1 Microscopy Technique
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Transcript of 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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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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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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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
Thank You