Brightfield microscopy
• Generally only useful for stained
biological specimens
• Unstained cells are virtually
invisible
Oblique Illumination
Phase Contrast
http://microscopy.fsu.edu/
primer/techniques/
phasegallery/chocells.html
Aberrations
• Spherical aberration
– Most severe
– Immersion fluid
• Field curvature
• Chromatic aberration
• Astigmatism, comma
• http://micro.magnet.fsu.edu/primer/lightandcolor/
opticalaberrations.html
Phenomenon of fluorescence
Probes.invitrogen.com
Jablonski diagram:
Absorption of photon elevates fluorophore to excited singlet state
S1’
Nonradiative decay to lowest energy singlet excited state S1
Decay to ground state by emission of a photon
Non-radioactive decay to triplet state leads to
photobleaching
Molecular
Expressions
website
Ideal fluorophore characteristics
• High quantum efficiency
• Slow photobleaching
• For live cells: excitation wavelengths nonphototoxic to cells
• Little overlap with autofluorescence
– Mammalian cells: Flavoprotein, pigment
– Plant cells: chlorophyll
Fluorescent proteins: GFP
Tsien Lab
(UCSD)
How we observe fluorescence
• Black light
– Not enough sensitivity
• Filters
– Bleed-through
• Darkfield fluorescence microscopy
Epifluorescence microscopy
Nikon
Microscopy U
Essence of epifluorescence microscope:
Dichroic mirror
www.microscopyu.com
Examples of Fluorescence
Confocal Microscopy
The term “confocal” means “having the same focus”
This is accomplished by focusing the condensor lens to
the same focal plane as the objective lens.
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Reduced blurring of the image from light scattering
Increased effective resolution
Improved signal to noise ratio
Clear examination of thick specimens
Z-axis scanning
Depth perception in Z-sectioned images
Magnification can be adjusted electronically
Laser
• Acronym: Light Amplification by Stimulated
Emission of Radiation
• Ordinary light emission: Comes from spontaneous
decay of excited state to ground levels
• Stimulated emission: molecule remains in excited
state until stimulated to emit by incoming light
that is insufficient to raise it to the next higher
excited state
Conventional Fluorescence
Confocal
The difference in resolution can be
significant in those specimens which are too
thick to fit entirely within the focal plane of
the lens.
Optical section of
an aphid showing
internal structure
of an intact animal
If this is coupled with a point source of
illumination and a matching point source of
detection
Pinhole 1
Pinhole 2
Specimen
Detector
Condenser
Lens
Objective
Lens
Modified from: Handbook of Biological Confocal
Microscopy. J.B.Pawley, Plennum Press, 1989
Arc Lamp
Fluorescent
Microscope
Excitation Diaphragm
Excitation Filter
Ocular
Objective
Emission Filter
Confocal
Principle
Laser
Excitation Pinhole
Excitation Filter
PMT
Objective
Emission
Filter
Emission Pinhole
In a confocal microscope only a relatively
small portion of the specimen is
illuminated at a time whereas in a
conventional fluorescence microscope a
much broader area is illuminated.
If the source of illumination is truly a point and
it is focused to a point then only a single point in
the specimen will imaged at any one time.
Either the specimen must be moved to create a
complete view or the beam must be scanned in a
raster pattern.
One way to
accomplish this is to
pass the illumination
through a series of
pinholes that have
been arranged in a
pattern. As this disk
is spun it will create
a raster pattern and the light coming back
through the pinholes will be confocal. The
pinholes in a spinning disk system act as both the
point sources and confocal apertures.
Spinning disk confocals:
1) Can image in “real”
time provided that the
disk is spun quickly
enough
2) Can use a variety of
light sources
3) Can be retrofitted to
many existing
fluorescence
microscopes
Spinning disk
confocals:
1) Are inefficient and
require a very
bright illumination
and fluorescence
2) Cannot use
sensitive light
detectors such as
photomultiplier
tubes
Scanning Galvanometers
Point Scanning
x
Mirrors control
beam movement
in X/Y raster
pattern
y
Laser out
To
Microscope
Laser in
Some scan mirror systems are able to be
rotated which can result in a rotation of
the raster pattern
By creating an image in a point by point
manner the confocal microscope functions as a
point scanning/signal detecting device and like
an SEM magnification can be increased by
scanning a smaller portion of the specimen
Imperfections in
conventional light
optics usually
restrict useful zoom
to 6X or less.
A modern confocal system consists of the microscope,
associated lasers, the scan head with detectors and
confocal apertures and a computer system that controls the
scanning, adjusts the illumination, collects the signal,
displays the images and stores the data for later image
processing and analysis.
A laser scanning
confocal microscope
has many components
including a way for
several different lasers
to provide excitation
wavelengths and
several separate
detectors for various
emission wavelengths
Although confocal microscopy
can be done in the reflected
(light backscattered) or even
transmitted mode most
systems are optimized for
fluorescence.
Light Sources - Lasers
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Argon UV ArUV
Solid State Violet
Argon
Ar
Krypton-Ar ArKr
Helium-Cad HeCd
Helium-Neon GreNe
Helium-Neon HeNe
351-364 nm
405 nm
488-514 nm
488-568-648 nm
442 nm
543 nm
633 nm
Excitation - Emission Peaks
Fluorophore
DAPI
FITC
Bodipy
Tetra-M-Rho
L-Rhodamine
Texas Red
CY5
EXPeak EM peak
358
496
503
554
572
592
649
461
518
511
576
590
610
666
% Max Excitation at
488 568 647 nm
0
87
58
10
5
3
1
0
0
1
61
92
45
11
0
0
1
0
0
1
98
Since the illumination wavelengths available
are often limited the selection of matching
fluorochromes is very important.
In a conventional
confocal scan head the
photons returning from
the specimen are
separated based on
their energies (color) by
passing them through a
series of filters and
collecting each on
separate PMTs
One significant advance is how
some systems separate the
emission spectrum (signal) by
wavelength and using slits
sample those specific
wavelengths using separate
PMTs.
One of the advantages of having separate control
over the collection of different emission spectra
is the ability to create evenly balanced double,
triple, and even quadruple labeled images. Each
of the signals can be collected simultaneously
and merged afterwards.
– Spectral properties of the available dyes limit the
experimental freedom.
– Often it is even difficult to clearly separate two
fluorescence markers.
– With more markers, the problem grows increasingly
complex.
Cross-talk between the FP variants at the excitation and emisson level
Fluorescent Proteins are essential for life science studies.
However, overlapping emission AND excitation spectra and
corresponding crosstalk makes combinations difficult for imaging!
(especially true for multiphoton imaging)
Heavy overlap!
GFP and YFP (Distance of emission peaks ca. 12nm)
A431 cells expressing GFP, Rab11-YFP
GFP
YFP
overlay
This type of separation is nearly impossible
to accomplish with conventional filters,
especially for weakly fluorescent samples
By collecting a series of images of the
specimen at different distances from the lens
(focal planes) a through-focus series or “Zseries” can be created.
The ability to collect data in the X,Y, and Z
dimensions enables one to create an image
of the specimen as if it were be observed
from an orthogonal plane.
Stereo pair
images can be
created from a
stack of confocal
images by a
technique known
as “pixel shifting”
In pixel shifting two separate 2-D projections
of the data set are created by shifting adjacent
image planes slightly out of registry creating a
pseudo-left and pseudo-right projection.
The two images can be colorize to produce
an anaglyph stereo pair. (PC12 cell stained for
microtubules)
Stereo Anaglyph
Two dimensional
projection of
focus series
Pacific coral in backscattered light mode
Zebrafish
embryo
Muscle cells
3D Image Reconstruction
y
z
x
By accessing information in all three dimensions
a 3-D reconstruction of the data is possible
3D Image Reconstruction
y
y
z
y
z
x
3D Image Reconstruction
y
y
z
z
x
y
Conventional
Volume Rendering
Confocal
Fly Brain
Optical sections
of cornea
Volume renderings can be manipulated as if
they were actual 3-D specimens
Applications
• Probe Ratioing
– Calcium Flux (Indo-1, Fluo-3)
– pH indicators (BCECF, SNARF)
Molecule-probe
Calcium - Indo-1
Magnesium - Mag-Indo-1
Calcium-Fluo-3
Calcium - Fura-2
Calcium - Calcium Green
Phospholipase A
- Acyl Pyrene
Excitation
351 nm
351 nm
488 nm
363 nm
488 nm
Emission
405, >460 nm
405, >460 nm
525 nm
>500 nm
515 nm
351 nm
405, >460 nm
“Exotic” Applications
• Release of “Caged” compounds
• Fluorescence Recovery After Photobleaching
(FRAP) (UV line)
• Fluorescence Resonance Energy Transfer (FRET)
“Caged” Photoactivatable Probes
Nitrophenyl blocking groups e.g. nitrophenyl ethyl ester
undergoes photolysis upon exposure to UV light at 340-350
nm
Examples
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Glutamate
Norepinephrine
IP3
cAMP
cGMP
ATP
Ca++
Applications
• Organelle Structure
• & Function
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Mitochondria (Rhodamine 123)
Golgi (C6-NBD-Ceramide)
Actin (NBD-Phalloidin)
Lipid (DPH)