Fluorescence Microscopy

This was a class presentation I made at BITS Pilani as part of an advanced course on Microbiology

1. Fluorescence
Microscopy
Aalap Tripathy, 2004P3PS208

2. Then… Now…

3. What I will discuss
•how living/non-living,
organic/inorganic
specimens, absorb and
subsequently re-radiate
light.
•We will learn that
fluorescence microscopy
is basically a method of
studying material which
can be made to
fluoresce, either in its
natural form or when
treated with chemicals
capable of fluorescing.
•Some Examples

5. A fluorescence microscope is basically a conventional light
microscope with added features and components that
extend its capabilities.
conventional microscope fluorescence microscope

uses light to illuminate the sample
and produce a magnified image of the
sample.

uses a much higher intensity
light to illuminate the sample

This light excites fluorescence
species in the sample, which then
emit light of a longer wavelength.

A fluorescent microscope also
produces a magnified image of the
sample, but the image is based on
the second light source

6. •general term applied to all
forms of cool light
•that does not derive energy
from the temperature of the
emitting body
TV Screen
Sonoluminescence is the
emission of light by
bubbles in a liquid excited
by sound.

7. Fluorescence v/s Phosphorescence
•If the luminescence is caused
by absorption of some form of
radiant energy, such as
ultraviolet radiation or X rays
(or by some other form of
energy, such as mechanical
pressure), and ceases as
soon as (or very shortly
after) the radiation causing
it ceases, then it is known
as fluorescence.
•If the luminescence
continues after the
radiation causing it
has stopped, then it is
known as
phosphorescence..
The term phosphorescence is often incorrectly considered synonymous with luminescence

8. Basic Concepts
•let excitation light radiate the specimen
•then sort out the much weaker emitted
light to make up the image.
•the fact that the emitted light is of lower
energy and has a longer wavelength is
used.
•The fluorescing areas can be observed in
the microscope and shine out against a
dark background with high contrast

10. Zeiss Axio Imager Z1
Objective lenses: Filtersets: Camera: Software: Imaging-Workstation:
ImageJ
Imaris 4.2
Monchrome
Color
Phase contrast
Oil

11. Why Prepare the Specimen
•Most Cells are colourless by nature
•Cellular components do not fluoresce
themselves. Fluorescent markers are
therefore introduced.

12. Preparation of Specimen
Tagging of Proteins
Fluorescent
Dyes
Immunofluorescence
Techniques
Fluorescent Dyes
Immunofluorescence
Tagging of Proteins
•taken up by the cells
•incorporated and
concentrated in specific
subcellular compartments
•living cells are then
mounted on a microscope
slide and examined in a
fluorescence microscope.
• use of antibodies to which
a fluorescent marker has
been attached.
• recognize and bind
selectively to specific
target molecules in the cell
•modify cells so that they
create their own fluorescing
molecules
•the location of that protein
can be studied. It is also
possible to watch the
movements of the proteins
and its interactions with other
cellular components inside
the cell

13. •fluorescein,
•DAPI,
•propidium iodide
•green fluorescent protein
(GFP)
•Texas Red
TYPES OF FLUOROPHORES
USED

14. irradiate the specimen with a desired and
specific band of wavelengths
then to separate the much weaker emitted
fluorescence from the excitation light

16. Cutaway diagram of a modern epi-fluorescence microscope

17. 1. Light source – epi-
fluorescence lamphouse
2. Light of a specific
wavelength (or defined
band of wavelengths), is
produced by passing
multispectral light from
an arc-discharge lamp
through a wavelength
selective excitation filter
3. Wavelengths passed
by the excitation filter
reflect from the surface
of a dichromatic (also
termed a dichroic)
mirror or beamsplitter
through the microscope
objective to bathe the
specimen with intense
light
Working of the Fluorescence Microscope

18. Working of the Fluorescence Microscope
4. If the specimen
fluoresces, the
emission light
gathered by the
objective passes back
through the
dichromatic mirror
5. It is Filtered by a
barrier (or emission)
filter, which blocks the
unwanted excitation
wavelengths

20. The “cube”

21. 1. Excitation light travels
along the illuminator
perpendicular to the
optical axis of the
microscope
2. The light then
impinges upon the
excitation filter where
selection of the desired
band and blockage of
unwanted wavelength
occurs.
Working in greater detail

22. 3. Fluorescence emission
produced by the
illuminated specimen is
gathered by the objective
4. Because the emitted
light consists of longer
wavelengths than the
excitation illumination, it
is able to pass through
the dichromatic mirror
and upward to the
observation tubes or
electronic detector.
Working in greater detail

23. The Dichroic Mirror
•The excitation light reflects off the
surface of the dichroic mirror into the
objective.
•The fluorescence emission passes
through the dichroic to the eyepiece or
detection system.
dichroic, two color

25. The Dichroic Mirror
dichroic, two color
•Each dichroic mirror has a set wavelength value
-- called the transition wavelength value -- which
is the wavelength of 50% transmission.
•reflects wavelengths of light below the transition
wavelength value (90%)
•transmits wavelengths above this value. (90%)
•Ideally, the wavelength of the dichroic mirror is
chosen to be between the wavelengths used for
excitation and emission.

26. Total Internal Reflection in Prism
Same Principle used in
Dichromatic beam splitter

27. Modern fluorescence microscopes are capable of
accommodating between four and six fluorescence cubes.
This is where the “turret’s” come into picture.
The “cube”
A specific combination of
excitation filter, emission
filter and dichroic mirror
are needed

28. The Filters
Excitation Filters
•to select the
excitation wavelength,
an excitation filter is
placed in the
excitation path just
prior to the dichroic
mirror.
Emission Filters
•In order to more
specifically select the
emission wavelength
of the light emitted
from the sample and
to remove traces of
excitation light
Fig: Light path through the filter
cube in a fluorescence
microscope.

29. Stoke’s Shift
Excitation 495 nm
Emission: 520 nm
The emission spectrum of an excited fluorophore is usually shifted to longer
wavelengths when compared to the absorption or excitation spectrum
•The intensity of the fluorescence is very weak in comparison
with the excitation light (10
-3
to 10
-5
).
•The emitted light re-radiates spherically in all directions.
•Dark background is required to enhance resolution.

30. Stoke’s Shift
As Stokes' shift values increase, it becomes easier to separate
excitation from emission light through the use of fluorescence filter
combinations.
Remember
Dichoric
Mirror ???

31. Data for Alexa Fluor 555
•absorbs light in the yellow-green region
•produces yellow-orange emission
•to achieve maximum fluorescence intensity
•a fluorophore is usually excited at wavelengths near or at the peak of the
excitation curve,
•And detected at widest possible range of emission wavelengths that include
the emission peak

32. The radiation collides with the atoms in the specimen and electrons are excited to a
higher energy level. When they relax to a lower level, they emit light.
Principle of Fluorescence
1. Energy is absorbed by the atom which becomes excited.
2. The electron jumps to a higher energy level.
3. Soon, the electron drops back to the ground state, emitting a photon (or a packet
of light) - the atom is fluorescing.

34. Visualizing The Cytoskeleton using
Fluorescence Microscopy
An Example of Fluorescent Dyes

35. Two cytoskeletal elements examined:
Why use Fluorescence Microscopy ?
• can visualize and localize individual proteins
within a cell.
Actin Microtubules

36. Let us test the effects of different drugs on the
cytoskeleton and cell shape
Nocodazole prevents
microtubule
polymerization.
Nocodazole
Taxol binds and
stabilizes microtubules,
Taxol
Latrunculin prevents
actin polymerization.
Latrunculin
TPA/PMA causes a
dramatic rearrangement
of actin filaments
TPA/PMA

37. Visualizing the cytoskeleton
using fluorescence microscopy
1) Prepare samples:
Fixation - kills and immobilizes cells
A. aldehydes - cross-link amino groups in proteins
(formaldehyde, glutaraldehyde)
B. alcohols - denature proteins, precipitate in place
(methanol)
Permeabilization - detergents make proteins
accessible to staining reagents (Triton X100)

38. 2) Staining
Actin - phalloidin covalently linked to rhodamine
(red) - binds to filamentous actin only
Microtubules - immunofluorescence
1
o
ab: rabbit anti-tubulin; 2
o
ab: fluorescein anti-rabbit

39. 3) Fluorescence microscopy
excitation
emission
fluorescent molecule
Fluorochrome Excitation wavelengthEmission wavelength
Fluoroscein 490 - blue 520 - green
Rhodamine 550 – green 580 - red
Hoechst (stains DNA) 345 - UV 455 - blue
wavelength
ex em
intensity

40. Microtubules
= green
DNA
= blue
interphase
mitosis

41. mitosis

42. Green Fluorescent Protein (GFP)
An Example of tagging proteins

43. Green Fluorescent Protein (GFP)
•is found in a jellyfish
•Why important - because it allows us to
look directly into the inner workings of
cells
•It is easy to find out where GFP is at
any given time: we just have to shine
ultraviolet light, and any GFP will glow
bright green
•So, we can attach the GFP to any
object that you are interested in
watching say
–a virus. Then, as the virus spreads
through the host, we can watch the
spread by following the green glow.
•engineer the cell with the genetic
instructions for building the GFP
protein, and GFP folds up by itself and
starts to glow.

44. These transgenic mice express
enhanced green fluorescent protein
under the control of a chicken beta-actin
promoter and cytomegalovirus enhancer

45. Why do this ??
developing transgenic mice to identify
critical neuronal subpopulations and
target them for electrophysiological
recordings and biochemical analyses.

46. Some Pictures

48. Cotton
A cross section of cotton
stained with Rhodamine B.
Mammalian Cells
Fluorescence double-labeling of mammalian
cells. The DNA in the cell nuclei are shown in
blue. Cytoplasmic fiber structures
(microfilaments) are shown in green.
Photo: Petra Björk, Stockholm University

49. Researchers tag
proteins with
fluorophores to
study the motion
of these
molecules.
However, this
creates an
extremely
complex motion
picture (for
example, in this
image different
colored particles
move
independently)

50. http://nobelprize.org/physics/educational/microscopes/fluorescence/fm.html
Control of a fluorescence microscope

51. Figure 3: Problems with Fluorescence microscopy

52. Summary
•sample you want to study is itself the light
source
•study specimens, which can be made to
fluoresce.
•The sample can either be fluorescing in its
natural form like chlorophyll and some
minerals, or treated with fluorescing
chemicals.

53. Future
•rapid expanding technique
•in the medical and biological sciences.
•certain antibodies and disease conditions
or impurities in inorganic material can be
studied with the fluorescence microscopy.

54. Some Pictures

55. Parainfluenza

58. Influenza

63. Fluorescent antibody detection

66. Fluorescent Antibody Staining

68. Fluoresence of chlorophyll-protein complex –
Cytochrome b
6
f
•a protein containing
a single chlorophyll a
molecule is excited
with polarized light
•fluorescence is
detected under 90O,
with a polarizer either
parallel or
perpendicular to the
original polarization
direction.

72. Resources
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland,
20657.
Michael W. Davidson - National High Magnetic Field
Laboratory, 1800 East Paul Dirac Dr., The Florida State
University, Tallahassee, Florida, 32310.