Fluorescence spectroscopy (RD)

Fluorescence spectroscopydoes 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 principles1. 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 vibrationallevels 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

A – photon absorption

VR – vibrational relaxation ~ 1 ps(excess energy - transferred from the molecule to the surroundings as heat)

IC – internal conversion 100 - 500 fs- between electronic states of the same spin multiplicity (no energy transfer to the surrounding)

ISC – intersystem crossing ns to µs- like IC but between electronic states of the different spin multiplicity

F – fluorescence emission – ns- 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

2. Jablonski diagram

A

VRIC

ISC

F

Electronic ground state

singlet states

triplet state

P

Microscopy (RD)

Light microscopy1 Microscopy: history

"Microscope" was first coined by members of the first "Academia dei Lincei" a scientific society which included Galileo

• simple microscopes (magnifying glass) - 2 µm resolution• best compound microscopes (objective and ocular) -around 5µm because of chromatic aberration

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…

first compound microscopes

embryology and early histology

famous microscopicalobservation

“Micrografia” 1665

first achromatic

lens

Spherical aberrations

problem - solved

1877 Abbe & Zeiss – oil immersion

* 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.

1886 Otto Schott: first

“achromatic” objective

Abbe’s Law

1.1 Earliest microscopes

1.2 Modern microscopes

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)

Köhler: the use of shorter wavelength light (UV) can improve resolution

2 DefinitionsAbsorption - intensity is reduced

depending on the color absorbed (the selective absorption of white light produces colored light)

Refraction - change of direction due to different optical density (light bends)

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

Diffraction - light rays bend around edges

Reflection and refraction: Snell’s law

short λ-s are “bent” more than long λ-s

colors separate - red is least refracted- violet most refracted

He sees the fish here

… but it is here

red filter

red lightno blue/green

Colors in white lightColors in white light Color of light absorbedColor of light absorbed

white

red

bluegreen

magenta

cyan

yellow

blue

blue

blue

blue

greengreen

green

green

redred

redredblack

gray green bluepink

θi = θr regardless of the surface materialθt depends on the composition of the

material n1sinθi = n2sinθt

velocity of light in a material is c/n

Microscopy (RD)

θtθi

θr

Incident beam

Reflected beam

Transmitted(refracted) beam

n1 n2

3 Thin lenses

idea of “burning glass”; photography

inverted and real

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

Magnification m = b/a

Lateral resolution d = 1.22 (λ/2NA)Rayleigh criterion

defines a “resolution element”in a medium of refractive index n

λ λ/n

intensity profile:

I↑ with NA↓

At the limit of resolution

Airy discs:(direct and diffracted light from small details)

numerical aperturewider angle of light received by the lens ⇒ greater resolving powerhigher 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

specimen

lens

angle = 2 α

Microscopy (RD)

4 Aberrations: result in faults in the image (monochromatic and chromatic)

Spherical (or geometrical) - related to the spherical nature of the lens: leads to “two” focal lengths

Solution: use the center part of a lensor a correcting lens:

Coma: streaking radial distortion for object points away from the optical axis

Astigmatism: a perfectly symmetrical image field is moved off axis, it becomes either radially or tangentially elongated

Chromatic aberrations: the wavelengths composing the white light are refracted according to their frequencies (dispersion)

results in colored fringes surrounding the image (blur)

longitudinal

axial

Solutions: Doublets (achromats) - each lens has a different refractive index and dispersiveproperties; bring 2 of the wavelength groups into a common focal planeApochromats - with fluorospar; bring 3of the wavelength groups into a common focal plane Triplets: 3 lenses cemented together

Microscopy (RD)

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)

5 Basic microscopy

Conventional microscope

focal lengthof objective= 45 mm

mechanicaltube length= 160 mm

object toimage distance = 195 mm

Bright fieldsource

Epi-illuminationsource

Inverted microscopeUpright microscope

Epi-illuminationsource

Bright fieldsource

Microscopy (RD)

Finite optics system

Sample

Intermediate Image

Objective

Other optics

Ocular

Sample

Primary Image Plane

Objective

Other optics

Ocular

Tube Lens

InfiniteImageDistance

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)

Microscopy (RD)

arc lampor laser

slitsample

Dark-field microscopy

“cardioid” condenser

objective

irisdiaphragm

Slit ultramicroscope

diffractedlight

Scattered light depends on: particle volumerefractive indexlight wavelengthangle of observation

Shape: - anisometric particles: fluctuate in orientation ⇒ twinkling effect- spherical particles: steady light

minimum detectable size: ≈ 50nm(for metal particles - 5 - 10nm)

specimen

6 Dark field microscopy

blocks of the central light rays and allows only oblique rays to illuminate the specimen

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

Objectives

7 Reflected light microscopy (incident light, epi-illumination, metallurgical microscopy)

Application: for imaging specimens that remain opaque even below thickness of 30µm

Optimal performance : with Köhler illumination

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)

9 Phase contrast microscopy (Zernike 1930)background light and light that interacts with the specimen take separate paths

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 the central spot

the central opaque darkfieldstop is replaced with a transparent, colored, circular stop

a transparent ring of a contrasting color

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

Another solution: slow down (negative or bright contrast)

conjugate to the rear focal plane of the objective

(phase shifter)

Needed accessories:

phase contrast condenser equipped with annuli with a rotating turret of annuli (with increasing magnification of the objective the annulus diameter should be increased)

a set of phase contrast objectives, each of which has a phase plate installed (a darkened ring on its back lens)

Microscopy (RD)

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

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

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

vibrate perpendicular to each other; with different direction

ray wave paths are altered in accordance with the specimen’s varying thickness, slopes, and refractive index

combines the two beams (the upper prism can be moved horizontally for varying optical path differences)

second polarizer (analyzer): makes the beams interfering, brings the vibrations of the beams of different path length into the same plane and axis

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)

Isotropic : the refraction index is equal in all directions

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

Polarized light microscopy

Incident light

Polarizer

Specimen

2nd Polarizer

Ocular lens no light gets through, except if its plane of polarization is shifted by passing through a birefringentstructure

11 Polarized light microscopy

plane polarized light

High-density columnar-hexatic liquid crystalline calf thymus DNA (10x)

High contrast: bright image on a dark background

Birefringent objects have regular arrays of non-spherical (elongated) structures e.g., mitotic spindle, muscle, etc.

Hair cross section from a mouse (20x)

crossedat right angles

Retardation = Thickness x Birefringence

Microscopy (RD)

captures the image projected directly onto a computer chip

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

12 Digital video microscopy

Features to be considered:

sensitivity of the camera and quantum efficiency (up to 70%)

signal to noise ratio (depends on cooling of the chip), spectral response,

dynamic range capability and speed of image acquisition and readout (10 – 20MHz), linearity or response, speed of response in relation to changes in light intensity

Binning : joining adjacent into super pixels to speed readout

a single pixel element is composed of four dyed photodiodes

pixel size: 4 – 14 µm

Microscopy (RD)