AFM analysis on Plant cell wall (NT1 (Nicotiana Tabacum) cell line) extract

Results and Conclusions

12.08.2011

Tejasvi Parupudi

Materials

• Instrument: Bruker SPM Dimension 3000

• Software used: NanoDrive 8.02

• Image Analysis software used: WsXM

• Mode used: Tapping

• Tip material used: OTESPA 1Ω Si

• Resonant frequency of oscillation: 322-368 kHz

Preliminary studies • In order to see the effect of a certain chemical species

on the cell separation, the studies on JTNT1 tobacco cell suspension were done

• Origins of the NT1 cell line are unclear but they were chosen because of ease of practice of the invention on those cultures and routinely used as model systems in laboratories

• It was observed that disrupting pectin synthesis to remove cell adhesive properties enables separation of cells

• Cell separation enhancement was observed for soybean cell cultures in presence of Colchicine (Umetsuet al., 1975)

• Tobacco suspensions normally contain cells in aggregate or small clusters

• These suspensions are highly heterogenous

• DAS-PMTI-1 was the chemical (invented) used to obtain single cells and all the information is from the Patent Publication No. : US 2010/0159598 A1 published on June 24, 2010

Technology behind the patent

Colchicine- a tubulin inhibitor• Colchicine inhibits mitosis in plant cells (tubulin

depolymerizing compound)• Simply involves addition of appropriate volume of

colchicine to the liquid medium during subculture stage• Colchicine binds to tubulin and prevents the assembly

of micotubules• To separate cells, it is necessary to have low

concentration of Colchicine and low treatment time of the culture with Colchicine

• Colchicine method is more preferred to obtain large volume of single cells with intact cell walls than the enzyme degradation method for at least two suspension cell types

Production of single cells with intact cell walls

• Single cells obtained by the Colchicine method are more stable due to presence of walls-hence do not require regeneration of cell walls

• It was observed that the cells have walls with a normal composition of xyloglucan/cellulose network (Hayashi and Maclachlan, 1984)

• No abnormal wall thickening but radial enlargement (Itoh, 1976)

Single cell isolation of JTNT1 suspension cells with intact cell wall in continuous Pectolyase(pectin degrading enzyme- 0.0005% to 0.005%) treatments sub-cultured in the medium for 7 days

Sample region over which scan was performed

• Area of the sample over which the scan was done : 9.6 µm2

• Scan parameters used:

Set Point: 5.2 V

Frequency: 334.246 kHz

P gain: 3.3

I gain: 1.85

Scan Rate: 0.5 Hz

Scan Range: 9.6463 µm

Samples/line: 256

Rotation: 900

Reason for rotating the scan : scan

at 00 had artifacts at many places

• Height data of a 9.6 µm x 9.6 µm scan showing fine features of cell wall at 0.5 Hz scan rate

712.76 nm

0.00 nm

1.9µm

4.56 V

-3.01 V

1.9µm

• Phase data of a 9.6 µm x 9.6 µm scan showing sharp features at the edges of the cell walls at 0.5 Hz scan rate

Tapping mode Scan over region of concentrated

deposition • Area of the sample over which the

scan was done : 50, 20, 11, 4.4 µm2

and 770 nm2

• Scan parameters used: Setpoint: 5.1 V 20 µm scans4.7 V for 50, 11, 4.4 µm scansFrequency: 334.359 kHzDrive Amplitude: 0.121 VP gain: 3, I gain: 1.4 for 20 µm scanP gain: 3.6, I gain: 1.2 for 50, 11, 4.4 µm and 770 nm scansScan Rate: 0.5 HzSamples/line: 256 for 50, 20 µm scans 512 for 11, 4.4 µm and 770 nm scan

The Auto tune option on the NanoDrive software allows us to do a tuning of the Cantilever between its range of frequencies

The line scan during the 20 µm scan for which the different ‘fits’ were examined

843.85 nm

0.00 nm

4.0µm

843.85 nm

0.00 nm

Observations: From the height image, it is clear that the scan area is completely filled with cell walls of microtubular structure. Due to different concentrations of the cell wall extract observed on different parts of the coverslip, the area corresponding to the above images is from a more concentrated area. Height features of almost 0.75 µm were observed from height profile during the scan. Towards the middle of the scan, the height was observed to decrease gradually to around 0.2 µm

20 µm x 20 µm scan area – plane 20 µm x 20 µm scan area – 3D plane texture shaded solid

Height profile of the 20 µm x 20 µm scan area showing a maximum feature height of 0.45 µm

from WsXM analysis

2520151050

400

350

300

250

200

150

100

50

0

X[µm]

Z[n

m]

1.05 µm

0.00 µm

10µm

50 µm x 50 µm scan area showing a network of cell walls with a high density indicating a lot of adhesion between the cell walls. The cell walls can also be observed to exhibit tubular structure with non uniform sizes

1.05 µm

0.00 µm

50 µm x 50 µm scan area’s 3D AFM image with non uniform distribution of sample features.

734.73 nm

0.00 nm

23.5 µm x 23.5 µm scan area 3D AFM and amplitude data image after zooming into an area with dense features from the 50 µm scan. A 0.5 µm high area at line 198 represents the white shiny portion in the height data image shown above

492.79 mV

-492.79 mV

4.7µm

510.30 nm

0.00 nm

2.2µm

510.30 nm

0.00 nm

From the 3D image and also from the height profile shown during the scan, it is clear that the maximum height of features was around 0.5 µm and the scan area which has features at lower heights indicate the remnants from the cellular matrix or deformities of the cover slip. Since the plant cell wall sample is of an extract of JTNT1 tobacco cells, it is possible that the low lying areas in the 3D data represent the grains corresponding to the cellular matrix that involve other cell organelles.

11 µm x 11 µm scan area – plane 11 µm x 11 µm scan area – 3D plane non texture shaded solid

435.00 nm

0.00 nm

880nm

4.4 µm x 4.4 µm scan area – plane

313.36 nm

0.00 nm

880nm

4.4 µm x 4.4 µm scan area – post equalization

Observation: The image after equalization shows a different scale bar indicating concentration of height in a particular area of the image (shown in the circle). The phase image of the same scan was observed to have interesting features in between the cell walls.

3.62 V

-5.97 V

880nm

252.41 mV

-252.41 mV

880nm

4.4 µm x 4.4 µm scan area – phase4.4 µm x 4.4 µm scan area –amplitude post equalization

Observation: The phase AFM image clearly shows many features including the roughness of the surface and the area of high concentration of particles. The amplitude image after equalization shows a significant ringing effect which might be due to the low gains or the set point. The area between the cell walls was observed in the 770 nm scan.

45.62 nm

0.00 nm

150nm

256.59 nm

0.00 nm

150nm

34.20 nm

0.00 nm

Observation: The raw height data (top left) was unclear due to interference with highest feature of the sample. So, it was equalized (above) and circular features can be seen. The 3D height data (left) shows the protrusions that were observed as circular shapes in the equalized image.

Navigator of the optical microscope shows the region over the sample over which the

scanning is done.

The entire scan area represents less

concentrated cell wall extract.

1.08 µm

0.00 µm

5.0µm

1.08 µm

0.00 µm

AFM images showing area of the sample with less concentrated deposition of extract 25µmx25µm scan area. Plane image (above) and 3D view (below)- scan rate was 0.75 Hz.

494.52 mV

-497.91 mV

5.0µm

0.49 V

-4.79 V

AFM images of Amplitude (derivative of height data) showing 3D view of the features (above) and after equalisation (below). Scan Area was 25 µm x 25 µm; scan rate: 0.75 Hz.

Scan parameters used to conquer the height feature of 3 µm in a area of 55 µm. High gains were used – P: 6.5; I: 5.4;

Set point: 4.6 V and a low scan rate of 0.5 Hz was used.

Forward and backward height data over a 75µm scan area showed the highest feature in two different orientations and this was observed to be due to the dragging of the tip in the trace or retrace

3.49 µm

0.00 µm

3.45 µm

0.00 µm

• Zoom over the highest feature (3.48 µm)of the sample with a reduced scan rate of 0.5 Hz- an area of 29.2 µm was scanned.

• The contour plot shows the area in reverse where the highest feature is represented by the darkest colour. It can be observed that the highest area has a very high concentration of cell wall extract compared to adjacent areas due to non-homogenous deposition during sample preparation.

302520151050

30

25

20

15

10

5

0

X[µm]

Y[µ

m]

3.48 µm

0.00 µm

Conclusions• The sample had non uniform distribution of cell

wall extract which was not favorable for imaging it with great accuracy.

• The cell walls can be clearly seen in all the images and this indicates that the extract contains maximum density of cell walls and less cellular matter

• The tubular structure of cell walls is confirmed from previous studies and analysis

• Cell walls that have been imaged can be tested for mechanical rigidity, permeability and composition to enhance our understanding of their strength and features For Eg: SERS can be done to find the composition corresponding to known peaks in the spectrum

References

[1] Patent No. US 8,012,752 B2[2] I.Horcas et al., Rev. Sci. Instrum., 78,

013705 (2007)