Microscopy

• closest distance on which human eye can focus

• 0,1 - 1,0 m

• average: 0,25m

• viewing angle of 2min

• two points in distance of 0,15mm can be viewed seperately at a viewing distance of 0,20m

• Vlens = tanb/tany = A’B’/AB = 250f

• 25cm = reference / conventional viewing distance

1) Spherical Aberration:

• parallel rays passing through fringes of lense

• are focused at different locations

• dependend of distnace from optical axis

  -> blurred image

2) Coma:

• point objects look like comets

• tail running to edge

• oblique bundling of parallel beam

3) Solutions:

• compound lenses with different color dispersion properties

• different type of glass: crown & flint

• ability to gather light & resolve fine sample details at fixed object distance

• NA = n*sin(sigma)

• n = 1: Luft

• n = 1,33: Wasser

• n = 1,56: Öl

  (refractive index)

• ability to distinguish between two distinct light sources as seperate items

• smallest distance between 2 points

• distinguishable as seperate items

• directly related to useful magnification & perception limit of sample detail

Classification:

• Purpose

• Microscopic Method

• Magnification

• Performance

• look down to see image

• high Magnification

• high Resolution at transmitted light sample

• short working distance

• designed for slide samples

  • cell biology

  • fixed samples

  • squeezed between slide & coverslip

• 1850

• popular technique: live cell imaging

• look up to see image: observed through bottom

• limited M

• lower R

• long working distance

• expensive

• for:

  • petridish sample

  • living cell incubation

  • cells at bottom of container

• 1957

• live cell imaging

• look down - top lit

• limited M, zoom in M

• low R

• very long working distance

• stereoskopic 3D effect = 2 path of light being entirely separated

1) Transmitted:

• developed first

• transmit light from source opposite of sample

• bright & darkfield

• phase contrast

• differential interference contrast from objective

2) Refracted light:

• incident light

• epi illumination

• industrial microscopy fluorescence

• for opaque samples

• dark background

• only object illuminated directly

• look from side into cone of light

• only light diffracted or scattered by object is seen

• fine structures

Vorteil:

• life samples, artefact free

Nachteil:

• low light level

• sensitive to contamination

• careful interpretation

• most used technique

• absense of contrast

• colored & non-viable

• light absorption in dense areas

• fine structures = not recognizable

Gut:

• simple to use

• only few adjustemts to microscope need

• easily adapted to new technologies

Schlecht:

• lack of contrast

• only inanimate, coloured specimen

• distortion due to use of aperture to adjust contrast

• requires coloring

• Microorg. without staining = reveals many cellular structures

• optics include: objectives & condenser

• Phase contrast techniques translates phase shifts into grey values

• perfectly aligned rings of annulus & phase plate with matching diameters

Double refraction:

• birefringence occurs in molecularly ordered materials: single ray of light split in 2 rays:

  • one unrefracted (O)

  • one reflected at angle (E)

• direction dependent dirfferences in refractive index

Anisotropic material:

• crystals

• ordered cellular structures: starch, cellulose fibers, actin filaments

Analyzer & Polaizer:

• polaroid sheets: incident waves parallel to transmission axis pass

• other rays: blocked

• image of light source falls into same plane as image of sample

Gut:

• enhanced contrast

• improved resolution

Schlecht:

• limited depth of field

• sample sensitivity

• Adjust image brightness

• put sample in obect holder

• turn gear knob to top stop

• bring objective to 10x in place

• close field diaphragm

• lower condenser

• center field diaphragm

• open field diaphragm

• focus image of fd with vertical adjuster

• in order to adjust aperture: take eyepiece off

• replace eyepiece in tube barrel

1) Application of prim. stain:

• crystal violet positive ions interact with negatively charged components of bacteria cell = cell stain blue

2) Application of Mordant:

• Iodine solution forms large crystal violet iodine complexes (CV-I) with cytoplasm & outer layer of cell

3) Decolorization:

• Ethanole / Ethanole-Acetone Mix interact with membrane lipids

  • loss of outer cell wall of gram negt cell = CVI whased off

  • dehydration of highly crosslinked peptidoglycan of gram post cell = CVI trapped inside

4) Application of counter stain:

• red dye safranin stains decolorized:

  • gram negt cells = red / pink

  • gram post cells = blue

• though wavelength of visible light

• light creates interference colors

• color gives idea about thickness of section

  -> preferred: color from silver to light gold (60-90nm)

• electrons instread of light

• higher energy = shorter wavelength = better resolution

• 0,05nm at 200kV (0,5A)

Gut:

• atomic resolution: 0.12nm (1,2A)

Schlecht:

• electrons need vacuum = no live cell imaging

• Electron gun: accelerates & generates electrons

• Condenser system: lenses to focus electrons into beam

• Image producing system: consists of sample & electromagnetic lenses to focus beam on fluorescent screen

• Images of sample by scanning surface with focused electron beam

• e-beam focused by 1 or 2 condeser lenses to spot about 0,4-0,5nm

• better resolution than: 1nm

• acceleration e to:

  • 0,2 - 40kV SEM

  • 300kV TEM

• Types:

  • tungsten filament

  • field emission gun

• cathodes form very sharp tip (100nm or less)

• very strong local field at tip: electrons can tunneö through potential barrier and become free

• tips remain at room T°C

• formed by elastically scattering of primary e with nuclei

• E >> 50eV

• worse image

• give info about samples components

• formed by elastic collision of primary e with shell

• E < 50eV

• highest quality

• greatst contrast

• elastically scattered e & unscattered e have same Energy after & before passing specimen

• unelastically scattered e have lower Energy after passing: E0 - deltaE

• use of omega filters to remove these electrons (with less energy)

• electrostatic charging of sample by electron beam

• prevention by ultrathin coating with gold, graphit, gold-palladium

• zB: insect

• need to be completely dry = high vacuum

• soft tissue/cells require fixation for stabilization

  = crosslinking with glutar di aldehyde

• only hard materials require little treatment

Gut:

• po > 60mbar & T°C > 0°C

• no charge of sample by electron beam

• biol. sample can be fresh & live

Schlecht:

• limited distance between sample & PLS (mm range)

• very good cytoplasm ultrastrict preservation

• may impair immunogenicity of proteins

• CL via schiff base reaction

• bifunctional agent

• rapid penetration

• good preservation of immunogenicity (freshly prepare from solit - paraformaldehyde)

• less effiecient crosslinking

• when goal is to: localize structures in cell using AB

• CL of unsaturated fatty acids in lipid proteins

• very good membrane preservation

• very good staining agent (high e density of Os)

Dehydration:

• for compatability with embedding resins: Ethanol or Acetone

  • 100% dehydration for epoxy

  • 70% for acrylis (LR white)

• causes shrinkage (minimalize by step by step dehydration)

• minimize lipid extraction by low T°C dehydration

Embedding:

Epon - epoxy resin - 60°C - hydrophobix - convt. ultrastructure

• extremely rapid freeting: 10^4-10^6 K/sec

• Vitrification (no ice crystals)

  
  

  1) Blotted grid plunged into cyrogen

  2) cyrogen precooled with liquid nitrogen -180°C

• lowering freezing point & supercooling T°C range

• 10^3 K/sec (vitrification)

• adequate freezing of samles up to 200micrometer without cryoprotectant

• substituation of vitreous ice in frozen specimens by acetone / methanol at below -90°C

• localisation of Antigens (proteins) in sectioned tissue

• using specific AB labelled with colloidal gold particles

• gold particles can be produced with narrow size distribution = multiple labelling possible

• emission of light by a substance that has absorbed light or electromagnetic radiation

• emission of light has longer wavelength than excitation light

Vorteil:

• high contrast

• specific labelling

• quantitative labelling

• labelling of living cells

• absorbtion / excitation (spectrum) maximum: stoke shift

• emission (spectrum) maximin: stoke shift

• molar absorption coefficient: how strongly fl. absorbs light (brightness)

• quantum yield (brightness)

• fluorescence lifetime

• photostability: before irreversible bleaching, blinking characterustics

• polarity (preferable water soluble)

Achromat:

• axial chromatic correction for 2 colors

• corrected for spericsl aberration

Fluorite (Semi-Apochromate):

• axial chromatic correction for 2 colors

• corrected for spericsl aberration (2-3 colors)

• fluorospar

• higher NA

• higher contrast

Apochromat:

• axial chromatic correction for 4-5 colors

• corrected for spericsl aberration (3-4 colors)

• more conjugated electron system

  -> small energy difference between states

  -> longer wavelength

• symmetric: small stokes shift

• heterocycles: longer wavelength

• rigid ring structure: no energy loss from excited state

1) Covalent:

• F chemically attached to protein

• use of different AA

  • Cystein: site specific

  • Lysine: stochastic

2) Non-Covalent:

• F not chemically attached to protein = intercolated into double helix

• F bound to actin filament = Organelle stains: Mito / ER-Tracker

• Calcium: calcium sensitive dyes (Fura-2) radiometric

• important immunochemical technique

• to determine location of Antigen in tissue by reaction with AB labelled with fluorescent dye

• Part of immunesystem: produced in mammals

Vorteil:

• many applications

• wide range of proteins

• wirde range of org. fluorophores

• easy to use

• localisation & expression of protein of interest can be determined

Nachteil:

• large label - 10nm

• may impair protein function

• for intracellular proteins = cells need to be fixed & permeabilized

• Direct: with fluorescently labelled primary AB that recognizes protein of interest

• Indirect: with fluorescently labelled secondary AB that recognizes primary AB

• fluorescent protein attached to protein of interest

  -> fluorophore genetically encoded

Gut:

• staining living cells

• stable cell lines with continious expression of fusion protein

Schlecht:

• not as bright & photostable

• background of wildtype protein

• rel. large GFP - 3nm

• may impair protein function

affected by:

• NA: higer NA = smaller PSF

• wavelength: short l = smaller PSF

unaffected by:

• Magnification

• Lichtmikroskopie

• Einsatz von Punktbeleuchtung & Lochblende (Pinhole) vor Detektor = hohe Auflösung & Kontrast

• Untersuchung von sehr dünnen opt. Schnittebenen

• single spot of focused light is rapidly transitioned along lateral axis of specimen

• no need for camera = point detector (PMT)

• fast scanning, collect single pixle in microseconds

• records pixel by pixel

Gut:

• thin optical sections through -50nm specimen

• high resolution 3D images of thick samples

• reduced background

• better signal-to-noise-ratio

Schlecht:

• slow

• not very efficient -> PTM detector 25% quantum yield, high intensity laser irradiation = sample photobleaching

• use of multiple pinholes & camera

• 2 rotating disk:

  • microlens disk

  • pinhole disk

• microlens focus light through 1000 pinhole in nipkow disk

Gut:

• CCD camera = very efficient (lower excitation intensity needed)

• fast - 1000 images / second = live cell imaging

Schlecht:

• pinhole crosstalk

• lower field uniformity

• higher background

• no single spot high power line illumination