Lecture 19
Principles of optical microscopy
Illumination conjugate planes
are shown in red; an image of
the lamp filament is in focus at
these planes
Imaging path conjugate planes
are shown in red; an image of
the specimen is in focus at
these planes
http://www.microscopyu.com/articles/formulas/formulasconjugate.html
Transillumination
Field diaphragm: will affect size of
field that is illuminated at the focal
plane
Condenser aperture: will affect
the numerical aperture of the
condenser
http://www.microscopyu.com/tutorials/java/conjugateplanes/index.html
Epi-illumination
Field diaphragm: will affect
size of field that is
illuminated at the focal
plane
Aperture diaphragm: will
affect the numerical
aperture of the objective for
illumination
Objective specifications
Brightfield or trans-illumination
microscopy
•
Simplest type of microscopy
•
Contrast provided by absorption
•
Biological specimens are not highly absorbing naturally
•
Use stains, which typically require fixation, i.e. cells no
longer alive
•
Used routinely in histopathology and hematology and
basic science studies for which looking at live specimen
is not crucial
Tissue histology
Blood cells
Phase contrast microscopy
• First microscopic method which allowed visualization of
live cells in action
• Nobel prize in physics was awarded to Frits Zernike in
1953 for its discovery
• It enhances contrast in transparent and colorless objects
by influencing the optical path of light
• It uses the fact that light passing through the specimen
travels slower than the undisturbed light beam, i.e. its
phase is shifted
Phase contrast microscopy
• Let S (red) be light passing through
medium surrounding sample and D
(blue) light interacting with
specimen. S and D typically
interfere to yield P (green), which is
what we can usually detect.
• P will be phase shifted compared to
S, but our eyes cannot detect phase
shifts.
• Phase contrast microscopy
effectively converts this phase shift
into an intensity difference we can
detect
Phase contrast microscopy
Phase
plate
•
•
•
•
Condenser annulus allows only ring of light to reach the condenser.
Light rays illuminating the sample but not interacting with it will go
straight through and be imaged along a ring at the back focal plane of
the objective
Light rays diffracted by the sample are phase shifted by a approximately
a quarter wavelength and will be scattered over a range of angles and
will generally not propagate in the exact forward direction
A phase plate at the back focal plane of the objective alters selectively
the phase and magnitude of the non-diffracted wave.
http://www.microscopyu.com/tutorials/java/phasecontrast/positivenegative/index.html (ex)
http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.htm (reference)l
Differential interference contrast
• Differential Interference Contrast
(DIC) microscopy converts phase
shift gradients across different
parts of a specimen into intensity
differences.
• Doesn’t suffer from some artifacts
seen in phase contrast
• Uses full NA of objective
http://www.olympusmicro.com/primer/techniques/dic/dicintro.html
DIC image of nematode embryo
• Mouse fibroblast embryo, 24.3 hour time
lapse video
http://www.microscopyu.com/moviegallery/livecellimaging/3t3/index.html
Principle of fluorescence
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.
Fluorescence Stoke’s shift
• Fluorescence emission peak wavelength is red-shifted
with respect to absorption peak wavelength
• This shift may vary typically from 5 to more than 100 nm,
depending on the electronic structure of the molecule
Advantages of fluorescence
• Highly sensitive method
• Simple implementation
• Highly sophisticated fluorescent probes
– Fluorescent dyes that accumulate in different cellular
compartments or are sensitive to pH, ion gradients
– Fluorescently tagged antibodies to specific cell features
– Endogenously expressed fluorescent proteins
» Really endogenous
NADH/FAD: enzymes involved in ATP production
structural proteins: collagen/elastin
amino-acids: tryptophan/tyrosine
» After gene modification
Green fluorescent protein and variants
Optical path of fluorescence microscope
Dichroic filter: reflects
excitation and transmits
fluorescence
http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html
You can image simultaneously or
sequentially the same sample at different
excitation emission wavelengths to look at
different cell components
• Cell nucleus stained
with blue Hoechst
dye
• Mitochondria stained
with Mitotracker red
• Actin cytoskeleton
stained with
phalloidin derivative
conjugated to Alexa
488 (green)
Photobleaching often limits the number
of exposures or the exposure time
Photobleaching is the irreversible photochemical destruction of the fluorescent
chromophores
Resolution is limited in thick specimens by
detection of out-of-focus fluorescence
• In a standard fluorescence
microscope, the excitation beam
illuminates uniformly a wide field of
the sample.
• If the sample is thick, fluorescence
will be excited within the focal
plane, but also within planes above
and below the focus.
• Some of this fluorescence will be
imaged onto the detector and will
result in a defocused-looking image
Human medulla
rabbit muscle pollen grain
fibers
Principle of confocal microscopy
In confocal microscopy two pinholes
are typically used:
OUT-OF-FOCUS PLANE
IN-FOCUS (OBJECT) PLANE
CONTAINING ILLUMINATED S POT
OUT-OF-FOCUS PLANE
"POINT"
S OURCE
OF LIGHT
CONDENS ER
LENS
BIOLOGICAL
S AMPLE
OBJECTIVE
LENS
– A pinhole is placed in front of the
illumination source to allow
transmission only through a small
area
– This illumination pinhole is imaged
onto the focal plane of the specimen,
i.e. only a point of the 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 fluorescence excited within the
focal plane of the specimen will go
through the detector pinhole
– Need to scan point onto the sample
"POINT"
DETECTOR
APERTURE
To create confocal image, scanning
is required
• Either specimen is scanned past excitation beam or laser beam is
scanned across specimen
• For biological experiments, it is most common to scan the laser
beam across focal plane using a combination of two galvanometricdriven mirrors
Optical train of a confocal
microscope
LAS ER
BEAM
BEAM
S PLITTER
TARGET
S URFACE
RAS TER
PLANE
MICROS COPE
OBJECTIVE
GALVANOMETRIC
S CANNER
RAS TER
LINE
POLYGON
S CANNER
CONFOCAL SCANNING LASER MICROSCOPE
Optical train of a confocal
microscope
AVALANCHE
PHOTODIODE
WITH PINHOLE
TARGET
S URFACE
RAS TER
PLANE
MICROS COPE
OBJECTIVE
GALVANOMETRIC
S CANNER
LAS ER
BEAM
BEAM
S PLITTER
RAS TER
LINE
POLYGON
S CANNER
CONFOCAL SCANNING LASER MICROSCOPE
LAS ER
BEAM
VIDEOTAPE
RECORDER
AVALANCHE
PHOTODIODE
WITH PINHOLE
BEAM
S PLITTER
VIDEO MONITOR
FRAME GRABBER
TARGET
S URFACE
RAS TER
PLANE
MICROS COPE
OBJECTIVE
GALVANOMETRIC
S CANNER
RAS TER
LINE
POLYGON
S CANNER
CONFOCAL SCANNING LASER MICROSCOPE
Elimination of out-of focus
fluorescence yields superior images
http://www.olympusfluoview.com/theory/confocalintro.html
A thick specimen can be optically scanned in three
dimensions and the images can be processed to yield
cross-sections along plane of interest, three dimensional
composites and animations
Pollen grain
http://www.olympusfluoview.com/java/scanningmodes/index.html
Hamster ovary Mouse intestine
cells
In vivo depth-resolved imaging is
possible
Tumor cells grown subcutaneously in mice, expressing Green Fluorescent
Protein
Blood vessels stained with Cy5-conjugated anti-PECAM antibody
Study interactions of tumor cells with their environment and potential
factors/drugs that affect processes, such as tumor growth or metastasis
Video rate microscopy captures
dynamic interactions
• Monitor cell-cell, cellenvironment
interactions in natural
environment to
understand animal and
human biology and
processes involved in
disease development
• Monitor dynamic
interactions
In Vivo Reflectance Confocal
Microscopy of human skin
ROTATABLE HEAD
MECHANICAL ARM
3-AXIS
TRANSLATION
STAGE
OBJECTIVE LENS
HOUSING
RING-AND-TEMPLATE
(attached to skin and
locks into the housing)
VivaScope by Lucid
Courtesy of S. Gonzalez
OPTIMUM RANGE PARAMETERS
FOR RCM OF HUMAN SKIN
•Wavelengths
•Refraction index medium
•Objective lenses
400-700 nm (visible)
800-1064 nm (NIR)
1.33 (water)
30 -100X, 0.7-1.2 NA
•Detector aperture diameter
100-200 µm
•Imaging Rate
10-30 frames/sc
•Illumination Power
up to 40 mW
•Tissue Stability
M-T clamping fixture
Reflectance-mode Confocal Microscopy
Live Normal Skin
“En face” SECTION
H&E
Confocal in vivo
SC
SG
SS
DEJ
VERTICAL
Hematoxylin &
Eosin stained
section of tissue
Rajadhyaksha M, González S, et al.
J Invest Dermatol 1999;113;293-303.
180 µm
100x, 1.2NA
Courtesy of S. Gonzalez
Elongated nuclei Monomorphism
Uniform Polarization of nuclei
60 x, 0.85 NA
x
y
20 µm
Stained in vitro section
In vivo confocal
250 µm
Courtesy of S. Gonzalez
OVERALL SENSITIVITY AND
SPECIFICITY
Criteria
Sensitivity %
Specificity %
Elongated monomorphic nuclei
100
71
Polarized nuclei
92
97
Inflammatory infiltrate
83
55
Increased vascularity
88
54
Pleomorphism
64
64
2 or more criteria
100
54
3 or more criteria
94
78
4 or more criteria
83
96
Results remained reliable across study sites and across Basal Cell
Carcinoma subtypes.
Combination of clinical photograph examination and reflectance confocal
microscopy evaluation significantly improved non-invasive diagnosis of BCC
Multiphoton microscopy
•At very high photon densities, it becomes possible for two or more photons to be
simultaneously absorbed
•Each multiple absorption induces a molecular excitation of a magnitude equivalent to
the sum of the absorbed photon energies
http://www.aep.cornell.edu/drbio/MPE/mpe.html
Multi-photon fluorescence: Basic principles
•
•
•
•
•
Multi-photon excitation is a nonlinear process
Because two photons are required for each excitation, the rate of two-photon
absorption depends on the square of the instantaneous intensity.
Because of the large intensities required, high power lasers providing very short
pulses (~100 fs) are used, so that peak intensity is high, but average power doesn’t
damage the specimen.
We have photon flux densities sufficiently high for multiple photons to arrive
“simultaneously” (in 10-15 s) at an excitable molecule (of 10-16 cm2 cross section)
only at the focus point of a beam.
The probability that a given fluorophore at the center of a focused beam absorbs a
photon pair during a single pulse is
2
2
1   * NA 
 
na   P Fp 
 hc 
 is the two - photon absorption cross - section
2
P is the average power
NA is numerical aperture
Fp is the repetition frequency

p2
p
2
is known as the two - photon advantage
Advantages of multi-photon
excitation
• With a single-photon source excitation occurs
throughout the beam profile
• With a two-photon source excitation events are limited to
the beam focus
• Focal point restriction of excitation automatically
provides 3-dimensionally resolved submicron information
• Photodamage is restricted to the focal plane
• Not necessarily to refocus the fluorescence through an
aperture
– Simpler, more efficient optical detection design
– Scattering in thick specimens degrades signal to a smaller extent
• UV absorbing molecules can be excited using practical
visible/NIR wavelength ranges
Second Harmonic Generation (SHG)
http://www.aep.cornell.edu/eng10_offsite.cfm?URL=http%3A%2F%2Fwww%2Edrbio%2Ecornell%2Eedu
• SHG can be thought off as the scattering equivalent of two-photon excited
fluorescence
• The emitted photons are at exactly half of the wavelength of the incident
radiation (as excitation  changes, emitted SHG signal  also changes)
• The SHG signal is phase matched to the incident radiation and it is
emitted in a highly directional fashion, which depends on the size, shape
and refractive index of the scatterers. (fluorescence is incoherent and
isotropic)
Instrumentation
http://www.aep.cornell.edu/eng10_offsite.cfm?URL=http%3A%2F%2Fwww%2Edrbio%2Ecornell%2Eedu
Multi-photon imaging is the method of
choice for looking at endogenous
fluorescence in thick biological specimens
Two-photon excited fluorescence (TPEF) is
particularly useful in imaging of endogenous
weak fluorescence
Second harmonic generation (SHG) yields
excellent intrinsic contrast for imaging of
asymmetric molecules, such as collagen
http://www.drbio.cornell.edu/Infrastructure/MPM_WWW/MPM_hist/home.htm
4-Pi, two-photon fluorescence
microscopy
• Combines localized excitation with
coherent fluorescence detection to
beat resolution limit
• Resolution achieved 80 nm
Mitochondrial network of a live yeast cell
Gugel et al. Biophys J 2004; 87:4146-4152