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 – 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 – – 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 solution: slow down (negative or bright contrast)

Needed accessories: Æ phase contrast condenser equipped with annuli with a rotating turret of annuli (with increasing magnification of the objective the annulus diameter should be increased) Æ a set of phase contrast objectives, each of which has a phase plate installed (a darkened ring on its back lens)

Microscopy (RD) 10 Differential interference contrast (DIC) (Nomarski 1950) “Nomarski optics”

second polarizer (analyzer): makes the combines the two beams interfering, brings the vibrations of beams the beams of different path length into the (the upper prism can same plane and axis be moved horizontally for varying optical path differences) ray wave paths are altered in accordance with the specimen’s varying thickness, slopes, and refractive index

vibrate splits the entering beam of polarized perpendicular to light into two beams traveling in each other; with slightly different direction (vary with different objective magnification: rotated by direction turret)

Contrast is highest at the edges of organelles, where the gradient of refractive index is steep

Phase contrast Differential Interference Contrast

The three-dimensional appearance is not representing the true geometric nature of the specimen, but is an exaggeration based on optical thickness. has a better resolution but is not suitable for accurate measurement of actual heights and depths.

Microscopy (RD) 11 Polarized light microscopy

Isotropic : the refraction index is equal in all directions

Anisotropic : birefringent - shift the plane of polarization Birefringence (double or bi-refraction):

Retardation = Thickness x Birefringence

Polarized light microscopy plane Incident light polarized light crossed Polarizer at right angles Specimen

2nd Polarizer no light gets through, except if its plane of polarization is shifted by Ocular lens passing through a birefringent structure

Hair cross section from a mouse (20x) High-density columnar-hexatic liquid crystalline calf thymus DNA (10x)

Birefringent objects have regular arrays High contrast: bright image on of non-spherical (elongated) structures a dark background e.g., mitotic spindle, muscle, etc.

Microscopy (RD) 12 Digital video microscopy captures the image projected directly onto a computer chip

expensive for the moment (a good camera = 20000 Euro); current resolution: up to 3900x3900px

Features to be considered: sensitivity of the camera and quantum efficiency (up to 70%) signal to noise ratio (depends on a single pixel element is cooling of the chip), spectral response, composed of four dyed dynamic range capability and speed of photodiodes image acquisition and readout (10 – 20MHz), linearity or response, speed of response in relation to changes in pixel size: 4 – 14 µm light intensity

Binning : joining adjacent into super pixels to speed readout

Microscopy (RD)