Fluorescence spectroscopy does not provide detailed structural information but: acute sensitivity to changes in structural and dynamic properties approaches: steady-state emission intensity: complexation and conformational phenomena; modest requirements in instrumentation time-resolved studies: kinetics
1. Basic principles 1. Franck-Condon principle: nuclei are stationary during transition; transitions occur to vibrationally excited states; QM: the intensity / probability of a vibronic transition is proportional to the square of the overlap between the vibrational wavefunctions of the two states 2. Emission occurs from the lowest vibrational level of the lowest excited singlet state because relaxation from the excited vibrational levels is much faster than emission 3. Stokes shift: emission is always of lower energy than absorption due to relaxation in the excited state (energy conservation) 4. Mirror image rule: emission spectra are mirror images of the lowest energy absorption band
2. Jablonski diagram A – photon absorption singlet states VR – vibrational relaxation ~ 1 ps (excess energy - transferred from the molecule to VR the surroundings as heat) IC triplet state IC – internal conversion 100 - 500 fs - between electronic states of the same spin ISC multiplicity (no energy transfer to the surrounding)
A F ISC – intersystem crossing ns to µs - like IC but between electronic states of the different spin multiplicity
F – fluorescence emission – ns P - like IC but involves the loss of energy as light (a photon)
P – phosphorescence – µs to seconds - like fluorescence but occurs between states of difference spin multiplicity; spin change ⇒ slower Electronic ground state
Fluorescence spectroscopy (RD)
Light microscopy 1 Microscopy: history "Microscope" was first coined by members of the first "Academia dei Lincei" a scientific society which included Galileo
“Micrografia” 1665 1.1 Earliest microscopes
Leeuwenhoek: incorrectly called "the inventor of the microscope" ; created a “simple” microscope with magnification about 275x using simple ground lenses (compound microscopes could only magnify up to 20-30x); discovered bacteria, sperm cells, blood cells…
famous 1886 Otto microscopical Schott: first observation first “achromatic” compound objective microscopes
first 1877 Abbe & embryology Spherical achromatic Zeiss – oil and early aberrations lens immersion histology problem - solved
Abbe’s Law
• simple microscopes (magnifying glass) - 2 µm resolution • best compound microscopes (objective and ocular) - around 5µm because of chromatic aberration
* Geometrical or Spherical aberrations- due to the spherical nature of the lens * Chromatic- arise from variations in the refractive indices of the wide range of frequencies found in visible light.
Microscopy (RD) 1.2 Modern microscopes
¾ Early 20th century “Köhler Illumination”: evenly illuminated field of view while illuminating the specimen with a very wide cone of light (uses both a field and an aperture iris diaphragm to configure microscope illumination) ¾ Köhler: the use of shorter wavelength light (UV) can improve resolution
Colors in white light Color of light absorbed 2 Definitions Colors in white light Color of light absorbed blue ¾ Absorption - intensity is reduced red green depending on the color absorbed (the blue red green selective absorption of white light green red blue produces colored light) yellow blue magenta green red filter cyan red green red light black red blue no blue/green white gray pink green blue
¾ Refraction - change of direction due to different optical density (light bends)
short λ-s are “bent” more than long λ-s He sees the fish here colors separate - red is least refracted - violet most refracted
… but it is here
¾ Dispersion - separation of light into its constituent wavelengths - the change of refractive index with wavelength, such as the spectrum produced by a prism or a rainbow ¾ Diffraction - light rays bend around edges ¾ Reflection and refraction: Snell’s law
Transmitted ¾ θ = θ regardless of the surface material Incident beam i r (refracted) beam ¾ θt depends on the composition of the θi material θt θ ¾ n1sinθi = n2sinθt n1 r n2 ¾ velocity of light in a material is c/n Reflected beam
Microscopy (RD) 3 Thin lenses