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 an object can be focused no closer than 25 cm from the eye (depending on your age) – normal viewing distance for 1x magnification focal distance idea of “burning glass”; photography Magnification m = b/a inverted and real numerical aperture wider angle of light received by the lens ⇒ greater resolving power higher NA ⇒ shorter working distance resolving power: ability of an objective to resolve two lines very close together NA = n sin(α); n - between the object and first objective element; α = 1/2 angular aperture of the objective; e.g. for λ = 550nm, n = 1, 40x objective, for narrow light beam i.e. closed field diaphragm sin(α) ≤ 0.65 ⇒ d > 0.5µm objective lens angle = 2 α Airy discs: (direct and diffracted light from small details) specimen intensity profile: I↑ with NA↓ Lateral resolution d = 1.22 (λ/2NA) Rayleigh criterion At the limit defines a “resolution element” of resolution in a medium of refractive index n λ λ/n Microscopy (RD) 4 Aberrations: result in faults in the image (monochromatic and chromatic) ¾ Spherical (or geometrical) - related to the spherical nature of the lens: leads to “two” focal lengths Solution: use the center part of a lens or a correcting lens: ¾ Coma: streaking radial distortion for object points away from the optical axis ¾Astigmatism: a perfectly symmetrical image field is moved off axis, it becomes either radially or tangentially elongated ¾Chromatic aberrations: the wavelengths composing the white light are refracted according to their frequencies (dispersion) Æ results in colored fringes surrounding the image (blur) axial longitudinal Solutions: Doublets (achromats) - each lens has a different refractive index and dispersive properties; bring 2 of the wavelength groups into a common focal plane Apochromats - with fluorospar; bring 3 of the wavelength groups into a common focal plane Triplets: 3 lenses cemented together Microscopy (RD) 5 Basic microscopy Conventional microscope mechanical tube length object to = 160 mm image distance = 195 mm focal length of objective = 45 mm Upright microscope Inverted microscope Epi- Bright field illumination source source Epi- Bright field illumination source source Eye pieces: look at the magnified (intermediate) virtual image and see it as if it were 25 cm from the eye; with inter-pupillary distance for personal focusing 5 to 15x magnification; Condenser: must focus the light onto the specimen fill the entire numerical aperture of the objective for objective with NA > 1.0 one needs oil on the condenser as well (except in inverted microscopes) Microscopy (RD) Finite optics system Ocular Intermediate Image Other optics Objective Sample Infinity optics system Main advantage: relatively insensitive to additional optics within the tube length Second: one can focus by moving the objective and not the specimen (stage) Ocular Primary Image Plane Tube Lens Infinite Other optics Image Distance Objective Sample Microscopy (RD) Slit ultramicroscope arc lamp or laser slit sample Dark-field microscopy objective diffracted light “cardioid” iris condenser diaphragm specimen minimum detectable size: ≈ 50nm (for metal particles - 5 - 10nm) Scattered light depends on: particle volume refractive index light wavelength angle of observation Shape: - anisometric particles: fluctuate in orientation ⇒ twinkling effect - spherical particles: steady light Objectives 6 Dark field microscopy Application: for imaging unstained specimens, which appear as brightly illuminated objects on a dark background When no specimen (and NAcondenser>NAobjective) dark field In terms of Fourier optics: removes the 0th order (unscattered light) from the diffraction pattern formed at the rear focal plane blocks of the central light rays and allows only oblique rays to illuminate the specimen 7 Reflected light microscopy (incident light, epi-illumination, metallurgical microscopy) Optimal performance : with Köhler illumination Application: for imaging specimens that remain opaque even below thickness of 30µm Amplitude specimens: absorption and diffraction by the specimen lead to readily discernible variations in the image (from black through shades of gray, or color) - Phase specimens: show little difference in intensity and/or color; their feature details are extremely difficult to distinguish; require special treatment or contrast methods Microscopy (RD) 8 Rheinberg illumination (100 year ago) medium power darkfield illumination using colored gelatin or glass filters to provide rich color Result: the specimen is in the color of the ring with a background of the color of a transparent ring of the central spot a contrasting color the central opaque darkfield stop is replaced with a transparent, colored, circular stop 9 Phase contrast microscopy (Zernike 1930) background light and light that interacts with the specimen take separate paths (phase shifter) phase objects: do not absorb light; slightly alter the phase of the diffracted light = retard it by about 1/4 wavelength (“out of phase”) because of specimen’s n Human eyes & cameras – insensitive to such phase difference Zernike’s: speed up the direct light undeviated light is advanced by the phase plate before interference at the rear focal plane of the objective conjugate to the rear focal plane of the objective Another