Presented to: Antresh sir Assistant professor Dept. of Biotechnology

Presented by:Mithilesh ChoudharyMphil-Phd BioinformaticsCBS BioinformaticsEnroll No. CUB 1403175001

What is microscopy? “observation and examination of minute objects which will provide a magnified image of an object not visible to the naked eye”Types: 3 main types Optical Microscopy Electron Microscopy Scanning Probe Microscopy (SPM)

Also known as scanning force microscope(SPM), invented in 1986 by Binning, quate and

Gerber.

Useful in obtaining 3D topographic information of insulating and conducting structure with

lateral resolution down to 1.5 nm and vertical resolution down to 0.05 nm.

Can operate in gas, ambient, and fluid environments and can measure physical properties

including elasticity, adhesion, hardness, 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

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.

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 AFM brings a probe in close proximity to the

surface

The force is detected by the deflection of a spring,

usually a cantilever (diving board)

Forces between the probe tip and the sample are sensed

to control the distance between the the tip and the sample.

The cantilever is designed with a very low spring

constant (easy to bend) so it is very sensitive to force.

The laser is focused to reflect off the cantilever and

onto the sensor

The position of the beam in the sensor measures the

deflection of the cantilever and in turn the force between

the tip and the sample.

Raster the Tip: Generating an Image

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.

Sca

nn

ing

Tip

Ras

ter

Mot

ion

Different modes of operation

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

AFM imaging is not ideally sharp

Easy sample preparation

Accurate height information

Works in vacuum, air, and liquids

Living systems can be studied

Limited vertical range

Limited magnification range

Data not independent of tip

Tip or sample can be damaged

Materials Investigated: Thin and thick film coatings, ceramics, composites,

glasses, synthetic and biological membranes, metals, polymers, and

semiconductors.

Used to study phenomena of: Abrasion, adhesion, cleaning, corrosion,

etching, friction, lubricating, plating, and polishing.

AFM can image surface of material in atomic resolution and also measure

force at the nano-Newton scale.

SEM/TEM AFM

Samples Must be conductive Insulating/ conductive

Magnification 2 Dimensional 3 Dimensional

Environment vaccum Vaccum/air/liquid

Time for image 0.1 to 1 minute 1 to 5 minute

Horizontal resolution 0.2 nm (TEM)5nm (SEM)

0.2 nm

Vertical resolution NA 0.05 nm

Field of view 100 nm (TEM)1 mm (SEM)

100 um

Dept of field Good Poor

Contrast on flat samples Poor Good

Abstracts :

OBJECTIVE OF THE STUDY: To characterize turtle erythrocyte membrane structure with molecular resolution in a quasi native state.

METHODS:

[1]. Isolation of Turtle erythrocytes

[2]. Preparation of the outer and inner leaflets of erythrocyte Membranes

[3]. Digestion of the inner leaflet of erythrocyte membranes with proteinase K

[4]. AFM imaging and force spectroscopy

Results:

(a). AFM imaging of the smooth outer surface of the turtle erythrocytes

Fig. 1. AFM topographic images of the smooth outer surface of the turtle erythrocytes.

(B). AFM imaging of the protein-covered inner leaflet of turtle erythrocyte membranes.

Fig. 2. Characterization of the protein-covered inner leaflet of the turtle erythrocyte membranes.

(C). Digestion of the inner leaflets of erythrocyte membranes by proteinase K

Fig. 3. Digestion of the inner leaflet of the turtle erythrocyte membranes by proteinase K.

(D). Asymmetric distribution of amino groups in the inner and outer leaflets of erythrocyte membranes

Fig. 4. Detection of exposed amino groups on both leaflets of the turtle erythrocyte membranes

Conclusion:

A large number of proteins are present on the inner leaflet of turtle erythrocyte

membranes, while fewer proteins are exposed on the outer leaflet of erythrocyte

membranes. This is because most proteins on the outer leaflet of the erythrocyte

membranes are glycosylated (Gao et al., 2013; Sage and Vazquez, 1967) and distributed

in a semi-mosaic pattern with no exposed amino groups.

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