Transcript Fluorescence, confocal microscopy

DIC, fluorescence and
confocal microscopy
Department of Mechatronics
GIST
Yong-Gu Lee
References:
1. “Fundamentals of light microscopy and electronic imaging,”
Chapter 10,11,12, Douglas B. Murphy, Wiley-Liss, 2001
2. http://www.bio.unc.edu/courses/2005Spring/Biol188/
A DIC microscope is a polarizing
microscope with condenser and
objective DIC Prisms
N-S
DIC Prism
DIC Prism
E-W
DIC (Differential Interference Contrast)
How does DIC differ from phase and polarizing?
Comparison of phase contrast to DIC for
cheek cell
What are 3 major features of a DIC image?
• Contrast is directional: maximum in one direction
and minimum in the orthogonal direction
• Contrast highlights edges; uniform areas have
brightness of background
• In direction of contrast, one edge is brighter, the
other darker than the background
The DIC
microscope is
a dual-beam
interferometer
made with
polarization
optics
(shear =
0.15-0.6nm
depending on
NA)
Shear
The DIC microscope is a dual-beam
interferometer made with polarization optics
The condenser DIC prism splits
illumination light into 2 divergent
orthogonal polarized beams
a
Prism is oriented with the optic axes at 45o to polarizer.why?
Divergent beams from condenser prism
pass through specimen as parallel beams
Image
intensity for
test specimen
with no
compensation
Image
intensity for
test specimen
with plus
compensation
Image intensity
for test
specimen with
minus
compensation
Comparison of
DIC image
intensity for
test specimen
with no, plus
and minus
compensation
Physical basis of fluorescence
• Molecules that are capable of fluorescing are
called fluorescent molecules, fluorescent
dyes, or fluorochromes. If a fluorochrome is
conjugated to a large macromolecule
(through a chemical reaction or by simple
adsorption), the tagged macromolecule is
said to contain a fluorophore, the chemical
moiety capable of producing fluorescence.
Fluorochromes exhibit distinct excitation and
emission spectra that depend on their atomic
structure and electron resonance properties.
Basic concept of absorption and emission
Jablonski diagram showing energy levels
occupied by an excited electron within
fluorescent molecule (chlorophyll a)
•
Chlorophyll a is unique in absorbing
blue and red wavelengths of the visual
spectrum. Blue photons are excited to
a higher energy level than are red
ones (straight upward arrows, left),
but the collapse to the ground state
by an electron excited by either
wavelength can occur through any of
the following three pathways:
Chlorophyll can give off a photon
(fluorescence emission, straight
downward pointing arrow); it can
release vibrational energy as heat
without photon emission (internal
conversion, wavy downward pointing
arrows); or its electron can enter an
excited triplet state (intersystem
crossing, dotted downward arrow),
which can make the molecule
chemically reactive. Electrons in the
triplet excited state can return to the
ground state through internal
conversion or by emission of
phosphorescence. Refer to the text
for details.
Stokes shift
Properties of fluorescent dyes
• An important criterion for dye selection is the molar
extinction coefficient, which describes the potential
of a fluorochrome to absorb photon quanta, and is
given in units of absorbance (optical density) at a
reference wavelength (usually the absorption
maximum) under specified conditions. The quantum
efficiency (QE) of fluorescence emission is the
fraction of absorbed photon quanta that is re-emitted
by a fluorochrome as fluorescent photons. QE varies
greatly between different fluorochromes and for a
single fluorochrome under different conditions. For
soluble fluorescein dye at alkaline pH, the quantum
efficiency can be as high as 0.9
Properties of fluorescent dyes cont’d
•
•
Quenching and photobleaching reduce the amount of fluorescence and are of
great practical significance to the microscopist.
Quenching reduces the quantum yield of a fluorochrome without changing its
fluorescence emission spectrum and is caused by interactions with other
molecules including other fluorochromes. Conjugation of fluorescein to a
protein usually causes a significant reduction in the quantum yield because of
charge-transfer interactions with nearby aromatic amino acids. Proteins such as
IgG or albumin that are conjugated with 5 or more fluorescein molecules, for
example, fluoresce less than when bound to 2–3 molecules, because energy is
transferred to nonfluorescent fluorescein dimers. Photobleaching refers to the
permanent loss of fluorescence by a dye due to photon-induced chemical
damage and covalent modification. As previously discussed, photobleaching
occurs when a dye molecule, excited to one of its electronic singlet states,
transits to a triplet excited state. Molecules in this state are able to undergo
complex reactions with other molecules. Reactions with molecular oxygen
permanently destroy the fluorochrome and produce singlet oxygen species (free
radicals) that can chemically modify other molecules in the cell. Once the
fluorochrome is destroyed, it usually does not recover. The rate of
photobleaching can be reduced by reducing the excitation or lowering the
oxygen concentration.
Basic concept of epi-fluorescence microscopy
Ploem-type epi-illuminator
Filter cubes
Basic design features
Exciter and barrier filters are
designed to separate emission
light from excitation light
Problems in filter Design: example
absorption and emission Spectra
The dichromatic mirror further
isolates the emission light from the
excitation light
Combined Transimttance
500
lambda
C
90%
A
500
lambda
B
A: Trasmittance
10%
400
500
400
500
400
500
90%
B: Trasmittance
10%
D
90%
90%
D: Trasmittance
C: Trasmittance
10%
10%
400
500
1
9
1
1
1
1
( A)  ( B)  (C )  ( D)  (C )  ( B)
10
10
10
10
10
10
1
9
1
1
1
9
9
( A)  ( B)  (C )  ( D)  (C )  ( B)  ( A)
10
10
10
10
10
10
10
Transmission
profiles of
filters in a
fluorescence
filter set
Confocal
laser
scanning
microscopy
Optical pathway in a confocal scan head
• EX and EM indicate
the paths taken by
the excitation and
fluorescence
emission
wavelengths.
• Photomultiplier
tube (PMT) detects
different
fluorescent
wavelengths
Pinhole aperture
The heart of confocal optics is the
pinhole aperture, which accepts
fluorescent photons from the
illuminated focused spot in the
raster, but largely excludes
fluorescence signals from objects
above and below the focal plane,
which, being out of focus, are
focused on the pinhole as disks of
much larger diameter. Because
the size of the disk of an out-offocus object is spread out over
such a large area, only a fraction
of light from out-of-focus objects
passes through the pinhole. The
pinhole also eliminates much of
the stray light in the optical
system. Examine the figure
carefully to see how the pinhole
blocks out-of-focal-plane signals.
Spatial filter
Tandem scanning confocal microscopy
•
The Yokogawa design features
two disks each with > 20,000
pinholes that rotate as a single
unified piece around a central
axis. The upper disk is fitted
with microlenses that focus
incident rays on a pinhole in the
second disk. The pinholes of
the disk are confocal with the
specimen and the surface of an
electronic imager such as a
charge-coupled device (CCD)
camera. A fixed dichroic mirror
positioned in between the
rotating disks transmits
excitatory wavelengths from the
laser while reflecting
fluorescent wavelengths to the
camera