Download - 2 Microscopy Techniques
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Microscopy techniques
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Why do we need microscopes?
• The human eye limit of resolution (limit to distinguish between two separate points) is 0.1mm (100μm)
• Most cells have sizes below this limit, so we use light microscopes to study them
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Resolution limitof LM
0,1 mm
0,2 μm
HUMAN EYE
Light microscope
To be continued……
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Light microscopy“light” microscope (LM) because it uses a source of light to study the specimens
Light passes through a very thin slice of tissue and the resulting image is magnified with the help of lenses inside objectives and oculars
http://blog.nationalmicroscope.com/microscope-parts-and-their-functions/
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Types of LM• Bright field microscope –
study of stained samples
• Phase – contrast (usually inverted) microscope – study of living cells
• Fluorescence microscope (inverted) – study of cell events with the help of fluorochromes
www.studyblue.com
www.novusbio.com
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Details of cell organization visible in LM are known as “structure”Unstained cells Stained cells
apbrwww5.apsu.edu
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Study of tissue slides in light microscopy
- magnification power
4x – slide orientation 40x - cell details
http://histologyolm.stevegallik.org/node/31
10x - tissue details
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Study of tissue slides in light microscopy- magnification power and its usefulness
• 4x and 10x – general examination of the specimen
• 20x and 40x – details related to tissue architecture and cellular organization (normal vs. pathologic)
• 60x and 100x (oil immersion objective) – details on cell morphology (structure of organelles and other intracellular elements)
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Electron microscopy
• If we want to look inside the cell, to see in-depth details about cell morphology, we have to use the electron microscope
• Details of cell organization visible in electron microscopy are known as “ultrastructure”
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Resolution limitof LM
0,1 mm
0,2 μm
HUMAN EYE
L.M.
E.M.
Theoretic resolution limit of EM
Gained resolution2nm
0,2 nm
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“electron” microscope because it uses an electron beam instead of a light beam
Electrons pass or not through the ultrathin sections; those who passed are further focused by magnetic lenses towards a viewing screen, forming a black and white image
www.nanocomposix.com
Electron microscopy
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Electron microscopy
If an electron touches the screen, it will create a light spot. The zones of the screen untouched by electrons (those electrons that have been scattered or reflected) will remain dark
Beam of electrons
Transmitted electrons
Reflected, scatteredelectrons
Specimen
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Electron micrographs depicting the sample morphology areblack&whiteimages!
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LM versus EM
Low magnification (10k-20k)40x
www.intechopen.com
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20x Medium magnification(30k-60k)
http://www.drjastrow.de/EMAtlasE.html
LM versus EM
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E.M. – high magnificationa DNA strand
Nano Lett., 2012, 12 (12), pp 6453–6458
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Scanning electron microscopy• the reflected/scattered
electrons can be “collected”, to obtain an image of the specimen surface
Beam of electrons
Transmitted electrons
Reflected electrons
Specimen
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Scanning electron microscopy
www.bio.davidson.edu
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https://www. fei.com
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How deep can we “see” inside cells?
(micro and nano-levels)
• Light microscopy shows us the nucleus and some information about organelles, under appropriate staining
• Electron microscopy shows us details of organelles, protein polymers or DNA strands (up to nano level)
• Can we see chemical structures of molecules?
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Resolution limitof LM
0,1 mm
0,2 μm
HUMAN EYE
L.M.
E.M.
Theoretic resolution limit of EM
Gained resolution2nm
0,2 nm
A.F.M.
www.oxford-instruments.com
Nature Chemistry 3, 273–278 (2011)
~10 pm
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Atomic force microscopy (AFM)
A “sensory tip” scans the sample and interacts with atoms of specimen’s surface
http://www.slideshare.net/AbeerKamal1/nanophysics-lec-1
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Atomic force microscopy (AFM)
The “sensory tip” moves up and down as it glideson the specimen’s surface, bending the arm
http://www.slideshare.net/AbeerKamal1/nanophysics-lec-1
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Atomic force microscopy (AFM)
Arm movement is recorded by a laser beam, reflected onto a photo detector, generating the image
http://www.slideshare.net/AbeerKamal1/nanophysics-lec-1
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Membrane protein complexes in AFM
Müller & Anderson, Biomolecular imaging using atomic force microscopy, Trends in Biotechnology Volume 20, Issue 8, 1 August 2002, Pages S45–S49
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Fig. 1 STM and AFM imaging of pentacene on Cu(111).
L Gross et al. Science 2009;325:1110-1114
Published by AAAS
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X-ray Crystallography
• a method in which x-ray diffraction patterns are used to determine the three-dimensional arrangement of atoms in a crystal.
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http://biologicmodels.com/category/about/
X-ray crystallography for proteins
Protein
The x-ray diffraction patterns are used to determine the three-dimensional arrangement of atoms in a crystalised (macro)molecules.
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Rules to operate a bright-field microscope• Select the objective with the lowest magnification• Place the slide on the microscope stage with the
coverslip towards the objective (facing upwards)• Lift the stage to the uppermost position (in the closest
position possible of the objective to the slide)• Focus the image using the coarse focus knob• Examine the entire sample• Switch to a higher magnification objective (diagnosis
objective)• Adjust focus using the fine focus knob ONLY!• Examine details and repeat the last two steps for a
higher magnification, if necessary (cytological objective)
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Summary• Light microscopy uses a light beam that crosses a
very thin specimen
• Resolution limit for LM is 0.2μm
• Cell organization as seen in LM are known as “structure”
• Electron microscopy uses a beam of electrons that crosses or not the tissue sample, based on its chemical composition
• “Ultrastructure” means details visible in EM