This document presents an overview of microscopy, focusing on atomic force microscopy (AFM) and its application in studying turtle erythrocyte membranes. It details AFM's components, operation modes, and advantages and limitations, along with the objective and methods involved in characterizing turtle erythrocyte membrane structures. The findings conclude that the inner leaflet has a high density of proteins, whereas the outer leaflet primarily features glycosylated proteins with fewer exposed amino groups.

1. Presented to:
Antresh sir
Assistant professor
Dept. of Biotechnology
Presented by:
Mithilesh Choudhary
Mphil-Phd Bioinformatics
CBS Bioinformatics
Enroll No. CUB 1403175001

2. 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)

3. 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

4. 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.

5. 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.

7. 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.

8. 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.
Scanning
Raster Tip
Motion

9. 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

11.  AFM imaging is not ideally sharp

12.  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

13. 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.

14. 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

15. Abstracts :

16. 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

17. 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.

18. (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.

19. (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.

20. (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

21. 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.

22. THANK U