllable depth of field
 Direct, noninvasive, serial optical sectioning of intact, thick, living
specimens with a minimum of sample preparation
 Cut exceptionally clean, thin optical sections of about 0.5 to 1.5 µm
out of thick samples measuring more than 50 µm, using either
reflection or fluorescence.
 Z-axis scanning and depth perception in Z-sectioned images
 Electronically adjusted magnification
ADVANTAGES
Source: https://www.azooptics.com/Article.aspx?ArticleID=653 Fig-Confocal z-stack images of an GFP modified silk fibroin 3D coffee stain

APPLICATIONS
LIVE CELL IMAGING
•FLUORESCENT PROBE TAGGING
•CHARACTERISATION OF PHARMACEUTICAL SYSTEMS
FRET-FLUORESCENCE RESONANCE ENERGY TRANSFER
FRAP-FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING
FLIM-FLUORESCENCE LIFETIME IMAGING MICROSCOPY
FIBER FISH (FLUORESCENCE IN-SITU HYBRIDISATION)

 Only the point being imaged is illuminated, which decreases excess
exposure to background cells decreasing the cell damage.
 Confocal imaging used to describe physiological processes in vivo in
the cornea, kidney and liver.
 Illustrate the healing of wounds in four dimensions (x, y, z, t) at a
cellular level called Tandem Confocal Microscopy (TSCM), a novel
paradigm for imaging in experimental biology.
 Used for imaging, qualitative analysis, and quantification of
endothelial cells of the cornea. kidney, liver, epididymis, muscle, and
adipose tissue.
 Confocal laser endomicroscopy is tool that carry out confocal
microscopic examination of the mucosal layer during ongoing
endoscopy, facilitating early diagnosis of gastrointestinal cancer or
bacterial infection
Sources: https://www.sciencedirect.com/science/article/pii/S0149763405801277
https://www.thieme-connect.de/products/ejournals/abstract/10.1055/s-2006-944813
LIVE CELL IMAGING

A variety of studies in neuroanatomy, neurophysiology
and morphological studies in various cells and tissues
by the use of fluorescent probes, fluorescent proteins
like GFP,RFP, etc.
Combination of direct fluorescence labeling with
different fluorescent probes for simultaneous
examination, increase assay efficiency and reduce the
number of animals used per experiment
Through the use of a fluorescent agent, fluorescein, it
has been possible to directly identify and evaluate areas
of corneal injury in vivo.
FLUORESCENT PROBE TAGGING
Source: https://www.researchgate.net/figure/Live-imaging-confocal-microscopy-of-
HEK293T-cells-after-co-expression-of-GFP-or_fig3_275587331

 Used in the characterization of many pharmaceutical systems,
including tablets, film coatings and colloidal systems.
 Used to study the interaction of biological barriers of the skin, eye
and intestinal epithelia, and the effectiveness of dosage forms at
delivering the drugs through these barriers.
For ex- The transcellular pathways via which different nano capsules
penetrate the cornea were identified through a series of in vivo and ex
vivo testing with confocal imaging. The imaging these pathways were
able to show the influence of different coatings of the nano capsules,
on both the rate of penetration and the biodistribution.
 powerful tool to probe the distribution of components within a
formulation, to characterize homogeneity of pharmaceutical
samples, to determine solid state of drug substances and excipients
as well as to characterize contaminations and foreign particulates.
 The use of confocal imaging was found to be an effective tool in
investigating surface defects in film-coated tablets and the film-
core interface.
CHARACTERISATION OF PHARMACEUTICAL SYSTEMS
Fig-Confocal microscopic image of
inhalation particles with optimized flight
properties, consisting of two components.
Component A is visualized in red whereas
component B is represented in green.Sources: https://www.news-medical.net/life-sciences/Applications-of-Confocal-Imaging.aspx
https://www.semanticscholar.org/paper/Confocal-Raman-Microscopy-in-Pharmaceutical-Haefele-Paulus/bd39ca017bfe2a4e79c4d3baf6283d78d7cd6f23

FRET has been used to measure distance between domains in a single protein and to quantify molecular
dynamics in biophysics and biochemistry, such as protein-protein interactions, protein–DNA interactions, and
protein conformational changes.
FRET-FLUORESCENCE RESONANCE ENERGY TRANSFER

Fluorescence Recovery After Photobleaching (FRAP)
Photobleaching of a region of interest (ROI) within the cell
analyzed allows the measurement of the fluorescence
recovery pace with molecules coming from the
surrounding, non-photobleached, area.
Baseline of fluorescence (a) is reduced after photobleach
(b). Over time, the fluorescent intensity in the
photobleached area increases as unbleached molecules
diffuse into this area (c). Later, there is a stabilization of the
amount of fluorescence recovery (d)
Source: https://www.researchgate.net/figure/Fluorescence-recovery-after-photobleaching-FRAP-a-Schematic-representation-showing-the_fig10_299859695

Fluorescence Lifetime Imaging Microscopy (FLIM)
• FLIM produces an image based on the differences in the excited state
decay rate from a fluorescent sample.
• A fluorescence imaging technique where the contrast is based on the
lifetime of individual fluorophores rather than their emission spectra.
The fluorescence lifetime is defined as the average time that a
molecule remains in an excited state prior to returning to the ground
state by emitting a photon.
• As the fluorescence lifetime does not depend on concentration,
absorption by the sample, sample thickness, photo-bleaching and/or
excitation intensity, it is more robust than intensity based methods.
• At the same time, the fluorescence lifetime depends on a wealth of
environmental parameters such as pH, ion or oxygen concentration,
molecular binding or the proximity of energy acceptors making it the
technique of choice for functional imaging of many kinds.
Source: https://www.picoquant.com/applications/category/life-science/fluorescence-lifetime-imaging-

Fiber FISH (Fluorescence in situ Hybridisation)
Fluorescence in situ hybridization (FISH) is a kind of cytogenetic
technique which uses fluorescent probes binding parts of the
chromosome to show a high degree of sequence
complementarity. Fluorescence microscopy can be used to find
out where the fluorescent probe bound to the chromosome. This
technique provides a novel way for researchers to visualize and
map the genetic material in an individual cell, including specific
genes or portions of genes. It is an important tool for
understanding a variety of chromosomal abnormalities and other
genetic mutations. Different from most other techniques used for
chromosomes study, FISH has no need to be performed on cells
that are actively dividing, which makes it a very versatile
procedure.
Source: https://www.creative-biolabs.com/fluorescent-in-situ-hybridization-FISH.html

The point spread function of the pinhole is an ellipsoid,
several times as long as it is wide. This limits the axial
resolution of the microscope. One technique of overcoming
this is 4Pi microscopy where incident and or emitted light
are allowed to interfere from both above and below the
sample to reduce the volume of the ellipsoid. An alternative
technique is confocal theta microscopy. In this technique
the cone of illuminating light and detected light are at an
angle to each other (best results when they are
perpendicular). The intersection of the two point spread
functions gives a much smaller effective sample volume.
From this evolved the single plane illumination microscope.
Additionally deconvolution may be employed using an
experimentally derived point spread function to remove the
out of focus light, improving contrast in both the axial and
lateral planes.
REFERENCE: Hirschfeld, V.; Hubner, C.G. (2010). "A sensitive and versatile laser scanning confocal optical microscope for single-molecule fluorescence at 77 K". Review of Scientific Instruments. 81 (11):
113705. Bibcode:2010RScI...81k3705H. doi:10.1063/1.3499260.
NOVEL DEVELOPMENTS IN CONFOCAL MICROSCOPY- 1) Improving Axial
Resolution
AXIAL RESOLUTION OF CONFOCAL
MICROSCOPY

There are confocal variants that achieve resolution
below the diffraction limit such as stimulated
emmisson depletion microscopy (STED). Besides,
this technique a broad variety of other (not
confocal based) super resolution techniques are
available like PALM, (d)STORM, SIM, and so on.
They all have their own advantages such as ease of
use, resolution, and the need for special
equipment, buffers, or fluorophores.
REFERENCE: Hirschfeld, V.; Hubner, C.G. (2010). "A sensitive and versatile laser scanning confocal optical microscope for single-molecule
fluorescence at 77 K". Review of Scientific Instruments. 81 (11): 113705. Bibcode:2010RScI...81k3705H. doi:10.1063/1.3499260
2) SUPER RESOLUTION

To image samples at low temperatures, two main approaches have been used, both based on
the laser scanning confocal microscopy architecture. One approach is to use a continuous flow
cryostat: only the sample is at low temperature and it is optically addressed through a
transparent window Another possible approach is to have part of the optics (especially the
microscope objective) in a cryogenic storage dewar. This is second approach, although more
cumbersome, guarantees better mechanical stability and avoids the losses due to the window.
References: Hirschfeld, V.; Hubner, C.G. (2010). "A sensitive and versatile laser scanning confocal optical microscope for single-molecule fluorescence at 77 K". Review of Scientific Instruments.
3. Low temperature operability

DISADVANTAGES OF CONFOCAL MICROSCOPY
1. It requires fluorescent samples.
2. it uses laser illumination which is quite expensive and of fewer wavelengths.
3. Instrument expensive to acquire and run.
4. Z-resolution typically > 500 nm.
5. Pinhole size :-
Diameter of source and detector pinholes cannot be varied independently.
Cannot change based on the objective.
6. low illumination- recent inclusion of microlenses in second disk improves efficiency.

LIMITATIONS OF CONFOCAL MICROSCOPY
1. Resolution limit due to signal to noise ratio caused by a relative small number of
detectable photons . Using more sensitive photo-detectors or increasing the intensity of
the laser point source may increase the resolution limit.
2. Photobleaching of the fluorescent probe : only a few minutes of illumination can alter
the molecular structure of a fluorescent dye and can decrease the intensity of
fluorescence significantly .
3. Phototoxicity of the fluorescent probe : when a fluorescent dye interacts with
excitation light , photons are absorbed by the dye and viable cells can get damaged due
to the photoreactive dye.
4. chromatic and spherical aberration : two different fluorescent dyes can create a visual
shift in an image which is a result of chromatic aberration of the lens used, causing an
optical artifact that impair confocal image quality. Using perfluorodecalin as mounting
can reduce such aberrations.

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