http://hyperphysics.phy-astr.gsu.edu

light and matter act as both waves and particles (very difficult to reconcile the two views, both are equally valid in different experimental setups)

light or e-

bright spot

dark spot(objective lens)

Matter (e-) or radiation (photons) in our probe interacts with matter (sample) as waves.

http://ase.tufts.edu/chemistry/sykes/stephen/images/diffraction2.JPG

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

from Murphy 2001

The figure at bottom right isa 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.

image of grating

One full wavelength λ, one full cycle, 360 degrees or 2 π

Relative phase difference of 1/4 λ or 1/2 π.

resultant energy in space

resultant energy in space

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 1/2 λ or π.

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)

Phase contrast (no stain necessary but a special instrument called phase contrast microscope is needed)

Biology Department Victoria Junior College Murphy 2001

Transmitted light microscopy

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

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.

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)

Photoelectric effect; Einstein mathematically demonstrates light as a particle Robert Millikan tried to disprove this model but instead, his experiments (below) confirmed it

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.

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.

Emmett Ientilucci, Ph.D.Digital Imaging and Remote Sensing LaboratoryChester F. Carlson Center for Imaging Science

dim

bright

brighter

all have same # of photons

http://www.aps.org/programs/outreach/history/historicsites/millikan.cfm

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

~ .1nm or 1 angstrom

nuclei

Electrons (valence )

Electrons (inner shell or atomic orbital)

Hepatitis virus 28nm (UC Davis)

invitrogen

atom

e- beam,

light

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?

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.

energy from probe

energy re-emitted from sample

SEM (ACCV=30kV) Fluorescence microscope (500nm laser)

type of probe

energy of each quanta in probe (in eV) 30keV

type of ‘sample electrons’ affected

radiation or matter emitted upon return of e- to ground state

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

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)

Goldstein, 1992

‘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

‘e- emissions’

all these occur both in SEM & TEM

Goldstein, 1992braking radiation characteristic (elemental) x-ray peaks

Characteristic x-ray production is defined by atomic orbital e- transitions

Each of these lines can correspond to a peak in a

spectrum like the one at left

Y a

xis

is #

of

xra

y p

ho

ton

s

X axis is energy of xray photons

Can we can do x-ray spectroscopy in SEM? How about optical confocal microscope?

EKα = EK – EL Ekβ = EK-EMso EKα<Ekβ<EK<E beam e-

EK or EM here is ionization energy, the energy difference between these

= the observed x-ray energies

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

absorption and excitation are often the same phenomenon, absorption does not necessarily lead to emission however.

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

Foster 1997

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

Beer / Lambert relationship

Sheffield Hallam University

School of Science and Mathematics

Transmittance: T = P / P0

% Transmittance: %T = 100 T

A = log10 P0 / PA = log10 1 / T

Absorbance:P is power (as in Watts not energy as in eV or Joules P= E / time)

Remember, we can model colored glass filters (top) or quantities of fluorescent molecule in a cell with this relationship

A = ε (pathlength) x (concentration)

ε is molar extinction coefficient (units: L / mole cm)

A is linearly related to pathlength and concentration, %T is not.

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm

optical filters with different pathlengths

labeled nuclei with different concentrations

light

P0 P1

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)

Emission is broad and centered at ~650nm

Chlorophyll a

Vibrational states

Electronic states

heatThese internal conversions are very fast such that almost

all emissions from almost all molecules come from the non-vibrationally excited S1 electronic state

S1

S2

Size of some fluorescent markers and associated bio-molecules

Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics 1/2005 X. Michalet

streptavidin maltose binding protein

invitrogen

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?

More detection channels usually means better spectral resolution.

The M.U. MRC-1024 has 3 emission channels (3 PMT detectors) with spectral resolution of 30-40nm as defined by our filters

598/40nmR

G

B

522/35nm

680/32nm

http://www.olympusmicro.com/primer/techniques/fluorescence/fluorhome.html

http://www.invitrogen.com/site/us/en/home/support/Tutorials.html

Remember the optional but useful tutorial on Olympus and molecular probes (invitrogen) sites below the probes one has audio too