1 Microscopy Technique

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Basic Microscopy Techniques

Instruments Department

NIKON SINGAPORE PTE LTD

Clement Khaw, Ph.D.


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From Genome to Human

Tissue

DNA

m-RNA

Ribosome

Genome

Protein

Protein

Functional Protein

Proteom

e

Cellome

Human

Microscope


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What is a Microscope?

The microscope performs three basic tasks

Produce a magnified image of the specimen

Separate the details of the magnified image

Render the details visible to the eye or camera


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Optical Techniques

Brightfield

Darkfield

Phase DIC

Epifluorescence


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BRIGHTFIELD


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Brightfield

Most common of all applications

Amplitude objects usually exhibit high natural

absorption, reflection & contrast. Biological materials are stained to produce these properties

Not recommended for unstained biological specimens ortransparent materials


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Darkfield


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Darkfield

a simple and popular method for making unstainedtransparent specimens clearly visible. Such objects

often have refractive indices very close in value to thatof their surroundings and are difficult to image inconventional brightfield microscopy.

For instance, many small aquatic organisms have arefractive index ranging from 1.2 to 1.4, resulting in a

negligible optical difference from the surroundingaqueous medium. These are ideal candidates fordarkfield illumination.


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DarkfieldBrightfield


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Phase Contrast


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Why cant we see living cellsin brightfield?

Little contrast (2-5%)

Very low light absorption

RI of cells similar tobackground

RI within the cells betweencytoplasm and nucleus issimilar

Phase Contrast


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Phase Objects- Transparent materials that absorb little lightbut produce a phase change in the light as it passes throughthe sample. The human eye cannot detect phase differences.

Phase changes are primarily due to thickness and RIvariations in the specimen structure

Live cells

Contrast-Ability to distinguish specimen detail when comparedto the background or adjacent features

Measured by the comparison between the highest andlowest intensity in an image

Positive contrast: Specimen darker than background

Negative contrast: Specimen brighter than background

Phase Contrast


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The specimen information is there but we cannot

see it.

Two main obstacles to overcome

Specimen information (higher orders of diffraction) is tooweak when compared to undiffracted signal

(background, 0 order)

Convert small phase shift in specimen detail to largechanges in intensity to visualize

Phase Contrast


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Converts minute changes in phase to large changes in

amplitude (grey values) which are viewed as

differences in contrast.

Phase Contrast


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Differential Interference Contrast

(DIC)


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DIC

Observation of transparent or low contrast specimens

Does not produce halo and has almost no affect onobjective performance in non DIC applications

Phase Contrast Issues

Halo obscures fine detail Phase plate reduces non phase specimen intensity by ~ 20-

30% depending on plate composition

Image sharpness with phase objectives is compromised innon phase applications


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Phase Contrast DIC


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DIC

DIC is a beam shearing interference system thatproduces a shadow-cast 3D image.

The shadow effect and contrast levels can be controlledby rotation of the polarizer or position of objective prism

DIC allows optical sectioning of the specimen

The 3D image is an optical illusion and is primarily basedon refractive index and path difference

Two types of prisms are used

Nomarski (Wollaston variation-Nikon system) & Wollaston

The shear is normally just below the resolution limit of the obj.

Does not work with plastic dishes or wells


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Phase Contrast DIC


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Why use Fluorescence?

Increased sensitivity

Improved signal-to-noise

Specificity Allows for imaging of single molecules

Allows for labeling biological specific structures, proteins or

even genes Allows for tagging of multiple structures in one cell

Viability

Can be used in live cells and tissues Quantification

Determine concentrations of specific proteins, calcium, or

measure pH


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Fluorescence

Def: The process by which a molecule absorbs the energy fromphotons of a certain wavelength and then emits photons of

light with a lower energy (longer wavelength).

- Higher energy (UV) to lower energy (IR).- Shorter wavelength (488nm) to longer wavelength (520nm)

- Higher frequency to lower frequency

> Emitted light is several orders weaker than the excitation energy> Stokes shift - the difference between excitation and emission

peaks

Sir George Stokes


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How Fluorophores work

A photons energy is rapidly absorbed (10-15 sec), andshifts one of the fluorophores electrons from a ground

state to an excited state

The electron then loses some of the gained energythrough smaller vibrational states (in the form of heat)

Electron hangs in the excited state for 10

-9

sec andthen emits a single lower energy photon


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Excitation and Emission


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Excitation Effect on Emission


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Fluorescence Phenomenon

First discovered in materials like Quinine, Fluorspar and otherminerals (natural or auto-fluorescence).

Seen in plant material (pollen grains, privet leaf)

Certain molecules have fluorescence properties (Fluorophores)

DAPI, FITC, TRITC, Alexa Fluors, Cy Dyes and can act as eitherstains or be tagged to cellular structures.

Fluorescence proteins (GFP) were discovered to occur injellyfish species in the 1950s but not utilized as a marker for

gene expression until the 1990s. Through molecular biology these proteins can be used to study gene

expression in non-fluorescence organisms.

Recently fluorescence proteins have also been found in coral species

as well.


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01

The Eyes of Science

Fluorescent Protein

History

1962Dr. Shimomura isolated GFP fromAequorea victoria

Journal of Cellular and Comparative Physiology. 1962 Jun;59:223-39.


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01

The Eyes of Science

Fluorescent Protein

History

1992

Dr. Douglas Prasher showed gene sequences of wild GFP (238 a.a.

26.9kDa). Gene 111 (2): 229-33, 1992.


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01

The Eyes of Science

Fluorescent ProteinHistory

Martin Chalfie 1994Expression in heterogeneous cells.

It has become a common Fluorescence technique.


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Fluorescent Protein

Roger Tsien


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01

The Eyes of Science

Fluorescent Protein

HEK 293 cell expressing GFP, YFP, CFP and RFP

Jean Livet et al., 2007, Nature450:56-62


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01

The Eyes of Science

Fluorescent Protein

Agar Plate of Fluorescent Bacteria Colonies expressing various fluorescent proteins.

Roger Tsiens Lab, UCSD


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The Enemy:

Photo-bleaching

Decrease in emission intensity after exposure

Exciting a molecule once has a probability Qb

of killing it

Each molecule will emit only a finite number of photons


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Photo-bleaching

Photo-stability varies between dyes

Wh t t d b t h t bl hi ?


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What to do about photo-bleaching?

Select fade-resistant dyes

Label densely

Decrease bleaching by anti-fade mounting media Glycerol Oxygen scavengers Free-radical scavengers

Note: some anti-fade agents quench some dyes.

Budget the photons you have Only expose when observing

Minimize exposure time & excitation power Use efficient filter combinations Use highly QE, low noise camera Use simple light path


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Fluorescent Labeling of Cells

Small Molecule Dyes

Fluorescent molecules which will bind to certain structuresin cells or other targets (e.g. DNA, Proteins, LipidMembranes) due to native structure or linkers

Immunofluorescence

- Fluorescence molecules which are bound to antibodies thatattach to specific proteins in the cells. (Alexa 488-AntiGoat)

Fluorescence Proteins- By cloning the genetic code to produce FPs into the cell it

will add the fluorescence molecule to the amino acid chainof your protein of interest so it can be located in the cell.


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B i b


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Brainbow

Fl t P t i /


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Fluorescent Proteins pros/cons

Pros

Can be easily introduced into live cells Minimally perturbative

Photoactivatible/photoconvertible versions exist

Avoids fixing / staining

Cons

Require genetically tractable system

Folding and maturation can be slow Some are pH and Cl- sensitive

Some have very complicated photophysics (strangephotoactivation / photobleaching behavior)

Wh i ll k !


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When it all works!

ik i i t


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www.nikonimagingcentre.com.sg


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http://www.microscopyu.com/

NIKON SINGAPORE PTE LTD

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1 Microscopy Technique