Fluorescent microscope Dr Nanda Kumar

1. PRINCIPLE AND APPLICATION
OF FLUORESCENT
MICROSCOPY
DR.NANDAKUMAR
Post graduate student
Department of Oral Pathology & Microbiology
SRM Dental College,
Ramapuram, Chennai, India

2. Introduction History of fluorescent microscopy
Basic terminologies Advantages and Disadvantages
Principle of fluorescent microscopy Features of fluorescent microscopy
Types of fluorescent microscopy Fluorescent microscopic techniques
Applications
Sample preparation for fluorescent microscopy
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3. INTRODUCTIoN
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Microscopy has played an important role in
determining the activity of cells, from the
very early appreciation of living animalcules
by Van Leeuwenhoek (Ford 1989) with a
simple microscope, to details of cellular
events with a variety of present-day
sophisticated imaging systems (Hell 2009).
The current drive is to watch living events
with ever more spatial and temporal
resolution

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FLUORESCENCE
“property of some atoms or
molecules to absorb light at a
particular wavelength and to
subsequently emit light of longer
wavelength after a brief interval. “

5. HISTORY OF FLUORESCENT MICROSCOPY
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1852
STOKES
AUGUST KHOLER
1904
REICHART &
HEIMSTADT 1911
1959
BRUMBERG
1967
PLOEM
1967
TOMPLINSON
1968
JOHNSON &
DOLLHOPF
1968
NAIRN

6. ‣ Photoluminescence describes the absorption and re-radiation, or emission,
of photons.
‣ Luminescence is the production of light at low temperature by cold-body
radiation, whereas incandescence is the production of light from a heated body
‣ Fluorescence is part of the family of luminescence.
‣ Fluorophore refers to a chemical group that confers fluorescence upon a
molecule. Similarly, a chromophore is the part of a molecule responsible for its
colour.
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7. × SELF LUMINOUS FLUOROPHORE
× SENSITIVITY
× SPECIFICITY
× VERSATILE
× MULTIPLE LABELLING POSSIBLE
× ECONOMICAL
× ANALYSE INTERACTION BETWEEN MOLECULES
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ADVANTAGES

8. ▸PHOTOBLEACHING
▸PHOTOTOXICITY
▸BLEED THROUGH/
CROSS OVER
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DISADVANTAGES

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PHOTOBLEACHING BLEED THROUGH/
CROSS OVER
PHOTOTOXICITY

10. PRINCIPLE OF
FLUORESCENT
MICROSCOPY
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‣ Photon #1 hits molecule
‣ Photon #1 absorbed, electron at ground level gains energy and jumps to higher level
‣ Electron loses some of that energy
‣ Electron jumps back down to ground level and emits photon #2
‣ Photon #2 has less energy, corresponding to a different color (wavelength of light)

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stoke’s shift
‣The Stokes Shift is named
after Irish physicist George
Gabriel Stokes
‣It is the spectral shift to
lower energy between the
incident light and the
scattered or emitted light
after interaction with a
sample.

14. fluorescent
microscopy
features
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NUMERICAL APERTURE
‣ Due to the wave nature of light, emitted photons are diffracted at the optical components of the microscope and
eventually appear as regularly spaced rings (Airy disks) at the detector unit.
‣ The NA of objectives that are used for fluorescence microscopy typically ranges from 0.2 to 0.95 for air objectives and
0.85 to 1.4 for oil objectives.

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RESOLUTION
The ability of a fluorescence microscope to resolve two point sources of light that are in close proximity is
essentially defined by the Rayleigh criterion
“RAYLEIGH CRITERION-SPECIFIES THE MINIMUM SEPERATION BETWEEN TWO LIGHT SOURCES,THAT
MAY BE RESOLVED INTO A DISTINCT OBJECT”

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POINT SPREAD FUNCTION
‣ The Point Spread Function (PSF) is the
three-dimensional representation of the
Airy pattern and a unique fingerprint of
each imaging system.
‣ In the lateral dimension, the PSF
appears as regular rings, while in the
axial dimension these rings appear as
elongated ellipsoids.

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NUMERICAL APERTURE, AIRY DISK
SIZE & RESOLUTION
‣ The NA of the optical
system fundamentally
defines its capability to
resolve details in the image.
‣ Larger collection angles
generate smaller Airy disks
of individual fluorophores
at the detector and
consequently produce
images with greater
resolutions.

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Types of
Fluorescent
Microscopes

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DIASCOPIC
FLUORESCENCE
First type of fluorescence
microscopy that used transmitted
light
Light from the illumination source
first passes through an excitation
filter and subsequently to the
specimen through a dark field
condenser.
Diascopic fluorescence is rarely
used now.

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ADVANTAGES DISADVANTAGES
• Good dark field, improved
contrast
• Any objective can be used as
long as the NA is less than
condenser
• Bright images at low
magnifications
• Difficult to align the condensers
• Not suitable for thick slides.
Specimen must be transparent
• Illuminates large area &
fluorescence tends to fade quickly

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EPISCOPIC FLUORESENCE
‣ In this type of fluorescent microscope, the
excitation light comes from above the
specimen through the objective lens.
‣ This is the most common form of
fluorescence microscopy today

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ADVANTAGES
1)Better resolution
2)Brighter images at high magnifications
3)Easy to combine a fluorescent image and a transmitted light image
4)Limited area excited that is viewed by objective lens

24. COMPONENTS OF
FLUORESCENT
MICROSCOPE
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LIGHT SOURCES
‣ For routine observation
‣ specific lifetime and proper storage
‣ Little change in light output
‣ long lifetime
‣ don’t require multiple filters

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FILTERS
‣ EXCITATION FILTER
‣ INTERFERENCE
FILTERS
‣ DICHROMIC MIRROR
‣ BARRIER FILTER

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FILTER CUBE

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CONDENSER AND OBJECTIVE
‣ BRIGHT
FIELD
CONDENSER
‣ DARK FIELD
CONDENSER
‣ QUARTS
FLUORITE
‣ LITHIUM
FLUORITE

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FLUORESCENT MICROSCOPE SAMPLE
ILLUMINATION AND DETECTION

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FLUORESCENT
MICROSCOPIC
TECHNIQUES

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WIDE FIELD MICROSCOPY
‣ Invented in 1932
‣ The sample is illuminated across the entire
field of view and emitted fluorescence is
collected in a detector unit .
‣ small light dose is sufficient to illuminate the
specimen
‣ imaging speed is increased,
‣ Photobleaching and phototoxicity decreases
‣ long-term imaging of living specimens.

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LASER SCANNING MICROSCOPY
‣ first used in 1987
‣ laser scans the specimen point-by- point and
emitted stray light from planes outside the focus is
removed by a pinhole near the PMT detector
therefore increasing the intensity of light
‣ Eliminates the need of tissue sectioning to image
thick specimens
‣ Used for high-resolution imaging of fixed specimens
of up to 100 mm in thickness
‣ strictly limited to fixed specimens.

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SPINNING DISK MICROSCOPY
‣ It consists of 2 rotating disc,where the first
disk consists of multiple micro-lens which
focuses light to the pinhole and to achieve
good confocal image.
‣ The second one consists of multiple pin-
hole,which clears the stray light.
‣ Reduced image resolution,because of the fixed
pin hole diameter
‣ Employed to study cellular behaviors

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MULTI PHOTON MICROSCOPY
‣ Pulsed lasers emits light not in a continuous mode,
but in the form of optical pulses/light flashes
‣ only small amounts of photons are necessary to
sufficiently illuminate the specimen
‣ Lasers with longer wavelength were used to excite
fluorophores
‣ used for long-term four-dimensional live imaging of
embryonic cell migration) and organogenesis

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LIGHT SHEET MICROSCOPY
‣ first used in 1993
‣ In light sheet microscopy, the illumination and
detection paths are perpendicular to each other
‣ The bleaching of fluorophores is strongly
reduced and phototoxic effects on cells in a life-
imaging setup are almost negligible
‣ Used for live imaging of dynamic and/or long-
term processes, such as whole-CNS functional
imaging or embryogenesis

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SR-SIM
‣ An extension of wide field microscope, which
provides double the resolution
‣ periodic grid translates and rotates in the
illumination path during the imaging procedure
to allow the laser light to attain the patterned
illumination
‣ increases sample light exposure and imaging
time, thereby limiting the ability to capture
dynamic processes
‣ works well for thin specimens

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STED
‣ utilizes two lasers: one excitation laser and a
depletion laser
‣ While the excitation laser activates fluorophores in
the focal volume
‣ The depletion laser simultaneously returns them
back from the excited state to the ground state.
‣ As a result, fluorescence signals are only detected
from the remaining small focal volume in the center.
‣ high laser power
‣ enhancing sample bleaching and phototoxic effects

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PALM/ STORM
‣ rely on photo switchable fluorescent dyes or proteins
that are, in a first step, stochastically activated by an
activation laser applied at low power
‣ After image acquisition, activated fluorophores are
photo bleached (PALM) or switched into a reversible
dark off- state (STORM) by an inactivation laser
‣ After image acquisition, activated fluorophores are
photo bleached (PALM) or switched into a reversible
dark off- state (STORM) by an inactivation laser
‣ require careful probe selection
‣ used for quantitative fluorescent measurement

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SAMPLE
PREPARATION FOR
FLUORESCENT
MICROSCOPY

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USED TO VISUALIZE
BIOMOLECULES(nucleic
acids and proteins)
DYNAMIC CELLULAR
PROCESSES(endosomal
transport signal
transduction)
ORGANELLES(nucleus
and golgi apparatus)
BEHAVIOUR OF SINGLE
CELLS AND CELL
POPULATION(cell
migration and wound
healing)
EVEN THE
DEVELOPMENT OF
ENTIRE ORGANISM

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FLUOROPHORES-BASED LABELING TECHNIQUES
DIRECT
INTERACTIONS
WITH
BIOMOLECULES
ANTIBODY-
ANTIGEN
BINDING

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TYPES OF FLUORESCENT LABELLING TECHNIQUES
SYNTHETIC FLUORESCENT STAINS AND PROBES
ANTIGEN-ANTIBODY BINDING (IMMUNOFLUORESENCE)
GENETICALLY ENCODED FLUOROPHORES

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SYNTHETIC FLUORESCENT STAINS & PROBES
‣ are typically applied to fixed cells or tissues
‣ Fluorescent stains and probes directly interact
with cellular components (e.g., by intercalation
into the DNA double helix) or may be fused to a
targeting molecule
‣ nucleic acids :e.g., Hoechst 33258 and DAPI
‣ lipids of biological membranes: e.g., NileRed,
FM dyes, and BODIPY
‣ organelles :LysoTracker, MitoTracker, and ER-
Tracker to label lysosomes, mitochondria, and
the ER

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IMMUNOFLUORESCENCE
‣ IF is an antibody-based staining technique using
immunoglobulin (e.g., IgG or IgM) coupled to synthetic
fluorescent dyes
‣ it is unsuitable for live imaging approaches as fixation and
membrane permeabilization is required prior to staining
‣ Each primary antibody is then targeted by multiple
secondary antibodies that are coupled to a synthetic
fluorescent dye (i.e., indirect IF). IF provides superior
specimen contrast
‣ Alternatively, primary antibodies can be directly labeled with
fluorophores (i.e., direct IF), reducing background staining
and the duration of the staining procedure.

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GENETICALLY ENCODED FLUORESCENT PROTEINS
‣ The endogenous expression of fluorescent proteins (FPs) provides a
genetically encoded avenue for the visualization of cellular components.
‣ FPs may be expressed un- der the control of regulatory elements (i.e., re-
porter FPs) or
‣ fused in-frame with the genetic sequence of a protein-coding gene to create a
tagged version of the target protein (i.e., fusion FPs).

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CONCLUSION
The era when optical microscopy was purely a
descriptive instrument or an intellectual toy is past
The modern fluorescence microscope combines
the power of high performance optical components
with computerised control of the instrument and
digital image acquisition to achieve a level of
sophistication that far exceeds that of simple
observation by the human eye.