Detection systems. 1Introduction 2Theoretical background Biochemistry/molecular biology 3Theoretical...

Detection systems

1 Introduction

2 Theoretical background Biochemistry/molecular biology

3 Theoretical background computer science

4 History of the field

5 Splicing systems

6 P systems

7 Hairpins

8 Detection techniques

9 Micro technology introduction

10 Microchips and fluidics

11 Self assembly

12 Regulatory networks

13 Molecular motors

14 DNA nanowires

15 Protein computers

16 DNA computing - summery

17 Presentation of essay and discussion

Course outline

Scale: 100 μm

Optical microscopy

Watch out!A cover slide!

Life under a microscope

History of microscopy


1673

1720

18801665


Today’s microscopy

Bright-field microscopy

Also called resolving power

Ability of a lens to separate or

distinguish small objects that are close

together

Light microscope has a resolution of 0.2

micrometer

wavelength of light used is major factor

in resolution shorter wavelength greater resolution

Microscope resolution

produces a dark image against a brighter

background

Cannot resolve structures smaller than

about 0.2 micrometer

Inexpensive and easy to use

Used to observe specimens and microbes

but does not resolve very small

specimens, such as viruses


has several objective lenses (3 to 4) Scanning objective lens 4X Low power objective lens 10X High power objective lens 40X Oil immersion objective lens 100X

total magnification product of the magnifications of the

ocular lens and the objective lens Most oculars magnify specimen by a

factor of 10


Microscope objectives

Working distance

Oil immersion objectives

Bright-field image of Amoeba proteus

Uses a special condenser with an opaque

disc that blocks light from entering

the objective lens

Light reflected by specimen enters the

objective lens

produces a bright image of the object

against a dark background

used to observe living, unstained

preparations

Darki-field microscopy

Dark-field image of Amoeba proteus

Microscope image

Fluorescence microscopy

Lamps Xenon Xenon/Mercury

Lasers Argon Ion (Ar) 353-361, 488, 514

nm Violet 405 405 nm Helium Neon (He-Ne) 543 nm, 633 nm Helium Cadmium (He-Cd) 325 - 441 nm Krypton-Argon (Kr-Ar) 488, 568, 647 nm

Excitation sources

Irradiance at 0.5 m (mW m-2 nm-1)

Xe Lamp

Hg Lamp

Arc lamp excitation spectra

Dichroic Filter

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter

EPI-Illumination

Fluorescent microscope

transmitted lightwhite light source

630 nm band pass filter

620 -640 nm light

Standard band pass filters


520 nm long pass filter

>520 nm light

Standard long pass filters


575 nm short pass filter

<575 nm light

Standard short pass filters

Chromophores are components of molecules

which absorb light

E.g. from protein most fluorescence results

from the indole ring of tryptophan residue

They are generally aromatic rings

Fluorescence

S0

S1

T0

transition involving emission/absorption of photon

radiationless transition

abso

rptio

n

+hν

fluor

esce

nce

-hν

inte

rnal

co

nver

sion

inte

rsys

tem

cr

ossi

ngin

tern

al

conv

ersi

on

Jablonski diagram

S0

S’1Energy

S1

hvex hvem

Simplified Jablonski diagram

Fluorescence

The longer the wavelength the lower the

energy

The shorter the wavelength the higher the

energy e.g. UV light from sun causes the

sunburn not the red visible light

Ethidium

PE

cis-Parinaric acid

Texas Red

PE-TR Conj.

PI

FITC

600 nm300 nm 500 nm 700 nm400 nm

457350 514 610 632488 Common Laser Lines

Some fluorophores

495 nm 520 nm

Stokes Shift is 25 nmFluoresceinmolecule

Fluorescence Intensity

Wavelength

Stokes shift

Change in the energy between the lowest energy peak of absorbance and the highest energy of emission

The rate of emission is dependent upon the time the

molecule remains within the excitation state (the excited

state lifetime τf)

Optical saturation occurs when the rate of excitation

exceeds the reciprocal of τf

In a scanned image of 512 x 768 pixels (400,000 pixels) if

scanned in 1 second requires a dwell time per pixel of 2 x

10-6 sec.

Molecules that remain in the excitation beam for extended

periods have higher probability of interstate crossings and

thus phosphorescence

Usually, increasing dye concentration can be the most

effective means of increasing signal when energy is not the

limiting factor (i.e. laser based confocal systems)

Material Source: Pawley: Handbook of Confocal Microscopy

Excitation saturation

Defined as the irreversible destruction of

an excited fluorophore

Methods for countering photo-bleaching Scan for shorter times Use high magnification, high NA

objective Use wide emission filters Reduce excitation intensity Use “antifade” reagents (not compatible

with viable cells)

Photo-bleaching

Not a chemical process

Dynamic quenching

Collisional process usually controlled by mutual

diffusion

Typical quenchers

oxygen

Aliphatic and aromatic amines (IK, NO2, CHCl3)

Static Quenching

Formation of ground state complex between the

fluorophores and quencher with a non-fluorescent

complex (temperature dependent – if you have

higher quencher ground state complex is less

likely and therefore less quenching

Quenching

Fluorophore EXpeak EMpeak

% Max Excitation at488 568 647 nm

Excitation and emission peaks

Material Source: Pawley: Handbook of Confocal Microscopy

FITC 496 518 87 0 0

Bodipy 503 511 58 1 1

Tetra-M-Rho 554 576 10 61 0

L-Rhodamine 572 590 5 92 0

Texas Red 592 610 3 45 1

CY5 649 666 1 11 98

FITC 488 525

PE 488 575

APC 630 650

PerCP™ 488 680

Cascade Blue 360 450

Coumerin-phalloidin 350 450

Texas Red™ 610 630

Tetramethylrhodamine-amines 550 575

CY3 (indotrimethinecyanines) 540 575

CY5 (indopentamethinecyanines) 640 670

Probe Excitation Emission

Probes for proteins

Hoechst 33342 (AT rich) (uv) 346 460

DAPI (uv) 359 461

POPO-1 434 456

YOYO-1 491 509

Acridine Orange (RNA) 460 650

Acridine Orange (DNA) 502 536

Thiazole Orange (vis) 509 525

TOTO-1 514 533

Ethidium Bromide 526 604

PI (uv/vis) 536 620

7-Aminoactinomycin D (7AAD) 555 655

Probes for nucleotides

GFP

GFP - Green Fluorescent GFP is from the chemiluminescent jellyfish

Aequorea victoria excitation maxima at 395 and 470 nm (quantum

efficiency is 0.8) Peak emission at 509 nm contains a p-hydroxybenzylidene-imidazolone

chromophore generated by oxidation of the

Ser- Tyr-Gly at positions 65-67 of the

primary sequence Major application is as a reporter gene for

assay of promoter activity requires no added substrates

Many possibilities for using multiple

probes with a single excitation

Multiple excitation lines are possible

Combination of multiple excitation lines or

probes that have same excitation and quite

different emissions e.g. Calcein AM and Ethidium (ex 488 nm) emissions 530 nm and 617 nm

Multiple emissions

Effective between 10-100 Å only Emission and excitation spectrum must

significantly overlap Donor transfers non-radiatively to the

acceptor PE-Texas Red™

Carboxyfluorescein-Sulforhodamine B

Non radiative energy transfer – a quantum

mechanical process of resonance between

transition dipoles

Energy transfer

FRETIntensity

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence Fluorescence

ACCEPTOR

Molecule 1 Molecule 2

Fluorescence resonance energy tranfer

Confocal microscopy

confocal scanning laser microscope

laser beam used to illuminate spots on

specimen

computer compiles images created from

each point to generate a 3-dimensional

image

Confocal microscopy

Reduced blurring of the image from light scattering

Increased effective resolution

Improved signal to noise ratio

Clear examination of thick specimens

Z-axis scanning

Depth perception in Z-sectioned images

Magnification can be adjusted electronically

Benefits of confocal microscopy

Fluorescent Microscope

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter

Objective

Laser

Emission Pinhole

Excitation Pinhole

PMT

EmissionFilter

Excitation Filter

Confocal Microscope

The different microscopes

767, 1023, 1279

511, 1023

00Start

Specimen

Frames/Sec # Lines1 5122 2564 1288 6416 32

Scan path of the laser beam

Resolution

comparison

PK2 cells

stained for microtubules

stained for microtubules (green) and nuclei (blue)

Copapod appendage

Eye of Drosophila

http://www.confocal-microscopy.com/WebSite/SC_LLT.nsf?opendatabase&path=/Website/ImageGallery.nsf/(ALLIDs)/63BCB66085E1015BC1256A7E003B6DD3

Fibroblast



Spirogyra crassa

SEM and TEM

electrons scatter when they pass

through thin sections of a specimen

transmitted electrons (those that do

not scatter) are used to produce

image

denser regions in specimen, scatter

more electrons and appear darker

Electron microscope

Transmission electron microscope

Provides a view of the internal structure of a

cell

Only very thin section of a specimen (about

100nm) can be studied

Magnification is 10000-100000X

Has a resolution 1000X better than light

microscope

Resolution is about 0.5 nm

transmitted electrons (those that do not

scatter) are used to produce image

denser regions in specimen, scatter more

electrons and appear darker


TEM of a plant cell

TEM of outer shell of tumour spheroid

No sectioning is required

Magnification is 100-10000X

Resolving power is about 20nm

produces a 3-dimensional image of

specimen’s surface features

Uses electrons as the source of

illumination, instead of light

Scanning electron microscope

Contrast

Incident Electron Beam

Contrast formation

Ribosome

Ribosome with SEM

http://cellbio.utmb.edu/cellbio/cit1.htm

SEM of tumour spheroid

Fly head

Detection systems. 1Introduction 2Theoretical background Biochemistry/molecular biology 3Theoretical...

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