Rasha mohamed mahmoud

GENERAL MICROBIOLOGY

Master of Microbiology

Microscopic examination using

Atomic force microscopy

and

Confocal scanning laser microscopy

• Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very high-resolution type of scanning force microscopy, invented in 1986 by Binning, quate and Gerber.

• AFMs can be operated in air, vacuum, and in liquids. Biological measurements in particular, are often carried out in vitro in biological fluids, friction and chemical functionality.

Ability of an AFM to achieve near atomic level resolution depends on three essential components:

1). Cantilever with sharp tip 2). Scanner that controls the x-y-z position 3). Feedback control and loop

How the AFM Works The AFM consists of a cantilever with a sharp tip (probe) at

its end that is used to scan the specimen surface. The cantilever is typically  silicon  or  silicon nitride  with a

tip radius of curvature on the order of nanometers. When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever according to  Hooke's law

Typically, the deflection is measured using a laser spot reflected from the top surface of the cantilever into an array of photodiodes

HOOKE’S LAW This states that "within the limits of elasticity the strain

produced by a stress of any one kind is proportional to the stress". The stress at which a material ceases to obey

Hooke's Law is known as the limit of proportionality. 

Cantilever with a sharp tip•The stiffness of the cantilever needs to be less the effective

spring constant holding atoms together, which is on the order

of 1 – 10nN/nm.

The tip should have a radius of curvature less than 20-50 nm

(smaller is better) a cone angle between 10-20 degrees.

AFM imaging is not ideally sharp

Scanner. • The movement of the tip or sample in the x, y, and z-

directions is controlled by a piezo-electric tube scanner, similar to those used in STM.

• For typical AFM scanners, the maximum ranges for are 80 mm x 80 mm in the x-y plane and 5 mm for the z-direction.

Feedback control. The forces that are exerted between the tip and the sample are measured by the amount of bending

(or deflection) of the cantilever. By calculating the difference signal in the photodiode

quadrants, the amount of deflection can be correlated with a height .

Because the cantilever obeys Hooke's Law for small displacements, the interaction force between the tip and the

sample can be determined.

The tip passes back and forth in a straight line across the sample (think old typewriter or CRT)

In the typical imaging mode, the tip-sample force is held constant by adjusting the vertical position

of the tip (feedback).

A topographic image is built up by the computer by recording the vertical position as the tip is rastered across the sample.

Raster the Tip: Generating an ImageSc

anni

ng

Ti p

RasterMotion

Mode of Operation Force of Interaction Contact mode strong (repulsive) - constant force or constant distance

Non-contact mode weak (attractive) - vibrating probe

Tapping mode strong (repulsive) - vibrating probe

Different modes of operation

Non-ContactLow damageLess sensitive to fine topographical detail

ContactCan damage soft samples through lateral forces (dragging material)

Tapping Mode AFM Similar to non-contact mode, but at bottom of travel the tip just ‘taps’ the sample surface

Tapping Mode AFM in Biology

erythrocyles

E-coli

Application in MicrobiologyThe AFM has been used to viewing and analyzing the ultra

structure of microbial cellsurface studies and it is used to investigated the property of

structure include to analyzingstructure of native membrane proteins at subnanometre

resolution, Function-relatedconformational changes in single proteins,Surface ultra

structure of living cells, Cellsurfacedynamics, and Morphology of biofilms. The physical

properties andbiomolecular interactions such as Stiffness of cell walls, Local

surface charge andh y d r o p h o b i c i t y, E l a s t i c i t y a n d conformational

properties of singlemolecules, Mechanical stability of supramolecular assemblies,

Unfolding pathways of membrane proteins, Molecular forces determining cell adhesion and cell aggregation also analy

Three-dimensional AFM image ofSaccharomyces cerevisiaeYeast cell immobilized in a porous membrane.

High-resolution deflection image of the surface of Phanerochaete chrysosporium Fungal Spores.

SCANNING -LASER

CONFOCAL MICROSCOPY

HOW DOES IT WORK?• A laser is used to provide the excitation light (in order to get very high intensities). The laser light (blue) reflects off a dichroic mirror. From there, the laser hits two mirrors which are mounted on motors; these mirrors scan the laser across the sample.

Confocal Scanning Laser Microscopy (SLCM)is a valuable tool for obtaining high resolution images and 3-D

reconstructions.

…..how does it work?•Dye in the sample fluoresces, and the emitted light (green) gets descanned by the same mirrors that are used to scan the excitation light (blue) from the laser. The emitted light passes through the dichroic and is focused onto the pinhole. The light that passes through the pinhole is measured by a detector.

•So, there never is a complete image of the sample -- at any given instant, only one point of the sample is observed. The detector is attached to a computer which builds up the image, one pixel at a time

3-D IMAGES

Mitosis

Tobacco BY2 cells in telophase

Tobacco leaf epidermal cells expressing

neurocapsule

Metaphase: tobacco BY2 chromosomes

Some of Microscopy Images

• studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues

…applications

It is particularly useful for,• evaluation of various eye diseases. Imaging, qualitative analysis and quantification of endothelial cells of the cornea.

-Advantages-Why is Confocal Microscopy Better?1. More Color Possibilities

Because the images are detected by a computer rather than by eye, it is possible to detect more color differences.

2. Three-Dimensional Reconstruction of Specimen 3D shadow projection Tight junctions (red) Cytoskeletal structures (green)

3.Improved Resolution

DisadvantagesDisadvantages

• Limited primarily to the limited number of excitation wavelengths available with common lasers, which occur over very narrow bands and are expensive to produce in the ultraviolet region.

• The harmful nature of high-intensity laser irradiation to living cells and tissues.

• The high cost of purchasing and operating multi-user confocal microscope systems.