Microscopy techniques

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

Resolution limitof LM

0,1 mm

0,2 μm

HUMAN EYE

Light microscope

To be continued……

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/

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

Details of cell organization visible in LM are known as “structure”Unstained cells Stained cells

apbrwww5.apsu.edu

Study of tissue slides in light microscopy

- magnification power

4x – slide orientation 40x - cell details

http://histologyolm.stevegallik.org/node/31

10x - tissue details

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)

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”

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

“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

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

Electron micrographs depicting the sample morphology areblack&whiteimages!

LM versus EM

Low magnification (10k-20k)40x

www.intechopen.com

20x Medium magnification(30k-60k)

http://www.drjastrow.de/EMAtlasE.html

LM versus EM

E.M. – high magnificationa DNA strand

Nano Lett., 2012, 12 (12), pp 6453–6458

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

Scanning electron microscopy

www.bio.davidson.edu

https://www. fei.com

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?

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

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

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

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

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

Fig. 1 STM and AFM imaging of pentacene on Cu(111).

L Gross et al. Science 2009;325:1110-1114

Published by AAAS

X-ray Crystallography

• a method in which x-ray diffraction patterns are used to determine the three-dimensional arrangement of atoms in a crystal.

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.

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)

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