CONFOCAL LASER SCANNING MICROSCOPE

By

SAKSHI PANDEY
Master of Pharmacy (Pharmaceutics) – 1 semester
Under the supervision of
Dr . Sunil K. Jain
Associate Professor

(Department of Pharmacy)

GURU GHASIDAS UNIVERSITY

SLT INSTITUTE OF PHARMACEUTICAL SCIENCE
BILASPUR C.G. - 495009
 CONTENT
 Introduction
 History and development
 Principle
 Working
 Advantage
 Disadvantage
 Application
 Reference
 INTRODUCTION
 Confocal laser scanning microscopy(CLSM), commonly known ads confocal
microscopy, is an optical imaging technique for increasing optical resolution
and contrast of a micrograph by means of using a spatial pinhole to block
out-of-focus light image formation.
 CLSM is a suitable tool used for investigation of various physicochemical
feature of pharmaceutical products and analysing their performance in
numerous in vitro and in vivo models.
 During last decade it has been particularly applied in the field of drug
delivery.
 CLSM allows the recognition of various interaction thar occurs between
micro-system or nano-system and cells.
 CLSM is helpful in distinguishing the cell interaction profiles of fluorescent
micro and nano pharmaceuticals and in getting the evidence of efficient
selective targeting through the use of suitable compounds.
 The cellular organization of nano-system is another important aspect that
has been intensely investigated with the help of this technique in order to
study the selective delivery of specific compounds, such as in the case of
various genetic materials.
 In this presentation these aspects will be described with the aim of providing
a brief report about the principle, working, advantage, disadvantage and
important application of CLSM in pharmaceutical science.

Confocal laser scanning microscope

PRINCIPLE
 In confocal microscope two pinholes are typically used.
 A pinhole is placed in front of the illumination source to allow transmission
only through a small area.
 This illumination pinhole is imaged into the focal plane of the specimen, i.e.
only a point of specimen is illuminated at one time.
 Fluorescence excited in this manner at the focal plane is imaged onto a
confocal pinhole placed right in front of the detector.
 Only florescence excited within the focal plane of the specimen will go
through the detector pinhole.
 Scanning of small sections is done and joined them together for better
view. This process is called as optical sectioning and 3D reconstruction.
 Confocal microscope is the modified version of wide field fluorescence
microscope.
 With confocal microscope point illumination can be achieved.
INSTRUMENTATION
The confocal microscope is made up of following parts;
 Objective lens (5X, 10X, 20X, 40X, 63X)
 Out-of-focus plane
 In-focus plane
 Beam splitters
 Detector
 Confocal pinhole (aperture)
 Helium-Neon diode laser (high intensity, monochromatic red light, with 670nm
wavelength).
 Oscillator mirrors
Schematic representation of confocal microscope
 How to prepare slides for CLSM?
 Take a clear glass slide.
 Place the specimen on it.
 In dark condition , fluorophores are poured on the specimen. In case if dilution of
fluorophores is required it is done using the media in which the specimen that is
to be examined is kept.
 Place the cover slip in such a way that no air bubbles get trapped between the
slide and cover slip.
 Ones the cover slip is placed properly, paint the edges of cover slip with
transparent nail polish, in order to fix the cover slip properly on the slide.
 Place the slide in dark before and after viewing.
 In CLSM slide is placed upside down for imagining.
 WORKING
 A specimen stained with fluorochrome is examined. When a beam of light is focused
at a particular point of fluorochromatic specimen, it produces illumination, that is
focused by objective lens, to the plane above the objective.
 The objective has aperture which blocks any stray light reaching the specimen.
 The specimen lies between the camera lens and the point of focus.
 The laser from the microscope scans over a plane on the specimen.
 The detector then measures the illumination producing an image of optical section.
 Scanning several optical section are collected in computerized system as data,
forming 3D image. This process is known as optical sectioning and 3D reconstruction.
 The image of the specimen formed when the microscope scanner scans the focused
beam across a selected area with the control of two high speed oscillating mirror. The
movement is facillated by galvanometer motor.
Image of macrophage phagocytosing yeast formed by confocal
microscope.
Fluorophores used in CLSM
 Fluorophores are basically staining dyes that shows fluorescence when interact
with light.
 It is used as staining agents in preparing slides for confocal microscope.
 Some of the fluorophores used in CLSM are:
 Alexa Fluor 488
 Alexa Fluor 555
 Benzanthrone dyes
Software used in CLSM
Software used in CLSM are:
 Olympus FV-300Fluo View confocal microscope workstation (Mc Guire)
 Leica TCS- SP2 AOBS invented confocal laser scanning microscope and
workstation
(Anatomy Lab, Sanger).
Confocal laser scanning image of mammalian cells.
 ADVANTAGE
 The advantage of confocal microscope is that it improves the outcome of the
image because it analyses the image from one optical point to another,
therefore there is no interference with scattered light from other parts of the
specimen.
 They have better resolution and each point of interest is visualized and
captured.
 It can be used to study live and dead cells.
 It illuminates uniformly across the focus points. It provides optical zoom feature.
 It adjust it’s magnification electronically, without changing the objectives, by a
factor known as the zoom factor.
 Specific location identity and 3D modelling can be achieved through confocal
microscopy technique.
 Confocal microscope removes all background noise.
 Gives fluorescence from exact fluorescence emitting region of the sample.
LIMITATIONS
 They have a limited number of excitation wavelengths, with narrow bands.
 They are expensive to purchase and manufacture (IIT Indore)
 Slow scanning speed.
 Limited use in dynamic tracking studies.
 Photobleaching from laser excitation.
 Lasers may damage live cells.
 APPLICATION
 CLSM represents a unique and specific tool that is able to predict the
morphology of fluorescent microparticles and nanoparticles and their behaviour
in in-vitro and in-vivo models.
 CLSM helps in detection of various components of pharmaceutical formulation.
For example: the use of suitable fluorescent probes favours the discrimination
between the different phases of emulsion.
 Through the use of CLSM the influence of the p H on the aggregation phenomena
of oil-in-water emulsion marked with Nile red.
 CLSM has also been employed to check the structure and stability of dosage
form. It allows detection of the distribution of water soluble or lipid soluble
fluorescent compounds in gel-like formulation.
 CLSM is connected to the investigation of morphology of drug delivery system
and their interaction with biological membranes or their cellular uptakes.
 In biomedical sciences, it is used in the analysis of the eye corneal infections, by
quantitatively and qualitatively analysing the endothelial cells of the cornea.
 CLSM is also used to identify the presence of the fungal elements in corneal
stroma, during keratomycotic infection, or rapid diagnosis and quick therapeutic
response.
 REFERENCE
 https://www.slideshare.net/RangineniPrada/confocal-microscopy
 https://www.researchgate.net/publication/266084439_Pharmaceutical_applications
_of_Confocal_Laser_Scanning_Microscopy
 https://www.academia.edu/22015831/Pharmaceutical_applications_of_Confocal_La
ser_Scanning_Microscopy
 https://slideplayer.com/slide/4934951/
 https://www.slideserve.com/toni/confocal-microscopy-powerpoint-ppt-presentation
 https://en.m.wikipedia.org/wiki/Confocal_microscopy
THANK YOU