Download lecture 5 radiation and matter

light and matter act as both waves and particles (very difficult to reconcile the
two views, both are equally valid in different experimental setups)
http://hyperphysics.phy-astr.gsu.edu
Matter (e-) or radiation (photons) in our probe interacts with matter (sample) as waves.
diffracting objects
slits on top and
grating on the bottom.
All samples can be
considered diffracting
objects.
Most samples are
irregular so they
make an irregular
diffraction pattern. A
grating consists of
regularly spaced
parallel lines so it
makes a regular
diffraction pattern.
bright spot
dark spot
http://ase.tufts.edu/chemistry/sykes/stephen/images/diffraction2.JPG
bright spot
dark spot
(objective lens)
The figure at bottom right is
a diagram of a grating being
imaged by a transmission, whole
image at once (not scanning) type
microscope. Notice that the
diffraction pattern from the grating
does appear at the back focal
plane of the objective lens. The
energy from this pattern is
focused onto the image plane as
an image of the object, in this
case the grating.
light
or eimage of grating
from Murphy 2001
One full wavelength λ, one
full cycle, 360 degrees or 2 π
Combine black ray or wave with grey one and derive resultant (orange). All are
same wavelength, only relative phase and amplitude changes. Remember, this
energy is not destroyed, only redistributed to another place in space (where is not
always obvious).
Relative phase difference of
1/4 λ or 1/2 π.
resultant energy in space
Relative phase difference
of 1/2 λ or π.
resultant energy in space
In a phase contrast microsccope, a ring at the back plane of the objective advances undiffracted rays (those that do not interact
with the specimen) by one quarter wavelength. This shift combined with a ~one quarter λ retardation at the sample leads to 1/2
λ difference or destructive interference at the image plane; differences in phase shifting properties of sample can become
amplitude differences.
Amplitude and phase contrast in light or electron transmission microscopy
Phase object leads to interference when the reference wave (does not
interact with object) and object wave recombine. This interference
appears as amplitude (brightness) differences at the image plane of the
objective lens.
Murphy 2001
Amplitude contrast (stained)
Transmitted light
microscopy
Phase contrast (no stain
necessary but a special
instrument called phase contrast
microscope is needed)
Biology Department Victoria Junior College
Murphy 2001
Amplitude contrast in transmission electron microscopy (in TEM, phase
effects are important in creating amplitude contrast after scattering)
Specimen is very thin (<100nm) so absorption is not important,
SCATTERING is important.
In TEM we talk about electron dense and electron rare regions of our
sample. These identifiers are almost entirely a function of the
differential staining (and often non-specific) that we get in preparation
of sample; staining with uranium and lead salts at lower left. Specific
staining is also possible as in the antibody labeling (attached to gold
particles) at lower right.
TEM micrograph of
human red blood cell
(RBC) with Fab'-1.4 nm
gold particles attached
(arrow).
Magnification=300,000 X.
Bar=30 nm. Proceedings
of the forty-ninth Annual
Meeting, Electron
Microscopy Society of
America; G. W. Bailey
(Ed.). San Francisco
Press, San Francisco, CA,
pp. 284-285 (1991)
TEM of thin section
(40nm) imaged in
Dr. Wang’s lab.
Dark areas have
been stained more
heavily with heavy
metal stains of lead
and uranium, these
areas are said to be
more e- dense and
scatter e- more than
the light areas. To
reiterate, scattering
(diffraction) is more
important than
absorption
Amplitude contrast in scanning electron microscopy (in SEM, phase effects
are not important in creating amplitude contrast, however the final minimum
spot size of the e- probe is limited by interference effects)
This sample is stained with
the same type of gold label
seen as black spots in the
TEM of the RBC (two slides
previous). In this SEM
image of a mouse egg, we
can consider the e- in our
probe to be particles, not
waves. These e- create the
backscattered e- and
secondary e- that make the
metallic gold spots(arrows)
appear brighter than the
organic egg. We do not
need to consider the wave
phenomena of diffraction and
interference when
interpreting this image.
Photoelectric effect; Einstein mathematically demonstrates light as a particle
Robert Millikan tried to disprove this model but instead, his experiments (below) confirmed it
http://www.aps.org/programs/outreach/history/historicsites/millikan.cfm
This quantum explanation
describes many of the phenomena
that we see in scanning microscopy
including visible light and x-ray
emissions in confocal and SEM and
the function of the PMT in confocal
and SEM emission detectors.
all have same # of photons
brighter
bright
dim
Emmett Ientilucci, Ph.D.
Digital Imaging and Remote Sensing Laboratory
Chester F. Carlson Center for Imaging Science
The individual photons must deliver enough
energy such that the e- can overcome the
work function of the material, this work
function changes with retarding V.
diagrams thanks again to GSU hyperphysics
Physical dimensions. Typical organic bond lengths are on the order of .1 nm,
most covalent bonds are between .1 and .4 nm
atom
Electrons (valence )
light
e- beam,
Electrons (inner shell or
atomic orbital)
~ .1nm or 1 angstrom
Hepatitis virus 28nm (UC Davis)
nuclei
invitrogen
Changes of energy, such as the transition of an electron from one orbit to another around the
nucleus of an atom, is done in discrete quanta. Quanta are not divisible and the term quantum leap
refers to the abrupt movement from one discrete energy level to another, with no smooth
transition. There is no ``in-between'‘ in quantum descriptions of matter energy interactions.
However, practically speaking, electrons, atoms, and molecules all have individual environments
that blurs the situation somewhat. For example, electrons in molecules have discreet electronic
states and a wider variety of vibrational states that are dependent on their local environment
(temperature, pressure, other species, etc.). Also, for example, an e- can give up some of its
energy to an atom and continue on to deliver the rest to a different atom.
What is the wavelength of a bluish green photon?
500nm
What is the frequency of this photon?
What is the potential energy (P.E.)of an electron held at 30kV potential, in Joules?
What is the wavelength of a 30keV electron (all P.E. converted to kinetic energy)?
How fast does the e- move in a vacuum?
What is the energy of a bluish green photon?
How fast does this photon move in a vacuum?
Typical molecular bond energy, for example C-C ~ 1-5 eV
(1.6 x 10-16 - 8 x 10-16 Joules or ~100kcal/mole of bonds)
Typical atomic inner shell ionization energy (for example Carbon K (1) shell ) ~ 1keV
energy re-emitted
from sample
energy from
probe
SEM (ACCV=30kV)
type of probe
energy of each quanta in probe (in eV)
type of ‘sample electrons’ affected
radiation or matter emitted
upon return of e- to ground state
30keV
Fluorescence microscope (500nm laser)
Electron penetration trajectories in a typical SEM beam
acceleration voltage
varied (10, 20, & 30kV),
same sample.
accV constant, carbon at
left, iron at right
Elastic and inelastic events in EM, a beam e- can lose its kinetic energy in
many different events, each of these is, at least in part, a quantized transfer
A 30kV ELECTRON CANNOT GENERATE A 31kV SIGNAL
•
Elastic: backscattered electrons are beam electrons that scatter at angles
up to 90 degrees from initial beam trajectory (by definition >50eV)
•
Inelastic:
secondary e- (continuous energy distribution by definition <50eV)
x-ray production: continuum (bremsstrahlung or braking) or
characteristic
Auger e- are of specific energies based on elemental composition (like
characteristic x-rays) these are not quantitatively measured in our
JEOL5310LV SEM
visible light fluorescence (cathodoluminescence)
‘e- emissions’
‘braking x-radiation’ is emitted as e- are slowed by the
fields around nuclei of material, this radiation
increases with sample atomic # because larger nuclei
lead to more dense Coulombic fields
Goldstein, 1992
all these
occur both in
SEM & TEM
Characteristic x-ray
production is defined
by atomic orbital etransitions
Y axis is # of xray photons
EKα = EK – EL Ekβ = EK-EM
so EKα<Ekβ<EK<E beam eEK or EM here is ionization energy,
the energy difference between these
= the observed x-ray energies
Each of these lines can
correspond to a peak in a
spectrum like the one at left
X axis is energy of xray photons
braking radiation
characteristic (elemental) x-ray peaks
Goldstein, 1992
Can we can do x-ray spectroscopy in SEM? How about optical confocal microscope?
Simple absorbance
without emission usually
involves only vibronic
states, not electronic
state transitions,
reflection involves
capture and return of a
photon the mechanism
of which are well beyond
me and this course
absorption and excitation are often the
same phenomenon, absorption does not
necessarily lead to emission however.
Foster 1997
Assume for now that we are talking only about how our probe (laser) and sample interact. In
CSLM, all of the above occur and can be important. Think about how each would effect a focused
laser as it probes the sample. In CSLM, we normally image in fluorescence mode
The following are important considerations:
penetration depth, signal intensity, spatial resolution and image formation
Visible light fluorescence fluorochromes (fluorescent
molecules have the following important properties)
• absorption spectrum- (UV – long red)
• emission spectrum- (spectrally down shifted in energy from absorption spectrum)
• Stokes shift- (spectral shift mentioned above, the energy ‘lost’ in this shift appears
as thermal or chemical changes)
• quantum efficiency-
(#photons emitted / #photons absorbed)
• extinction coefficient (ε)-
(A = ε x concentration x path length)
A varies in a linear fashion with concentration [Molarity] and path length (cm)
• fluorescent yield -
this is of greatest practical importance and is a combination of
ε and quantum efficiency (quantum yield )
Assuming that the above parameters are the
Extinction coefficient and how we measure absorbed light in sample and filters
Transmittance: T = P / P0
% Transmittance: %T = 100 T
P is power (as in Watts not
energy as in eV or Joules
P= E / time)
Absorbance: A = log10 P0 / P
A = log10 1 / T
Beer / Lambert relationship
P0
A = ε (pathlength) x (concentration)
light
P1
optical filters with
different pathlengths
ε is molar extinction coefficient
(units: L / mole cm)
A is linearly related to pathlength and
concentration, %T is not.
Sheffield Hallam University
labeled nuclei with
different concentrations
Remember, we can model colored glass filters (top)
or quantities of fluorescent molecule in a cell with this relationship
School of Science and Mathematics
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm
S2
S1
Chlorophyll a
Emission is broad and centered at ~650nm
Vibrational states
Electronic states
These internal conversions are very fast such that almost
heat all emissions from almost all molecules come from the
non-vibrationally excited S1 electronic state
molecular e- transitions in chlorophyll define
absorption and emission spectra (Murphy text & G.
Weber and F. W. J. Teale, "Determination of the absolute quantum yield of
)
fluorescent solutions," Trans. Far. Trans., 53, 646-655, 1957
Size of some fluorescent markers
and associated bio-molecules
streptavidin
maltose binding protein
Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics 1/2005 X. Michalet
invitrogen
More detection channels usually means
better spectral resolution.
Nature Biotechnology 2006
How many emission channels are being displayed in these panels? What color is the light
signal that is coming from dKiema570-nu?
The M.U. MRC-1024 has 3 emission channels (3 PMT detectors) with spectral
resolution of 30-40nm as defined by our filters
R
598/40nm
G
522/35nm
B
680/32nm
Remember the optional but useful tutorial on Olympus and molecular probes
(invitrogen) sites below the probes one has audio too
http://www.olympusmicro.com/primer/techniques/fluorescence/fluorhome.html
http://www.invitrogen.com/site/us/en/home/support/Tutorials.html