hyperphysics.phy-astr.gsu

1. 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

2. 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 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. light or e- image of grating from Murphy 2001

3. 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.

4. 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

5. Transmitted light microscopy Phase contrast (no stain necessary but a special instrument called phase contrast microscope is needed) Amplitude contrast (stained) Biology Department Victoria Junior College Murphy 2001

6. 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)

7. 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

8. 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.

9. 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.

10. diagrams thanks again to GSU hyperphysics

11. 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 Electrons (inner shell or atomic orbital) e- beam, ~ .1nm or 1 angstrom Hepatitis virus 28nm (UC Davis) nuclei invitrogen

12. 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?

13. 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) 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

14. Electron penetration trajectories in a typical SEM beam acceleration voltage varied (10, 20, & 30kV), same sample. accV constant, carbon at left, iron at right

15. 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)

16. all these occur both in SEM & TEM ‘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

17. Characteristic x-ray production is defined by atomic orbital e- transitions EKα = EK – EL Ekβ = EK-EM so EKα<Ekβ<EK<E beam e- EK or EM here is ionization energy, the energy difference between these = the observed x-ray energies Y axis is # of xray photons Each of these lines can correspond to a peak in a spectrum like the one at left X axis is energy of xray photons characteristic (elemental) x-ray peaks braking radiation Goldstein, 1992 Can we can do x-ray spectroscopy in SEM? How about optical confocal microscope?

18. 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

19. 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 εand quantum efficiency (quantum yield ) Assuming that the above parameters are the

20. 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) A = log10P0/ PA = log10 1 / T Absorbance: Beer / Lambert relationship P0 P1 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. light optical filters with different pathlengths labeled nuclei with different concentrations Remember, we can model colored glass filters (top) or quantities of fluorescent molecule in a cell with this relationship Sheffield Hallam UniversitySchool of Science and Mathematics http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm

21. heat Vibrational states Electronic states S2 S1 Chlorophyll a Emission is broad and centered at ~650nm moleculare- 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) These internal conversions are very fast such that almost all emissions from almost all molecules come from the non-vibrationally excited S1 electronic state

22. streptavidin maltose binding protein Size of some fluorescent markers and associated bio-molecules Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics 1/2005 X. Michalet invitrogen

23. 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?

24. 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 680/32nm B

25. 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