INTERNSHIP REPORT

ON
A. Training on Instruments, Biotechnology Techniques in Biomedical Research
(i) Autoanalyzer
(ii) Blood Cell Counter Machine
(iii) Pathology Microscope
(iv) Field Emission Scanning Electron Microscope
(v) X-Ray Diffractometer
(vi) Atomic Force Microscopy

B. Biomedical Research towards Detection and Magnitude of Diseases”

The analysis was done using Kidney/Gall Stones using confirmed KFT Data
and confirmed six stones to do FESEM, X-RAY Diffractometer, and AFM analysis.

DURATION: - 04/07/2022 - 30/07/2021

By

MUKUL KUMAR
At

Government Medical College, Saharanpur(UP) and

Indian Institute of Technology Roorkee, Saharanpur Campus
 DECLARATION

I, Mr. Mukul Kumar, hereby declare that this report is being submitted in
fulfilment of the Summer Internship/Training Program at IITR-GMC Academy
under MOU between Indian Institute of Technology, Roorkee & Government
Medical College, Saharanpur (UP). The work was carried out by me under the
guidance of Dr. Rakesh Sharma at Government Medical College, Saharanpur and
Dr. Yuvraj Singh Negi at Indian Institute of Technology Roorkee.

I further declare that to my knowledge, the structure and content of this report
are original and have not been submitted before to any purpose.

MUKUL KUMAR
B. E. 2ND Year (4TH Semester)
Biotechnology Engineering
UIET, PANJAB UNIVERSITY
 ACKNOWLEDGEMENT

Training is an integral part of engineering curriculum providing engineers with first hand
and practical aspects of studies. It gives me great pleasure in completing my summer training
at IIT-Roorkee Saharanpur Campus and Government Medical College Saharanpur and
submitting the report for the same.

I would like to thank Director Prof. J. K. Goswamy, UIET for providing me an esteemed
permission to do work at IITR-GMC Academy for training. I would also like to thank to
Principal Dr Arvind Trivedi at GMC, Saharanpur and Professor Yuvraj Singh Negi at Indian
Institute of Technology Roorkee, Saharanpur campuses for my timely training in their
institutes.

For instrument training and patient data analysis, I appreciate the spared time and
efforts of Dr Nitu Goyal and Mr Tiwari (Pathology), Dr Swati Singh (Blood Bank), Dr
Prabhakar Singh Bains (Clinical Chemistry), Dr Devinder Bohra (Medicine) at GMC,
Saharanpur and Dr Roby Gupta, Ekta Hospital, Sharanpur. For stone analysis at Indian
Institute of Technology Roorkee, I acknowledge the training by Prof. Y.S.Negi (Dean), Prof.
Dr. Gaurav Manik (Polymer and Process Engineering), Dr Anurag and assistance of Rahul
Sharma, Balvir Chauhan at Bioinstrumentation Center.

I take privilege to express my sincere thanks to Dr. Mary Chatterjee and Dr. Anupreet Kaur
for giving me the opportunity to work in IIT-R Saharanpur and GMC Saharanpur institutions
and supporting me constantly and channelize my work towards more positive manner.

I express my deepest gratitude to Dr. Rakesh Sharma & Dr. Yuvraj Singh Negi under whom
I have completed this training and Mr. Rahul Sharma who helped me to carry out sample
results o FESEM, X-Ray and AFM.

MUKUL KUMAR
 GOVERNMENT MEDICAL COLLEGE,
SAHARANPUR
And
Indian Institute of Technology –ROORKEE,
SAHARANPUR CAMPUS

It is to certify that Mr MUKUL KUMAR, B.Engg student at

department of Biotechnology, UIET, Panjab University,
Chandigarh was resident summer trainee during 4.7.2022 to
30.7.2022. He successfully completed trainee period and
accomplished with learning cum using Bioinstruments at our
place under supervision of Professor Rakesh Sharma at
Government Medical College, Saharanpur.
He analysed six samples of renal stones and two gall stones
using SEM, X-Ray Diffraction, Atomic Force Microscopy under
supervision of Prof Gaurav Manik, Head, Polymer and Process
Engineering at IITR Saharanpur campus.
He also completed data sheets for covid comorbidities in
patients using our data base.

Dean,
Indian Institute of Technology Roorkee and
Saharanpur Campus, Saharanpur(UP)

Principal,
Government Medical College,
Saharanpur(UP)
 A. Training Report of Bioinstruments

1. AUTOANALYZER
AUTOMATION

Using the concept of ‘Automation’ functions independently using software intelligence

and adaptability at less manpower and nonstop supervision for the following analysis
process:
1. Loading the patient samples
2. Processing the correct samples for correct tests
3. Identifying and proper labeling of the sample
4. Delivery of sample in proper reaction condition and within time.
5. Preparation of sample for test
6. Sample aspirating after tests are over.
7. Computer Based Analysis
8. Electronic Reporting
9. Entering in data base register

The benefits are:

1. Reduced workload; 2. Increases turnaround time; 3. Hundreds of tests done in less
time; 4. No repetition and high accuracy; 5. Improved reproducibility (repeatability); 6.
less sample and reagents

In a clinical laboratory, autoanalyzers do: 1. Transport of specimen; 2. Processing of

specimen; 3. Loading of specimen into auto analyzer; 4. Assessment of results.
PROCEDURES
1.Sample collection
The use of glucometer is one such example where just by pressing a button on finger tip , the fin ger is pricked with least pain and analysis is also done then and there. However, not all tests can be done in glucometer. So, automation in sample collection main ly refers to improved, faster and least discomfort causing techniques of collection. Such as: robotic system, vacutainers etc. Blood collected using a vacutainer. Here the phlebotomist need not pull the syringe; blood gets sucked in due to negative pressure filling the vaccum.
Different types of vacutainer for serum collection and for plasma collectio n using different types of anticoagulants. The identification is done with the colour of caps used.

2.Sample identification by Labeling and bar coding

The laboratory in formation system comes in to take part here. It first generates a unique identity or hospital number for each new patient. A record is maintained thereafter for him/her. All sample collected have to bear the name and details of the patient alo ng with this unique identity (hospital number). The same is used while entering the details into auto analyzer software and result is also published with this number and other details. Some advanced labs are using computer generated bar coding technolo gy for labeling samples. It has the advantage that it can be scanned and read by barcode reader accurately so transcriptional error (mistake in writing manually)is avoided. Bar coding of the patient, unique sample identity is attached to his/her wrist. Even when patient is sle epin g or unconscious, nurse can read the code for patient identification.
Bar coding of blo od bag in hematology, stores entire detail of type of blood, its components, storage condition, time of storage etc just by bar codes.

3.Sample delivery
Most of the laboratories rely on human pick up system or conveyer belt system. Though cheaper, it may lead to human error, delays etc. Pneumatic tube systems (use of pressurized gas to move the tubes containin g samples) are used in some laboratories. However, care has to be taken that acceleration and decele ration should not damage any sample.
In very advanced laboratory mobile robots are used
The pneumatic tube systems at right sample which can fit into all pneumatic tube systems.

4.Sample preparation
Most of the labs depend on technicians for sample processing (such as serum separation) as soon as sample arrives. However introduction of automatio n can reduce the workload on technicians and save their time and expertise for analysis purpose. Therefore, now days many semi automated devices are developed which can analyze whole blo od itself. The automated sample processors come in 2 types:
 Stand alone automated sample processors and
 Independent automated sample processors.
These automated sample processors can do the following tasks: sortin g of samples, removin g caps, separating samples, bar coding etc. having an automated sample processor solves the task of bar coding and sample delivery via conveyer belt system.

Technical Details:
Integrated System in Autoanalyzer
In integrated automation system as single equipment, different functionality of different
softwares are integrated to give solution. However, maintenance is expensive.
The autoanalyzers are of different types:
(a) Continuous flow processing: Based on this principle a continuous flow analyzer (CFA)
is made of different modules, such as: Sampler, Pump Mixing coil, Heater/incubator,
Sample treatment chamber (dialysis, distillation etc), Signal detector, Read out device
(data generator).
This provides one analysis per analyte for one sample at a time. The flowing carrier
solution passes through small tubes continuously for Continuous flow processing. Here
sample is injected into a flowing carrier solution. The sample mixes with diluents and
reagent and it is sent through the tubing and mixing coils. The machine prevents carry
over effect between different samples by injecting bubbles of air. The air bubbles literally
create separate space for different reactions to take place inside the tubing and mixing
coils. The tubing passes the samples from one apparatus to the other. There are
different apparatus for different functions, such as ion exchange, heating, incubation, and
finally recording of the signal. The flow conditions are regulated. When reaction is taking
place, the optical density of the colour formed is read and results are obtained.

(b) Discrete processing: In this type of auto analyzer, each sample is provided a discrete
space. It means each analysis even for same analyte or sample takes place at different
cups for discrete processing. In discrete processing analyzer the same patient sample
will be sucked by the instrument and poured into 3 different cups. Then reagents for
protein, albumin and creatinine and diluents (if needed) will be added. Mixing will be
done. Cups will be read at different times to give results. Exact amount of sample and
reagent is aspirated and mixed. So there is no loss of excess reagents used for flow as
in continuous flow processing. As each analysis is done in different cups and read in
different cuvettes, there is no carry-over effect at all.
Based on this principle the auto analyzers developed into two different varieties such as
centrifugal analyzer and random access analyzer.
Centrifugal analyzer: Sample and reagents is pipetted into different chambers on a rotor.
The centrifugal force is used for transfer and mixing of sample and reagents. Rotor
moves the final product up to the optical system for final reading. This is time saving for
batch analysis because all cuvettes can be read at a time. But its disadvantage is that
only one test can be performed at a time.
Random access analyzer: Each sample can be analyzed for multiple tests, and multiple
samples for one test also can be done by giving appropriate commands to computer
software. It is the most versatile of all type analyzers.

REPORTING
The ‘hospital information system’ can be linked to automated instruments to report
authorities’ desktops as information on reporting officer’s computer for approval of the
results to display in all computers in the hospital. Unique hospital number directly gives a
print out of the report.
This minimizes the time delay in reporting.

AUTOANALYZER / FULLY AUTOMATED BIOCHEMISTRY RANDOM ACCESS AUTOANALYZER /

STAT ANALYZER

• Fully Automated Random Access Autoanalyzer is a multi-channelled; computer based

sophisticated instrument or device with microprocessor and storage capacity.
• It has a good optical system which enables precision and accuracy of results.
• As it is multi-channelled, it does several parameters on a single serum sample at a time as per
feedback information in the computer.
• It can display, print and memorise the results and has facility to show the graphs of the
reaction.
• Sample and reagents are mixed, incubated and monitored as per the programmed data and
results are displayed.
• It can perform linear, nonlinear, fixed time, kinetic modes at wavelength from 340 to 630 nm.
• Results are available in short period, that is turnaround time (TAT) is less.
• Error messages alert the operator regarding mistakes like non-availability of reagents, wrong
filter or any other anomalies.
• In these analyzers, many reagents (8-20 or more) can be pipette one after another, so that
various biochemical determinations can be performed on one specimen, according to the
number of tests ordered for the patient.

FULLY-AUTOMATED AUTOANALYZER (SELECTRA XL)

SEMI-AUTOMATED ANALYZER ABOTT ARCHITECT c8000

Features: Photometric, Potentiometric, Turbidimetric
w Maximum Throughput : Up to 1,200 tests/hour
w Sample Types : Serum, Plasma, Whole Blood, Urine, CSF
w Sample Tubes : Height: 72–102 mm , Diameter :  9.6 –16.1 mm
w Sample Cup (Dead Volume) : Yes (50 µL)
w Sample Capacity : 215
w Sample Barcode Types : Code 39, Codabar, Interleaved 2 of 5, Code 128
w Sample Result Storage : 50,000
w Sample Volume : 1.5–35 µl, Average: 7.2  µL
w Automatic Dilution : Yes
w Sample Probe Carryover : <1000  ppm WB to WB, ≤0.1  ppm WB to Serum
w Assays Capacity Onboard : 65 plus patented ISE (Na+, K+, and Cl-)
w Reagent Type : >97% liquid ready-to-use
w Reagent Onboard Stability : 5–65 days, Average : 38 days
w Calibration Frequency : 1–60 days, Average : 25 days
w Sample, Clot and Bubble Detection : Yes
w Reagent Pressure Monitoring : No
w Sample Interference Measurement : Yes, hemolysis, icterus, and lipemia
w System Control Center : 1 SCC, with color touchscreen monitor, keyboard, and mouse
w Onboard Maintenance Records : Yes
w Online Error Code Help : Yes
w Host Interface : Bidirectional, serial RS-232 interface, host query option available
w Remote Diagnostics : AbbottLink
w Dimension (H x W x D) : 48" x 79" x 49“, 121.9 x 200.6 x 124.5 cm
w Weight : 1,425 lbs, 646.4 Kg
w Electrical Requirements : AC 180-264 V, 47-63 Hz, 20 amp
w Water Requirements : Deionized water 25 liters/hour during normal operation
w Heat Output* : 3400 BTU/hr, running mode
w Sample Loading : RSH + Carouse
FULLY- AUTOMATED AUTOANALYZER SELECTRA XL

FEATURES
w Two rotors each with 24 positions for 25 ml bottles and 8 positions for 5 ml bottles.   
w All positions can be assigned as R1 and R2.             
w Adapters for 5 ml bottles in 25 ml positions 
w Reagent disk compartment is cooled to approx. 12°C below ambient temperature.            
w Preheated reagent needle with level detection and integrated mixer. 
w Sample rotor containing            
w 80 barcode read samples positions            
w Inner ring for 20 calibrators and 10 controls             
w STAT and pediatric samples            
w Continuous loading.             
w Internal barcode reading             
w Primary tubes (13 or 16 mm OD)  
w Sample probe with level detection and integrated mixer.   
w Semi-disposable rotor with 48 cuvettes, Path length 7 mm.             
w Minimum measuring volume 220 µl.             
w Measuring temperature 37°C, controlled by Peltier elements   
w Kinetic measurement with linearity check.             
w Bichromatic end point measurement with or without bichromatic reagent blank and/or sample blank correction.

w Two point measurement.             

w Graphic plot of all measuring points.             
w Automatic rerun with sample reduction.             
w Non-linear calibration curves   
w Up to 15 different controls can be defined, 3 per test.             
SPECIFICATIONS
w Light Source Quartz-iodine lamp 12V-20W
w Throughput 360 samples/hr (400 with ISE)
w Height 115 cm
w Length 77 cm
w Width 117 cm
Additional Specifications
w Photometric range: -0.1 to 3.0 Absorbance

WORKING OF AUTOANALYZER
• Autoanalyzer is programmed for the specific test.
• The reagents for the specimen are automatically pipette by reagent probe.
• Analyzer identifies various reagent and specimen.
• Photometric determinations are carried out by the autoanalyzer.
• Values of these repeated tests are displayed on the computer screen and memorise as well as
printed on paper, after the specific test incubation period.

ADVANTAGES OF AUTOANALYZER
• Large number of samples can be tested in a short time.
• It gives precise and accurate results.
• Although Autoanalyzer are expensive but in the long run they prove to be cost effective,
because the amount of specimens and reagents required can be as low as 100 and 5 micro-litres
respectively.
• Allows Laboratories to process much larger workload a comparable in the number of staff
number.
• In Autoanalyzer, the laboratory staff members do not come in contact with specimens and
reagents (bio-hazardous material) and hence working on autoanalyzer is very safe.

DISADVANTAGES OF AUTOANALYZER
• Autoanalyzer occupy larger space in the laboratory.
• Autoanalyzer is quiet costly.
• Autoanalyzer may require highly trained person to maintain them properly so that they can do
the job efficiently.
Sometimes the results from autoanalyzer are not easy to understand without the help of a
skilled technician who knows exactly what he’s reading (i.e. The Knowledge of experienced
technician will help to understand the result obtained from autoanalyzer).
2. COMPLETE BLOOD COUNTER MACHINE
Complete Blood Count Analyzer is computerised, highly specified equipment for the complete
blood count which is perhaps the most common routine-based line investigation. It analyses the
parameters like RBC (Red Blood Cell Counts), WBC (White Blood Cell Counts), Hemoglobin level,
Hematocrit level.

RBC Indices
 Red Blood Cell Distribution Width (RDW)

WBC differential Count in percentage and absolute values

Platelet Indices
 Platelet Distribution Width
 Mean Platelet Volume
 Platelet Large Cell Ratio
 Platelet Crit

Automated CBC analysers are of 2 types based on the type of differential white cell count :
(i) 3-part differential Cell Counter
(ii) 5-part differential Cell Counter

(i) 3-part differential Cell Counter

 It functions on the principle of Electrical Impedence to determine the size and volume of the
cells.
 It differentiates WBC’s into Neutrophils, Lymphocytes and mixed cell count.
 It is unable to distinguish between Monocytes, Eosinophils, and Basophils.

(ii) 5-part differential Cell Counter

 It functions on the principle of flow cytometry and volume conductivity, scatter to determine
granularity, diameter, and inner complexity of blood cells.
 It differentiates WBC’s into neutrophils, lymphocytes, easinophils, Monocytes and basophils.

Principles of CBC Analyzer: Automated CBC Analyzer aspirates the required quantity of blood. It
quantifies, classifies and describes the cell population using both electrical and optical
techniques. The three main technologies used in CBC analyzers are:
(i) Photometric Method for Hemoglobin Estimation
RBC’s are lysed and Hemoglobin released is converted to a coloured measurable compound.
A monochromatic light source is then passed through the solution. The absorbance of the
solution is measured by a photo sensor in the hemoglobin flow cell calculating the
hemoglobin concentration.
(ii) Electrical Impedance (Coulter Principle)
It is used to count white blood cells, red blood cells and platelets. As they pass through an
aperture each cell is drawn through the aperture, a change in Electrical resistance occurs
generating Voltage pulse. The number of pulse during a cycle corresponds to the number of
cells counted whereas amplitude of each pulse is directly proportional to the cell volume.
The different cell components are depicted in respective histogram as per their size
distribution.
(iii) Flow Cytometry by VCS (Volume Conductivity Scatter)
This is used in the 5 part analysers for white blood cell differential counts. This technique is
more expensive, due to expensive reagents. It gives detailed information about the
morphology of white blood cells.
 Besides using Impedance principle, 5-part differential cell counters employ flow cytometric
principles. Through hydrodynamic focusing, cells are passed through an aperture in a single
file. A laser beam then focuses on these cells as they pass in a single file. Absorbance and
scattered light generated by each cell is measured at multiple angles to determine the cell’s
granularity, diameter, and inner complexity. Based on size, granularity and nuclear
structure, the 5 types of white blood cells are separated in three-dimensional space and
depicted as scattergram. Scattergram is 3 dimensional pictoral display of WBC based on
their size and granularity causing light scatter.

Fluorescent flow cytometry

It is used in some instruments where fluorescent reagents are used to identify specific cell
populations. Principle is same as that for VCS, and in addition uses Fluorescent dyes to reveal the
nucleus plasma ratio of each stained cell. The added advantage is analyses of platelets,
nucleated RBC’S and reticulocytes.
3-part CBC analyzer parts:

(i) Display or graphic screen where menu and functions are displayed. In menu display, we
see bars for sample ID number, the mode: Whole mode & pre dilution mode, analyzing status.
(ii) Probe which is used for aspirating sample, Quality control, for maintenance purpose.
(iii) Trap Chamber prevents from flowing into the vaccum pimp of the compressor when an
error occurs within the instrument.
(iv) Waste Chamber collects waste from transducer.
(v) Detector blocks incorporate RBC transducer, WBC transducer, Hemoglobin of flow cell.
(vi) Sample rotor Valve/Tray makes volumetric measurement of aspirated blood.

Operation of CBC Machine

As per the sample collection guidelines, whole blood is collected in EDTA anti-coagulated tubes.
EDTA can be of two types: K2 EDTA and K3 EDTA. In K2 EDTA, anticoagulant is in form of dry
powder and in K3 EDTA it is present in liquid form which enables easier mixing in the blood and
the anticoagulant. Blood collected has a tendency to produce more artefacts. Hence, K 2 EDTA
tubes are preferred. Tubes must be inverted several times to mix blood and anticoagulant.
Always check the anticoagulant before taking blood samples. If anticoagulant is absent or spills
out, the sample will clot.

Reagents

(i) Lysing Solution: Reagents lyses the RBC’s for accurate counting of WBC’s and Hemoglobin
level measurement. Expiry of lysing Solution is 90 days from the date of opening. Date of
opening should be noted on bottle. Lysing solution should be maintained at a temperature of
2OC to 350C.
(ii) Diluents: Cell-pack which is an isotonic solution used for appropriate dilution for cell
count. It stabilises the cell membrane and conducts current, as cell pass through aperture and
focus in one stream. Expiry of diluents is 60 days from the date of opening. Cell-pack is usually
maintained at a temperature of 50C to 300C.
(iii) Cleaning Solution: It is used to clean the instrument which removes residuals of lysing
reagents, cellular residues and blood proteins from the hydraulic systems detector block, blood
aspiration tube and flow cell. Used at time of shutdown and several maintenance.
All reagents come with a material safety Data Sheet (MSDS) which should be read carefully and
filled. It gives information of composition and instructions for handling, storage, first aid and
managing any adverse incident due to the reagent. 5–part analyser use addition reagents for the
differential count e.g. Fluorescent dyes which are used to stabilize the cells for identification.

Operating the machine

Inspect the connection of tubing and cards to see that they are not broken and the power cord is
properly plugged in the outlet. Inspect the trap chamber for any waste and check if printer paper
is sufficient. Wear protective equipments.

Switch on the machine. Start up process begins and this usually takes 4 to 5 minutes. In the start
process, machine will do self check analysis of internal parts to check whether they are working
properly or not.

Procedure

Do the maintenance first as per the manufacture’s guideline. If any other maintenance is due,
the machine will display the message on the screen. Operator will have to proceed with the
cleaning transducer first before the machine can be run for the day.

Background check is done. Auto rinse is done 3-5 times automatically by the machine followed
by background check and limits should be within permeable limits set by the manufacture. If
background count of any parameters falls out of these limits, the message ‘Background Error’ is
displayed with alarm sound. Machine gives background error due to reasons like dirty aperture,
dirty flow cell, Air Bubble in probe, faculty reagents due to bacterial growth or contamination. If
machine shows no error in self analysis, the machine is ready. Run Quality Control.

After running Quality Control, collect the blood sample as per guidelines. Mix the sample gently
by inverting 8-10 times. Place the sample on the rotor and mixer for 3-5 minutes. Keep inverting
gently till aspiration.

Then Modes are performed on CBC analyser, e.g. Whole Blood Mode in which tube is set for
sample probe. Press the start button. Buzzer sound twice, removes tube carefully to avoid
bending the probe. Do not remove when screen shows aspirating, air can enter giving wrong
results. When LCD screen displays ready, prepare next sample.

Results: Analytical parameters are displayed:WBC Count, RBC Count, Hemoglobin, HCT
(Hematocrit), MCV (Mean Corpuscular Volume), Mean Corpuscular Hemoglobin (MCH), Mean
Corpuscular Hemoglobin Concentration (MCHC), MPV (Mean Platelet Volume) and
Lymphocytes- LYM% * M*D%/100.

Similarly mixed cell and neutrophils absolute count, are calculated as RDW (Red blood cell
distribution width) through which RBC’s are distributed around the MCV. PDW (Platelet
distribution width) is seen high in rapid turnover of platelets seen in dengue.
 Results include histogram/graphs of RBC’s, WBC’s and platelets.

Shut down
At the end of the day, shut down process should be followed by the operator. Don not switch of
the machine without performing the procedures:
1. Press shutdown key “Aspirate the cell Clean” will be displayed on the screen.
2. Open the cap of cell clean reagent. Set the cell clean reagent vial to the sample probe.
3. Press the start button. Cleaning process occurs for about 5 minutes.
4. Shutdown will be displayed. Now, switch of the machine.
Storage and discarding the samples
As per the regulation, labs are required to store samples for defined time duration for repeat
testing if required. All Labs also needs to validate the adequacy of sample storage, requirements
like Temperature, and time duration, by repeat sample studies. The % difference between the
running is 24 hours.
Waste Management
All blood samples should be discarded as per biomedical waste management rule. Waste fluids,
instrument must be disposed off after disinfecting with hypochlorite for at least 30 minute.
Safety & Precaution
 Always wear protective PPE and gloves when processing with the instrument and during
maintenance.
 After completion of work, wash hands. Follow the WHO guidelines for hand washing.
 Make sure the reagents used with the instrument are kept level or below the main unit of the
instrument.
 Do not put reagents on top of the instrument.
 As far as possible, keep the waste container at a lower level to enable gravity drain and prevent
back flow.
 Avoid direct contact with reagents. They can cause irritation to the eyes, skin and mucous
membrane.
 Diluents are good electrical conductor. If Diluent is spilled near electrical cables or appliances,
there is risk of electric shock.
 Switch off the instrument and unplug it and wipe up the liquid.
 Cleaning agent should not come in contact with skin or clothing.
A complete blood count test measures several components and features of patient blood,
including:
 Red blood cells, which carry oxygen
 White blood cells, which fight infection
 Hemoglobin, the oxygen-carrying protein in red blood cells
 Hematocrit, the proportion of red blood cells to the fluid component, or plasma, in your blood
 Platelets, which help with blood clotting
Abnormal increases or decreases in cell counts as revealed in a complete blood count may
indicate the medical condition that calls for further evaluation.
Why it's done
A complete blood count is a common blood test that's done for a variety of reasons:
 To review overall health: It is done as part of a routine medical examination to monitor general
health and to screen for a variety of disorders, such as anemia or leukemia.
 To diagnose a medical condition: If experiencing weakness, fatigue, fever, inflammation, bruising
or bleeding, a complete blood count may help diagnose the cause of these signs and symptoms.
Doctor suspects you have an infection; the test can also help confirm that diagnosis.
 To monitor a medical condition: If one has been diagnosed with a blood disorder that affects
blood cell counts, complete blood counts is done to monitor your condition.
 To monitor medical treatment: A complete blood count may be used to monitor the health if
taking medications that may affect blood cell counts.
Preparation
If blood sample is being tested only for a complete blood count, patient can eat and drink
normally before the test. If the blood sample will be used for additional tests, patient may need to
fast for a certain amount of time before the test.
For a complete blood count, a sample of blood is taken by inserting a needle into a vein in your
arm, usually at the bend in your elbow. The blood sample is sent to a lab for analysis.
Results
The following are normal complete blood count results for adults:
Red blood cell count
Male: 4.35-5.65 trillion cells/L (4.35-5.65 million cells/mcL)
Female: 3.92-5.13 trillion cells/L (3.92-5.13 million cells/mcL)
Hemoglobin
Male: 13.2-16.6 grams/dL (132-166 grams/L)
Female: 11.6-15 grams/dL (116-150 grams/L)
Hematocrit
Male: 38.3-48.6 percent
Female: 35.5-44.9 percent
White blood cell count
3.4-9.6 billion cells/L (3,400 to 9,600 cells/mcL)
Platelet count
Male: 135-317 billion/L (135,000 to 317,000/mcL)
Female: 157-371 billion/L(157,000 to 371,000/mcL)
• L = liter
• mcL = microliter
• dL = deciliter
Outcome
Results in the following areas above or below the normal ranges on a complete blood
count may indicate a problem.
• Red blood cell count, hemoglobin and Hematocrit: The results of red blood cell
count, hemoglobin and hematocrit are related because they each measure aspects of
red blood cells. If the measures in these three areas are lower than normal, patient
have anemia. Anemia causes fatigue and weakness. Anemia has many causes, including
low levels of certain vitamins or iron, blood loss, or an underlying condition. A red
blood cell count that's higher than normal (erythrocytosis), or high hemoglobin or
hematocrit levels, could point to an underlying medical condition, such as
polycythemia vera or heart disease.
 White blood cell count: A low white blood cell count (leukopenia) may be caused by
a medical condition, such as an autoimmune disorder that destroys white blood cells,
bone marrow problems or cancer. Certain medications also can cause white blood cell
counts to drop. If white blood cell count is higher than normal, patient may have an
infection or inflammation. Or, it could indicate that patient have an immune system
disorder or a bone marrow disease. A high white blood cell count can also be a reaction
to medication.
 Platelet count: A platelet count that's lower than normal (thrombocytopenia) or
higher than normal (thrombocytosis) is often a sign of an underlying medical condition,
or it may be a side effect from medication.

3. PATHOLOGY MICROSCOPE

Introduction

 The function of the pathology microscope is to enlarge, magnify (1 x 10 3 times) and capture
the images of the microscopic cells. The efficiency of microscopes is indicated by its resolution.
Resolution is ability of microscope to distinguish two close related points.
 In compound microscope, multiple lenses are used.

Principles of Microscope
An object placed on the stage is magnified through the objective lens. When the target is
focused, a magnified image can be observed through the ocular lens.

WORKING OF MICROSCOPE

Microscope works on three principles of physics:

 Magnification
 Resolving power
 Numerical aperture

 Magnification

In compound microscope, the magnification is the product of magnifying power of both the
lenses. The magnifying power of lens depends on focal length. Lower the focal length, higher is
the magnifying power of lens.
 Resolution

The resolving power is the ability to distinguish two closely placed points. The resolution power
of lens allows us to observe the details of an object. The resolution of microscope can find out by
using Abbe’s equation.

d = 0.5 /n sin 

where,
d – distance between two closely distant points
λ – wavelength of light
n sin θ – numerical aperture
 The microscope with higher magnification has small d value. λ is the wavelength of light,
shorter is the wavelength; higher is the resolution. The wavelength of visible light is
from 300 to 700 nm. The best resolution for light microscope is obtained in the range
of 450 to 500 nm.

‘n’ is the refractive index of medium

 Refractive index is the ability of the medium to bend the light. The angle of cone of light is
affected by the refractive index of medium. The refractive index of air is 1. ‘θ’ is the half of the
angle of the cone of light that enters the microscope. The value of ‘Sin θ’ cannot be more than 1
because angle of entering cone of light cannot be more than 90° and value of sin 90 is 1.
d= 0.5 x 450 nm
d= 225 nm 0r 0.2 μm

PARTS OF MICROSCOPE

(i) Stand
(ii) Body
(iii) Optical System

(i) Stand
 It is horse-shaped and made up of solid iron.
 It provides balance and stability to the instrument.
 It also supports the optical parts with perfect alignment.
(ii) Body
1. Stage
 It is platform to support specimen under observation.
 The stage has the central aperture for the passage of light rays.
 The microscope slide is kept on the stage by means of clips.
2. Substage
 It is below the stage.
 It moves up and down with the help of screws.
3. Condenser
  It is used to concentrate the light.
4. Irish Diaphragm
 It regulates the amount of light falling on the specimen.
5. Mirror
 It reflects the light on specimen. It has two surfaces.
 Plain Surface: It is used for sunlight.
 Concave Surface: It is used for artificial light.

(ii) Optical System

It consists of:
 Body Tube: It consists the objective lens and eye piece.
 Coars Adjustment: It is used to raise and lower the objective lens.
 Fine Adjustment: It is used for the delicate focusing.
 Noise Piece: It consist the socket to receive objects of different magnification power i.e.
X10, X40, X100.
 Objective Lens: It forms of a magnified image depending upon the magnify power of
objective lens i.e. X10, X40, X100.
 Eye Piece: It magnifies further the magnified image formed by the objective lens.

4. FIELD EMISSION SCANNING ELECTRON MICROSCOPY(FESEM)

 A Field Emission Electron Microscope (FESEM) is microscope that works with electrons
instead of light. These electrons are librated by a field emission source. The object is
scanned by electrons according to a zig-zag pattern.
 A FESEM is used to visualize very small topographic details on the surface or entire or
fractioned objects. Researchers in biology, chemistry and physics apply this technique to
observe structures that may be as small as 1nm. The FESEM is may be employed for
example to study organelles and DNA material in cells, synthetical polymers, and coatings
on microchips.

PRINCIPLE OF FESEM
• Electrons are liberated from a field emission source and accelerated in a high electric
field gradient. Within the high vacuum column these so-called primary electrons are
focused and deflected by electronic lenses to produce a narrow beam that bombards the
object. As a result secondary electrons are emitted from each spot on the object. The
angle and velocity of these electrons relates to the surface structure of the object. A
detector catches the secondary electrons and produces an electronic signal. This signal is
amplified and transformed to a video scan-image that can be seen on a monitor or to a
digital image that can be saved and processed further
 FIELD EMISION MICROSCOPY SETUP
PREPERATION

 In order to be observed with SEM objects are made conductive for current. This is done by
coating them with an extremely thin layer (1.5-3.0 nm) of gold or gold-palladium. Further
 on, objects must be able to sustain the high vacuum and should not alter the vacuum, for
example by losing water molecules or gases. Metals, polymers and crystals are usually little
problematic and keep their structure in the SEM. Biological material, however requires, a
prefixation, e.g. with gold slush nitrogen (cryo-fixation) or with chemical compounds.

 This particular microscope is foreseen of a special cryo-unit where frozen objects can be
fractured and coated for direct observation in the FESEM. Chemically fixed materials need
first to be washed and dried below the critical point to avoid damage of the fine structures
due to surface tension. Coating is then performed in a separate device.

SPUTTER COATER

SOURCE OF ELECTRON

• In standard electron microscopes electron are mostly generated by heating a tungsten

filament by means of a current to a temperature of about 2800 C. Sometimes electrons
are produced by a crystal of lantanumhexaboride (LaB6) that is mounted on a tungsten
filament. This modification results in a higher electron density in the beam and a better
resolution than with the conventional device.

• In a Field emission electron microscope no heating but a so-called “cold” is employed. An

extremely thin and sharp needle (tip diameter 10 m) functions a cathode in front of a
primary and secondary anode. The voltage between cathode and anode is in the order of
magnitude 0.5-30KV. Because the electron beam produced by the FE source is about 1000
times smaller than in a standard microscope, the image quality is markedly better. As a
field emission necessitates an extreme vacuum () in the column of the microscope, a
device is present that regularly decontaminates the electron source by a current flash. In
contrast to a conventional tungsten filament, a FE tip last theoretically for a lifetime,
provided the vacuum is maintained stable.
LENSES AND APERTURES

The electron beam is focused by the electro-magnetic lenses (condenser lens, scan coils,
stigmator coils and objective lens) and the apetures in the column to a tiny sharp spot.

1. Condenser Lens
The current in the condenser determines the diameter of the beam: a low current result in a
small diameter, a higher current in a larger beam. A narrow beam has the advantage that the
resolution is better, but the disadvantage that the signal to noise ratio is worse. The situation is
reversed when the beam has a larger diameter. The condenser lens consists of mostly out of two
parts.

2. Scan Coils
The scan coils deflect the electron beam over the object according to a zig-zag pattern. The
formation of the image on the monitor occurs in synchrony with this scan movement. The scan
velocity determines the refreshing rate on the screen and amount of noise in the image (rapid
scan = rapid refreshing = low signal = much noise). The smaller the scanned region on the object,
the larger the magnification becomes at a constant window size. Scan coils often consist of upper
and lower coils, which prevent the formation of a circular shadow at low magnification.

3. Objective Lens
The objective lens is the lowest lens in the column. The objective focuses the electron beam
on the object. At a short working distance the objective lens needs to apply a greater force to
deflect the electron beam. The shortest working distance produces the smallest beam diameter,
the best resolution, but also the poorest depth of field.

4. Stigmator Coils
The stigmator coils are utilized to correct irregularities in the x and y deflection of the beam
and thus to obtain a perfectly round-shaped beam. When the beam is not circular, but ellipsodial,
the image looks blurred and stretched.

5. Object Chamber
After the object has been covered by a conductive layer it is mounted on a special holder.
The object is inserted through an exchange chamber into the high vacuum part of the
microscope and anchored on a movable stage. In the virtual FESEM the object can be moved in
horizontal and vertical direction on the screen by operating the arrows in the POSTION BOX. In
the real microscope the object can be repositioned in the chamber by means of a joy stick that
steers in left-right axis, or forward and backward. In addition, the object can be tilted, rotated
and moved in Z direction. The “secondary electron emission” detector is located at the rear of
the object holder in the chamber.

IMAGE FORMATION
When the primary bombards the object, secondary electrons are emitted from the object surface
with a certain velocity that is determined by the levels and angles at the surface of the object. The
secondary electrons, which are attracted by the Corona, strike the scintillator (fluorecing mirror)
that produces photons. The location and intensity of illumination of the mirror vary depending on
the properties of secondary electrons. The signal produced by the scintillator is amplified and
transduced to a video signal that is fed to cathode ray tube in synchrony with the scan movement
of the electron beam. The contrast in the ‘real time’ image that appears on the screen reflects the
structure on the surface of the object. Parallel to the analog image, a digital image is generated
which can be further processed
5. X-RAY DIFFRACTOMETER

 Van Laue in 1912 suggested that X-rays can be diffracted by crystals. Later, Friendrich and
Kripping successfully demonstrated that diffraction by X-ray and ZnS crystal.

 The interplanar spacing (d) in a crystal is of the order of 1 A0 and wavelength (l) is also of
same order i.e. d = lx-ray = 1 A0, so X-rays can be diffracted by crystals.

 When X-ray fall on crystal the electrons of atom of crystal start vibrating with frequency of
incident X-rays.

 The electrons get accelerated. The accelerated electrons (charges) re-emit radiated radiations
of frequency of incident X-rays. The emitted radiation will interfere constructively and
destructively and we will get diffraction pattern on screen which provides valuable information
regarding structure and properties of crystal.

BRAGG’S LAW

Ÿ In 1912, W. L. Bragg gave a very simple formula for a geometrical condition which must be
satisfied if X-rays are to be diffracted by parallel set of planes. This simple formula is called 2 d
sinq = nl

P R
Q S
 A
    
d C  D
    
B
Consider a set of parallel planes. Let the planes be at a distance d from each other. Let the
planes be at a distance d from each other. Let a parallel beam of X-ray of wavelength ‘l’ be
incident on these parallel planes at angle ‘q’.

The reflected rays AR & BS will interfere constructively if the path difference CB + BD is equal to
integral multiple of l.
 i.e. CB + BD = l (i)

In triangle ACB, sinq = CB/AB  CB= d sinq (ii)

Similarly, BD= d sinq (iii)

Using (ii) & (iii) in (i), we get

d sinq + d sinq = nl

2d sinq = n l

INSTRUMENTATION OF X-RD POWDER DIFFRACTION

X-RD DIFFRACTOMETER INSTRUMENTATION

• X-ray diffractometers consist of three basic elements: an X-ray tube, Sample Holder, and
an X-ray detector.

• X-ray are generated in a cathode ray tube by heating a filament to produce electrons,
accelerating the electrons toward a target by applying a voltage, and bombarding the
target materials with electrons.

• When electrons have sufficient energy to dislodge inner shell electrons of the target
material, characteristic X-ray spectra are produced. These spectra consist of several
components, the most common being Ka and Kb. Ka consist Ka1 and Ka2. Ka1 has slightly
shorter wavelength and twice the intensity as Ka2.

• The specific wavelengths are characteristic of the target material (Cu, Fe, Mo, Cr).
Filtering, by foils or crystal monochrometers X-rays needed for diffraction.

• Ka1 and Ka2 are sufficiently close in wavelength such that a weighted average of the two
is used. Copper is the most common target material for single-crystal diffraction, with
CuKa radiation = 1.5418 A0 .

• These X-rays are collimated and directed onto the sample. As the sample and detector
are rotated, the intensity of the reflected X-rays is recorded. When the geometry of the
incident X-rays impinging the sample satisfies the Bragg Equation, constructive
interference occurs and a peak in intensity occurs.

• A detector records and processes this X-ray signal and converts the signal to a count rte
which is then output to a device such as printer or computer monitor.

• The geometry of an X-ray diffractometer is such that the sample rotates in the path of
the collimated X-ray beam at an angle q while the X-ray detector is mounted on an arm
to collect the diffracted X-rays and rotates at an angle 2q.

• The instrument used to maintain the angle and rotate the sample is termed as
goniometer. For typical powder patterns, data is collected at 2q from ~ 5o to 700 angles
that are present in X-ray scan.

TEMPERATURE CONTROL UNIT

 X-Ray Diffractometer UPS, 3 PHASE, 15KVA

STEP DOWN TRANSFORMER CHILLER – 12A, 200V

SAMPLE COLLECTION AND PREPERATION

Determination of an unknown requires: the material, an instrument for grinding, and a sample
holder.

Ÿ Obtain a few tenths of a gram (or more) of the material, as pure as possible.

Ÿ Grind the sample to a fine powder, typically in a fluid to minimize inducing extra strain
(surface energy) that can offset peak positions, and to randomize orientation. Powder less than
~10 μm (or 200-mesh) in size is preferred.

Ÿ Place into a sample holder or onto the sample surface:

w Smear uniformly onto a glass slide, assuring a flat upper surface.
w Pack into a sample container.
w Sprinkle on double sticky tape.

Ÿ Care must be taken to create a flat upper surface and to achieve a random distribution of
lattice orientations unless creating an oriented smear.

Ÿ For analysis of clays which require a single orientation, specialized techniques for preparation
of clay samples are given by USGS.
For unit cell determinations, a small amount of a standard with known peak positions (that do
not interfere with the sample) can be added and used to correct peak positions.

Data Collection, Results and Presentation

Ÿ Data Collection

The intensity of diffracted X-rays is continuously recorded as the sample and detector rotate
through their respective angles. A peak in intensity occurs when the mineral contains lattice
planes with d-spacing appropriate to diffract X-rays at that value of θ. Although each peak
consists of two separate reflections (Kα1 and Kα2), at small values of 2θ the peak locations
overlap with Kα2 appearing as a hump on the side of Kα1. Greater separation occurs at higher
values of θ. Typically these combined peaks are treated as one. The 2λ position of the diffraction
peak is typically measured as the center of the peak at 80% peak height.

Ÿ Data Reduction
Results are commonly presented as peak positions at 2θ and X-ray counts (intensity) in the form
of a table or an x-y plot (shown above). Intensity (I) is either reported as peak height intensity,
that intensity above background, or as integrated intensity, the area under the peak. The
relative intensity is recorded as the ratio of the peak intensity to that of the most intense peak
(relative intensity = I/I1 x 100).
Ÿ Determination of an Unknown
The d-spacing of each peak is then obtained by solution of the Bragg equation for the
appropriate value of λ. Once all d-spacing have been determined, automated search/match
routines compare the ds of the unknown to those of known materials. Because each mineral has
a unique set of d-spacing, matching these d-spacing provides an identification of the unknown
sample. A systematic procedure is used by ordering the d-spacing in terms of their intensity
beginning with the most intense peak. Files of d-spacing for hundreds of thousands of inorganic
compounds are available from the International Centre for Diffraction Data as the Powder
Diffraction File (PDF). Many other sites contain d-spacing of minerals such as the American
Mineralogist Crystal Structure Database. Commonly this information is an integral portion of the
software that comes with the instrumentation.

Ÿ Determination of Unit Cell Dimensions

For determination of unit cell parameters, each reflection must be indexed to a specific hkl.

Strengths and limitations of X-ray Powder Diffraction

Strengths
Ÿ Powerful and rapid (< 20 min) technique for identification of an unknown mineral.
Ÿ In most cases, it provides an unambiguous mineral determination.
Ÿ Minimal sample preparation is required.
Ÿ XRD units are widely available.
Ÿ Data interpretation is relatively straight forward.
Limitations
Ÿ Homogeneous and single phase material is best for identification of an unknown.
Ÿ Must have access to a standard reference file of inorganic compounds (d-spacing, hkls).
Ÿ Requires tenths of a gram of material which must be ground into a powder.
Ÿ For mixed materials, detection limit is ~ 2% of sample.
Ÿ For unit cell determinations, indexing of patterns for non-isometric crystal systems is
complicated.
Ÿ Peak overlay may occur and worsens for high angle 'reflections’.

APPLICATIONS

X-ray powder diffraction is most widely used for the identification of unknown crystalline
materials (e.g. minerals, inorganic compounds). Determination of unknown solids is critical to
studies in geology, environmental science, material science, engineering and biology.
Other applications include:
Ÿ Characterization of crystalline materials.
Ÿ Identification of fine-grained minerals such as clays and mixed layer clays that are difficult to
determine optically.
Ÿ Determination of unit cell dimensions.
Ÿ Measurement of sample purity.

With specialized techniques, XRD can be used to:

Ÿ Determine crystal structures using Rietveld refinement.Ÿ Determine of modal amounts of
minerals (quantitative analysis).
Ÿ Characterize thin films samples by:
w Determining lattice mismatch between film and substrate and to inferring stress and strain.
 Determining dislocation density and quality of the film by rocking curve measurements.
 Measuring superlattices in multilayered epitaxial structures.
w Determining the thickness, roughness and density of the film using glancing incidence X-ray
reflectivity measurements make textural measurements, such as the orientation of grains, in a
polycrystalline sample.
6. ATOMIC FORCE MICROSCOPY (AFM)

Ÿ AFM is a very powerful microscope which was invented by IBM scientist Gerd Binning in 1982
for which he received Nobel Prize in physics in 1986.
Ÿ It is very important tool to image and manipulate matter at nanoscale. It is also known as scanning
force Microscope (SFM). AFM is modification of scanning tunelling microscope (STM).
Ÿ AFM has the advantage of imaging almost any type of surface, including polymers, ceramics,
composites, glass, and biological samples.
Ÿ AFM allows accurate and non-destructive measurements of the topographical, electrical,
magnetic, chemical, optical, mechanical, etc. Properties of a sample surface with very high
resolution in air, liquids or ultrahigh vacuum.
CONSTRUCTION OF AFM
AFM CONSTRUCTION

(i) Cantilever: The main component of AFM is cantilever having a sharp tip at the end.
(ii) Tip: A very fine tip of size of the order of nanometer is composed of Silicon (Si) or Silicon
Nitride and is attached at the end of cantilever.
(iii) Platform: The sample under investigation is placed on the platform.
(iv) Piezoelectric Tube (XYZ Scanner): The platform containing sample is mounted on the
piezoelectric tube which can move sample in X, Y, Z direction.
(v) Laser: It helps in cantilever deflection measurement, laser is reflected from back of
cantilever due to deflection in cantilever while scanning the sample.
(vi) Photodetector: It consist of several photodiodes which converts laser light into electric
current which helps in imaging sample surface.

PRINCIPLE OF AFM

AFM works on a principle that a fine sharp tip of the order of nanometer attached to cantilever
when brought into close to a sample surface, the Van der Waals forces between the tip and
sample produces a defection of the cantilever according to Hook’s Law (F = -kx).
The deflection is measured by laser spot reflected from the tip of cantilever into photodetector.
WORKING OF AFM

AFM works in two modes:

Ÿ Contact Mode: This mode is used for imaging solid surfaces. In this method, tip is dragged
across the sample means tip comes in contact with sample surface. The force between the
sample and the tip displaces the cantilever. It helps in evaluation of sample friction.
Ÿ Non-Contact Mode: This mode is used for imaging soft surfaces like biological surfaces. In this
mode, tip does not make any contact with sample surface. The tip is kept at a constant height
of few nm above the sample surface. Van der Waals forces between tip and sample surface
helps the scanning software to construct topographic image of sample surface.

l Advantage of Non-Contact Mode over Contact Mode: There is no degradation of sample or tip which is
observed after taking numerous scans with contact mode.

ADVANTAGE OF AFM OVER STM

Ÿ AFM Provides 3-D image of sample.

Ÿ It does not require vacuum.
Ÿ It has high resolution.
Ÿ It can image non-conducting surface like glass and polymer.

DISADVANTAGE OF AFM
Ÿ The scanning speed of AFM is less than STM. It can take several minutes for a typical scan.
TRAINING REPORT
EXPERIMENTAL WORK
1. Patients Data Analysis:
Comorbities identified among COVID-19 patients:
Pneumonitis>Diabetes mellitus>Hypertension>Fatty Liver
See attached Tables 1-4 for Statistical Analysis of patient data
2. Stone Analysis by FESEM, AFM and X-Ray Diffraction
Analysis
a. Gross analysis: Rings, monolayers of crystallization
b. Crystallization, shapes of crystal, size and molecular
orientation: see SEM and AFM Figures 1-4.

c. Composition of stones: Calcium Oxalate, Potassium

and Carbon Urate peaks: See XRD figures
1. Patients Data Comorbidity Analysis
TABLE 1: DM

TABLE2: HTN
TABLE3: DM & SPO2 < 95%

TABLE4: DM2
 FIGURE 1: Stone analysis by pathology Microscope

PATIENT BLOOD BLOOD CREATININE BLOOD

NAME UREA UREA (PRE- UREA
(PRE- (POST- SURGERY) (POST-
SURGERY) SURGERY) URGERY)
1. 48.10 24.3 1.32 0.85
2. 96 29.1 2.02 0.74
3. 64.62 51.40 1.33 1.31
4. 32.87 24.00 0.99 0.91
5. 32.07 27.8 1.110 0.88
6. 42.04 26.19 1.01 1.00
HIGH FIELD IMAGES

1. 2.

3. 4.
5.

6.
FIGURE 2: Stone Analysis by FESEM (Field Emission
Scanning Electron Microscope)
Sample 1: Stone 1
Image at Magnification: 5 * 103 x
Image at Magnification: 100O x

Image at Magnification: 10 * 103 x

 EDAX REPORT

kV: 12 Mag: 76 Takeoff: 36.7 Live Time(s): 41 Amp Time(µs):

8

Sum Spectrum

eZAF Smart Quant Results

Element Weight % Atomic % Net Int. Net Int. Error

CK 43.88 55.09 169.7 0.01

OK 41.7 39.3 106.4 0.02

NaK 0.53 0.35 2.9 0.39

MgK 0.17 0.11 1.4 0.65

KK 0.35 0.13 1.7 0.62

CaK 13.36 5.03 47.6 0.04

Sample 2: Stone 2

Image at Magnification: 2.52 * 103 x

Image at Magnification: 5 * 103 x

Image at Magnification: 25 * 103 x
 EDAX REPORT

Mag: 54 Takeoff: 37.2 Live Time(s): 41 Amp Time(µs): 3.

38 84

Sum Spectrum

eZAF Smart Quant Results

Element Weight % Atomic % Net Int. Net Int. Error

CK 12.39 19.8 56.5 0.03

OK 52.59 63.13 178.1 0.01

NaK 0.55 0.46 3.7 0.37

MgK 0.27 0.21 2.9 0.62

KK 0.32 0.16 2.4 0.6

CaK 33.89 16.24 169.5 0.02

Sample 3: Stone 3
Image at Magnification: 10 * 103 X
Image at Magnification: 500 X

Image at Magnification: 3 * 103 x

 EDAX REPORT

Mag: 41 Takeoff: 34.7 Live Time(s): 30.7 Amp Time(µs):

97

Sum Spectrum

eZAF Smart Quant Results

Element Weight % Atomic % Net Int. Net Int. Error

CK 11.58 18.6 61.5 0.03

OK 53.32 64.26 220.9 0.01

NaK 0.57 0.48 4 0.38

MgK 0.18 0.15 1.9 0.67

KK 0.39 0.19 2.1 0.63

CaK 33.95 16.33 116.1 0.02

Sample 4: Stone 4
Image at Magnification: 5 * 103 x
 Image at Magnification: 1 * 103 x

Image at Magnification: 10.2 * 103 x

 EDAX REPORT

kV: 10 Mag: 41 Takeoff: 34.7 Live Time(s): 30.7 Amp Time(µs):

97

Sum Spectrum

eZAF Smart Quant Results

Element Weight % Atomic % Net Int. Net Int. Error

CK 11.58 18.6 61.5 0.03

OK 53.32 64.26 220.9 0.01

NaK 0.57 0.48 4 0.38

MgK 0.18 0.15 1.9 0.67

KK 0.39 0.19 2.1 0.63

CaK 33.95 16.33 116.1 0.02

Sample 5: Stone 5
Image at Magnification: 5.02 * 103 x
Image at Magnification: 500 x

Image at Magnification: 10 * 103 x

 EDAX REPORT

Mag: 24 Takeoff: 35 Live Time(s): 30.7 Amp Time(µs):

07

Sum Spectrum

eZAF Smart Quant Results

Element Weight % Atomic % Net Int. Net Int. Error

CK 26.36 37.54 142.6 0.02

OK 47.91 51.22 193.5 0.01

NaK 0.47 0.35 3.3 0.63

MgK 0.36 0.25 3.7 0.47

KK 0.5 0.22 2.5 0.62

CaK 24.39 10.41 79.5 0.03

Sample 6: Stone 6
Image at Magnification: 5 * 103 x

Image at Magnification: 15 * 103 x

Image at Magnification: 500 x

EDAX REPORT
kV: 10 Mag: 14 Takeoff: 34.4 Live Time(s): 41 Amp Time(µs):
43

Sum Spectrum

eZAF Smart Quant Results

Element Weight % Atomic % Net Int. Net Int. Error

CK 6 11.33 14.1 0.07

OK 36.07 51.09 125.3 0.02

NaK 1.47 1.45 10.2 0.14

MgK 2.76 2.58 27.4 0.06

PK 19.16 14.02 147.6 0.01

KK 0.44 0.26 2 0.61

CaK 34.1 19.28 106.2 0.02

FIGURE 3: X-RAY DIFFRACTOMETER

 Sample 1: Stone1

10000 Meas. data:stone-1/Data 1

8000

6000

4000
Intensity (counts)

2000

0
1 2 3 4 5
Q (1/ang.)
Peak list

No. 2-theta(deg) d(ang.) Height(counts) FWHM(deg) Size(ang.)

1 10.093(12) 8.757(10) 1543(39) 0.554(11) 150(3)

2 13.785(13) 6.418(6) 869(29) 0.502(18) 166(6)
3 14.313(10) 6.183(4) 521(23) 0.43(6) 196(29)
4 14.76(2) 5.995(8) 1013(32) 0.65(5) 129(10)
5 15.527(12) 5.702(4) 1215(35) 0.576(13) 145(3)
6 18.06(2) 4.908(6) 696(26) 0.43(11) 197(51)
7 18.56(3) 4.776(7) 428(21) 0.32(13) 263(104)
8 20.04(2) 4.427(5) 227(15) 0.51(7) 164(23)
9 20.853(10) 4.256(2) 565(24) 0.44(3) 193(11)
10 23.11(3) 3.846(4) 845(29) 0.93(3) 91(3)
11 27.53(4) 3.238(5) 2130(46) 0.6(3) 148(66)
12 27.798(12) 3.2067(14) 4200(65) 0.67(14) 129(27)
13 28.216(15) 3.1601(16) 3842(62) 0.50(6) 170(21)
14 29.310(11) 3.0447(11) 2253(47) 0.67(6) 128(11)
15 31.63(2) 2.827(2) 661(26) 0.54(5) 159(14)
16 32.08(3) 2.788(3) 690(26) 0.57(12) 152(31)
17 32.93(2) 2.7179(16) 954(31) 0.51(7) 168(24)
18 34.74(3) 2.580(2) 394(20) 0.41(4) 212(20)
19 35.31(5) 2.540(4) 375(19) 0.6(5) 145(126)
20 35.891(12) 2.5000(8) 345(19) 0.29(4) 304(40)
21 37.95(3) 2.3692(18) 590(24) 0.54(3) 162(8)
22 39.10(2) 2.3019(13) 153(12) 0.22(6) 403(114)
23 40.63(8) 2.219(4) 349(19) 0.94(9) 94(9)
24 45.50(3) 1.9918(11) 173(13) 0.41(8) 218(43)
25 47.015(11) 1.9312(4) 331(18) 0.36(4) 252(25)
26 47.80(2) 1.9012(8) 123(11) 0.22(7) 413(133)
27 48.54(3) 1.8741(12) 402(20) 0.44(3) 206(16)
28 50.29(4) 1.8129(12) 356(19) 0.38(3) 240(21)
29 52.96(6) 1.727(2) 103(10) 1.7(3) 55(10)
30 58.18(12) 1.584(3) 186(14) 1.37(14) 69(7)
31 59.89(5) 1.5432(12) 290(17) 0.74(7) 129(11)
32 66.18(4) 1.4109(8) 94(10) 0.71(13) 140(26)
33 68.01(3) 1.3773(6) 252(16) 0.52(3) 193(11)
34 72.03(3) 1.3099(5) 108(10) 1.18(10) 87(7)
35 81.130(19) 1.1845(2) 134(12) 0.48(6) 228(28)
36 82.73(5) 1.1656(6) 131(11) 0.64(7) 174(20)
37 84.60(7) 1.1445(8) 115(11) 0.54(6) 208(23)
 Sample 2: Stone2

Meas. data:stone-2/Data 1

15000

10000
Intensity (counts)

5000

0
1 2 3 4 5
Q (1/ang.)
Peak list
No. 2-theta(deg) d(ang.) Height(counts) FWHM(deg) Size(ang.)
1 9.714(2) 9.0974(19) 4925(70) 0.131(7) 637(33)
2 9.893(6) 8.933(6) 5704(76) 0.347(6) 240(4)
3 13.289(10) 6.657(5) 1112(33) 0.334(10) 250(7)
4 14.032(13) 6.306(6) 981(31) 0.409(18) 204(9)
5 14.642(13) 6.045(5) 806(28) 0.332(17) 252(13)
6 15.417(4) 5.7426(15) 3736(61) 0.336(4) 250(3)
7 17.777(9) 4.985(2) 1461(38) 0.343(8) 245(6)
8 18.36(5) 4.827(14) 157(13) 0.20(5) 411(97)
9 19.8384(18) 4.4716(4) 3539(59) 0.099(8) 850(65)
10 20.859(7) 4.2550(14) 3020(55) 0.348(6) 242(4)
11 22.797(7) 3.8976(11) 1827(43) 0.285(17) 297(18)
12 23.230(9) 3.8259(14) 1102(33) 0.31(2) 269(19)
13 23.914(8) 3.7179(13) 1170(34) 0.166(14) 510(44)
14 25.89(4) 3.438(5) 404(20) 0.29(3) 296(33)
15 27.288(5) 3.2654(6) 3607(60) 0.301(7) 283(7)
16 27.669(3) 3.2214(3) 11185(106) 0.338(5) 253(4)
17 28.199(4) 3.1620(4) 10239(101) 0.516(5) 165.8(17)
18 29.60(6) 3.015(6) 224(15) 0.25(7) 347(103)
19 30.34(2) 2.9437(19) 262(16) 0.11(5) 803(380)
20 30.91(3) 2.891(3) 554(24) 0.21(4) 400(76)
21 31.432(3) 2.8437(3) 1066(33) 0.09(2) 996(249)
22 32.0553(13) 2.78985(11) 2492(50) 0.071(14) 1214(236)
23 32.136(6) 2.7830(5) 1854(43) 0.150(17) 575(63)
24 34.679(14) 2.5846(10) 1856(43) 0.361(14) 241(9)
25 35.70(2) 2.5133(15) 597(24) 0.296(19) 295(19)
26 36.81(2) 2.4398(14) 631(25) 0.30(7) 288(64)
27 37.06(2) 2.4239(13) 405(20) 0.20(3) 435(67)
28 37.834(11) 2.3760(6) 1487(39) 0.332(10) 264(8)
29 38.48(5) 2.337(3) 133(12) 0.5(3) 195(115)
30 39.892(2) 2.25800(14) 2822(53) 0.048(3) 1846(117)
31 40.002(4) 2.2520(2) 3213(57) 0.107(6) 824(44)
32 40.112(6) 2.2461(3) 1315(36) 0.018(14) 4778(3559)
33 40.529(5) 2.2239(2) 1320(36) 0.060(6) 1484(152)
34 40.635(10) 2.2184(5) 656(26) 0.066(14) 1347(288)
35 40.955(19) 2.2018(10) 665(26) 0.29(2) 307(22)
36 41.79(3) 2.1596(16) 343(19) 0.32(4) 278(36)
37 42.71(4) 2.1155(19) 387(20) 0.52(5) 170(17)
38 44.54(5) 2.033(2) 180(13) 0.32(5) 282(42)
39 45.43(7) 1.995(3) 134(12) 0.45(7) 201(30)
40 45.92(2) 1.9745(9) 304(17) 0.20(6) 454(143)
41 46.077(7) 1.9683(3) 384(20) 0.177(16) 510(46)
42 47.656(9) 1.9067(3) 408(20) 0.28(3) 321(29)
43 49.41(4) 1.8430(14) 319(18) 0.32(5) 284(40)
44 50.27(5) 1.8134(18) 210(14) 0.40(10) 232(56)
45 51.936(14) 1.7592(5) 343(19) 0.50(4) 184(16)
46 53.81(11) 1.702(3) 136(12) 0.45(10) 209(47)
47 55.00(6) 1.6682(17) 187(14) 0.29(6) 327(63)
48 56.63(4) 1.6239(11) 246(16) 0.43(6) 221(32)
49 57.46(2) 1.6024(6) 520(23) 0.46(3) 204(15)
50 58.42(9) 1.578(2) 163(13) 1.3(3) 75(18)
 Sample 3: Stone3

Meas. data:stone-3/Data 1

10000

5000
Intensity (counts)

0
1 2 3 4 5
Q (1/ang.)
Peak list
No. 2-theta(deg) d(ang.) Height(counts) FWHM(deg) Size(ang.)
1 13.330(10) 6.637(5) 305(17) 0.19(3) 446(73)
2 14.811(3) 5.9764(13) 6673(82) 0.334(4) 250(3)
3 15.201(7) 5.824(3) 1495(39) 0.222(12) 377(20)
4 17.95(3) 4.937(7) 360(19) 0.25(2) 339(33)
5 19.51(2) 4.546(5) 432(21) 0.22(2) 383(41)
6 22.914(13) 3.878(2) 665(26) 0.29(2) 295(21)
7 23.415(12) 3.7961(19) 911(30) 0.345(16) 246(11)
8 24.2828(18) 3.6623(3) 10404(102) 0.2875(15) 295.2(16)
9 27.2(3) 3.28(4) 114(11) 1.8(6) 47(14)
10 27.82(2) 3.205(2) 615(25) 0.30(3) 284(25)
11 28.630(10) 3.1154(11) 1391(37) 0.344(13) 249(9)
12 29.617(14) 3.0138(14) 1254(35) 0.28(3) 307(35)
13 30.005(4) 2.9756(4) 7003(84) 0.312(8) 275(7)
14 30.630(11) 2.9163(10) 1985(45) 0.466(13) 185(5)
15 31.391(9) 2.8474(8) 1882(43) 0.334(13) 258(10)
16 31.727(12) 2.8180(10) 652(26) 0.035(12) 2442(843)
17 31.804(16) 2.8113(14) 320(18) 0.036(18) 2414(1234)
18 32.01(4) 2.794(3) 435(21) 0.45(10) 190(43)
19 35.49(3) 2.528(2) 448(21) 0.24(7) 356(100)
20 35.905(7) 2.4990(5) 3716(61) 0.305(10) 286(9)
21 36.58(4) 2.455(3) 354(19) 0.49(14) 180(53)
22 37.04(2) 2.4252(14) 601(25) 0.35(3) 253(21)
23 38.129(5) 2.3582(3) 7285(85) 0.340(4) 258(3)
24 39.782(8) 2.2640(4) 2753(52) 0.368(6) 240(4)
25 40.714(11) 2.2143(6) 1081(33) 0.274(11) 324(13)
26 42.33(3) 2.1334(14) 434(21) 0.33(3) 272(24)
27 43.565(9) 2.0758(4) 2385(49) 0.414(8) 216(4)
28 44.67(2) 2.0270(9) 202(14) 0.17(2) 519(66)
29 45.35(2) 1.9982(9) 494(22) 0.4(2) 226(136)
30 45.763(10) 1.9810(4) 2016(45) 0.32(2) 280(20)
31 46.434(8) 1.9540(3) 2045(45) 0.451(14) 200(6)
32 46.919(5) 1.9349(2) 2166(47) 0.403(7) 225(4)
33 48.026(7) 1.8928(2) 1316(36) 0.366(6) 248(4)
34 48.882(13) 1.8617(5) 507(23) 0.22(3) 408(61)
35 49.284(10) 1.8474(4) 1146(34) 0.39(4) 237(23)
36 49.962(9) 1.8239(3) 1286(36) 0.532(10) 172(3)
37 50.824(6) 1.7950(2) 1469(38) 0.330(6) 279(5)
38 52.589(7) 1.7388(2) 1390(37) 0.393(6) 235(4)
39 53.66(3) 1.7067(9) 194(14) 0.33(13) 286(116)
40 54.11(4) 1.6934(12) 353(19) 0.46(6) 203(26)
41 56.02(2) 1.6401(6) 302(17) 0.38(2) 244(14)
42 56.70(3) 1.6220(8) 154(12) 0.27(4) 356(49)
 Sample 4: Stone4

8000
Meas. data:stone-4/Data 1

6000

4000
In te n sity (c o u n ts )

2000

0
1 2 3 4 5
Q (1/ang.)

Peak list
No. 2-theta(deg) d(ang.) Height(counts) FWHM(deg) Size(ang.)
1 6.22(3) 14.21(7) 560(24) 0.64(8) 129(17)
2 8.41(10) 10.50(12) 155(12) 0.36(14) 230(87)
3 22.3(2) 3.98(4) 93(10) 1.4(2) 59(8)
4 25.787(13) 3.4520(18) 1169(34) 0.542(12) 157(3)
5 27.96(3) 3.188(4) 481(22) 0.86(4) 99(5)
6 30.44(4) 2.934(3) 422(21) 0.55(8) 157(22)
7 31.16(4) 2.868(4) 1443(38) 0.54(6) 161(17)
8 31.76(3) 2.815(2) 2569(51) 1.52(12) 57(4)
9 33.81(5) 2.649(4) 457(21) 0.76(7) 115(10)
10 34.482(16) 2.5989(12) 350(19) 0.48(3) 181(12)
11 39.67(5) 2.270(3) 404(20) 1.29(5) 69(3)
12 41.35(6) 2.182(3) 192(14) 0.74(8) 120(12)
13 46.49(5) 1.952(2) 296(17) 1.42(12) 64(5)
14 49.43(5) 1.8422(16) 332(18) 1.18(9) 77(6)
15 53.18(2) 1.7208(6) 482(22) 0.504(17) 184(6)
16 61.93(11) 1.497(2) 85(9) 0.72(14) 134(26)
17 63.72(7) 1.4594(15) 256(16) 1.97(7) 49.5(17)
18 76.99(13) 1.2376(18) 121(11) 2.53(12) 42(2)
19 87.60(8) 1.1129(8) 139(12) 1.48(7) 78(4)
 Sample 5: Stone5

Meas. data:stone-5/Data 1

10000
I n t e n s it y ( c o u n t s )

5000

0
1 2 3 4 5
Q (1/ang.)

eak list
No. 2-theta(deg) d(ang.) Height(counts) FWHM(deg) Size(ang.)
1 14.073(4) 6.2877(16) 716(27) 0.051(7) 1631(216)
2 14.5733(17) 6.0731(7) 7128(84) 0.088(4) 946(39)
3 14.670(3) 6.0333(12) 7146(85) 0.111(8) 757(57)
4 14.854(5) 5.959(2) 1935(44) 0.109(13) 765(89)
5 24.167(17) 3.680(3) 740(27) 0.272(18) 312(20)
6 25.70(5) 3.463(7) 236(15) 0.34(6) 249(45)
7 29.721(6) 3.0035(6) 1378(37) 0.100(9) 855(75)
8 29.951(9) 2.9809(9) 2703(52) 0.323(11) 266(9)
9 30.55(3) 2.924(3) 345(19) 0.24(4) 364(59)
10 31.44(2) 2.8434(19) 578(24) 0.31(3) 277(23)
11 31.93(6) 2.801(5) 384(20) 0.61(11) 142(25)
12 35.735(13) 2.5105(9) 835(29) 0.234(12) 372(19)
13 36.85(6) 2.437(4) 124(11) 0.35(7) 250(50)
14 38.017(10) 2.3650(6) 1087(33) 0.315(9) 279(8)
15 39.608(16) 2.2735(9) 371(19) 0.38(2) 229(15)
16 40.611(10) 2.2197(5) 519(23) 0.203(16) 436(34)
17 42.257(16) 2.1369(8) 173(13) 0.19(9) 459(205)
18 45.460(7) 1.9936(3) 813(29) 0.075(8) 1196(132)
19 45.695(14) 1.9838(6) 1067(33) 0.311(18) 290(17)
20 46.30(9) 1.959(3) 170(13) 0.5(3) 200(118)
21 46.697(17) 1.9436(7) 336(18) 0.32(4) 286(36)
22 47.84(6) 1.900(2) 179(13) 0.23(5) 402(97)
23 49.12(5) 1.8533(18) 158(13) 0.39(5) 235(31)
24 50.04(9) 1.821(3) 84(9) 0.30(9) 303(88)
25 50.61(2) 1.8021(7) 387(20) 0.27(2) 340(25)
26 52.38(3) 1.7452(9) 270(16) 0.27(4) 341(52)
27 57.82(3) 1.5933(7) 280(17) 0.27(4) 354(50)
 Sample 6: Stone6

10000 Meas. data:stone-6/Data 1

8000

6000
Intensity (counts)

4000

2000

0
1 2 3 4 5
Q (1/ang.)
Peak list
No. 2-theta(deg) d(ang.) Height(counts) FWHM(deg) Size(ang.)
1 9.97(12) 8.86(11) 74(9) 1.4(4) 59(15)
2 14.21(3) 6.228(13) 504(22) 0.46(5) 183(19)
3 14.764(5) 5.9952(18) 3146(56) 0.279(6) 300(6)
4 15.068(10) 5.875(4) 788(28) 0.228(13) 367(21)
5 19.56(15) 4.53(4) 154(12) 2.3(5) 37(9)
6 19.831(5) 4.4733(10) 597(24) 0.161(17) 523(57)
7 22.66(7) 3.921(12) 310(18) 1.22(17) 69(10)
8 23.38(5) 3.802(8) 518(23) 0.69(9) 124(15)
9 24.202(3) 3.6744(5) 5516(74) 0.289(3) 293(3)
10 28.53(3) 3.126(3) 471(22) 0.27(3) 314(35)
11 29.60(2) 3.015(2) 617(25) 0.24(3) 353(42)
12 29.946(6) 2.9814(6) 3180(56) 0.276(7) 311(8)
13 30.58(3) 2.921(2) 706(27) 0.44(2) 197(11)
14 31.303(12) 2.8552(11) 621(25) 0.90(4) 96(5)
15 31.9944(5) 2.79502(4) 2852(53) 0.022(3) 3854(555)
16 32.034(9) 2.7917(8) 2079(46) 0.235(7) 368(11)
17 35.857(11) 2.5023(7) 1372(37) 0.276(9) 316(11)
18 36.923(17) 2.4325(11) 304(17) 0.62(7) 142(17)
19 37.081(8) 2.4224(5) 428(21) 0.22(3) 395(55)
20 37.327(5) 2.4071(3) 426(21) 0.023(11) 3849(1808)
21 37.951(13) 2.3689(8) 1843(43) 0.145(12) 604(49)
22 38.053(7) 2.3628(4) 2889(54) 0.241(8) 363(12)
23 39.717(15) 2.2675(8) 895(30) 0.41(3) 217(16)
24 40.137(12) 2.2448(6) 610(25) 0.180(19) 490(50)
25 40.653(18) 2.2175(9) 449(21) 0.28(2) 318(23)
26 42.435(5) 2.1284(3) 787(28) 0.22(2) 398(35)
27 43.468(16) 2.0801(7) 839(29) 0.31(2) 285(18)
28 45.35(2) 1.9982(9) 265(16) 0.26(2) 350(34)
29 45.705(11) 1.9834(5) 900(30) 0.308(11) 292(10)
30 46.274(12) 1.9603(5) 1071(33) 0.407(15) 222(8)
31 46.859(14) 1.9372(5) 713(27) 0.416(18) 217(9)
32 47.73(2) 1.9041(9) 510(23) 0.32(19) 280(162)
33 47.92(3) 1.8967(13) 315(18) 0.27(11) 332(128)
34 49.26(4) 1.8484(13) 366(19) 0.65(4) 140(9)
35 49.823(17) 1.8287(6) 519(23) 0.47(2) 195(8)
36 50.755(17) 1.7973(6) 446(21) 0.312(17) 294(16)
37 52.483(4) 1.74211(12) 1241(35) 0.061(5) 1521(114)
38 52.623(11) 1.7378(3) 740(27) 0.21(2) 443(49)
39 53.941(18) 1.6984(5) 154(12) 0.32(5) 293(49)
40 57.92(5) 1.5907(13) 180(13) 0.57(13) 165(38)
41 58.731(2) 1.57078(5) 3058(55) 0.085(2) 1122(27)
42 58.892(4) 1.56689(9) 1510(39) 0.089(4) 1074(46)
43 59.45(5) 1.5534(12) 255(16) 0.85(17) 113(23)
44 60.302(10) 1.5336(2) 280(17) 0.35(4) 278(29)
45 61.454(10) 1.5076(2) 307(18) 0.31(5) 308(47)
46 70.13(7) 1.3408(11) 55(7) 1.4(2) 75(14)
47 78.54(4) 1.2169(6) 71(8) 0.67(13) 160(30)
48 80.86(8) 1.1877(10) 182(13) 0.83(9) 131(14)
FIGURE 4: ATOMIC FORCE MICROSCOPY (AFM)
Sample 1: Stone1
At 2µm
At 3µm
Sample 2: Stone2
At 2µm
At 3µm
Sample3: Stone3
At 2µm
At 3µm

stone-1-
Results
Image Raw Mean 605 nm
Image Mean 0.000012 nm
Image Z Range 354 nm
Image Surface Area 4.43 µm²
Image Projected Surface Area 4.00 µm²
Image Surface Area Difference 10.7 %
Image Rq 50.5 nm
Image Ra 41.6 nm
Image Rmax 354 nm
Raw Mean 606 nm
Mean 0.000012 nm
Z Range 354 nm
Surface Area 4.43 µm²
Projected Surface Area 4.00 µm²
Surface Area Difference 10.7 %
Rq 50.5 nm
Ra 41.6 nm
Roughness Rmax 354 nm
Skewness 0.472
Kurtosis 3.33
Rz 103 nm
Rz Count 11.0
Peak Count 11.0
Valley Count 14.0
Max Peak ht (Rp) 249 nm
Average Max Height (Rpm) 71.5 nm
Maximum Depth (Rv) -249 nm
Average Max Depth (Rvm) -24.9 nm
Line Density 0.996 /µm
Box X Dimension 2.00 µm
Box Y Dimension 2.00 µm

stone-2-

Results
Image Raw Mean -135 nm
Image Mean -0.000019 nm
Image Z Range 531 nm
Image Surface Area 4.90 µm²
Image Projected Surface Area 4.00 µm²
Image Surface Area Difference 22.4 %
Image Rq 114 nm
Image Ra 95.3 nm
Image Rmax 531 nm
Raw Mean -134 nm
Mean 0.500 nm
Z Range 531 nm
Surface Area 4.86 µm²
Projected Surface Area 3.97 µm²
Surface Area Difference 22.5 %
Rq 114 nm
Ra 95.3 nm
Roughness Rmax 530 nm
Skewness 0.0851
Kurtosis 2.21
Rz 441 nm
Rz Count 2.00
Peak Count 2.00
Valley Count 2.00
Max Peak ht (Rp) 275 nm
Average Max Height (Rpm) 166 nm
Maximum Depth (Rv) -275 nm
Average Max Depth (Rvm) -275 nm
Line Density 0.500 /µm
Box X Dimension 1.99 µm
Box Y Dimension 1.99 µm

stone-3-

Results
Image Raw Mean -845 nm
Image Mean 0.111 nm
Image Z Range 426 nm
Image Surface Area 4.85 µm²
Image Projected Surface Area 4.00 µm²
Image Surface Area Difference 21.3 %
Image Rq 67.3 nm
Image Ra 54.7 nm
Image Rmax 426 nm
Raw Mean -846 nm
Mean 0.146 nm
Z Range 418 nm
Surface Area 4.84 µm²
Projected Surface Area 3.98 µm²
Surface Area Difference 21.4 %
Rq 67.2 nm
Ra 54.6 nm
Roughness Rmax 419 nm
Skewness -0.0232
Kurtosis 2.64
Rz 362 nm
Rz Count 4.00
Peak Count 6.00
Valley Count 4.00
Max Peak ht (Rp) 254 nm
Average Max Height (Rpm) 121 nm
Maximum Depth (Rv) -254 nm
Average Max Depth (Rvm) -192 nm
Line Density 1.50 /µm
Box X Dimension 1.99 µm
Box Y Dimension 2.00 µm
Project Report:
Scanning Electron Microscopy, X-Ray Diffraction Analysis and Atomic
Force Microscopy of the Ring Structures in Human Renal Stones
MUKUL KUMAR
Mentors: Professor Rakesh Sharma* and Professor Yuvraj Singh Negi**
*Government Medical College, Saharanpur (UP) and **Indian Institute of Technology, Roorkee

Background: In Saharanpur district, whole upper crust is abdundant in calcified and alkaline clay.
As a result, kidney stones are a painful affliction that will affect almost >30-40% of the population
suffers during their lifetimes. More than 50% of the stones are diagnosed in adolescents and adults
specially inhabiting in industrialised belt in city. Major stones are composed of calcium oxalate and
urate crystals as their major constituent. Both the monohydrate form of calcium oxalate known as
whewellite, and the dihydrate forms[1]. The mineral name wedellite is very common in human kidney
stones. In intravascular scanning (IVC) examination, calcium oxalate dihydrate forms of stone are seen
as distinct bipyramids and is usually found on the outside of mixed stones.
Scope: The mechanism for kidney stone formation is still controversial and the role of various promoters
or inhibitors in the growth and aggregation of crystallites is still the subject of ongoing research.
Aim: The progress of stone formation and its size is indicated by abnormal kidney function tests.
The kidney stone examination dictates the stone crystallization process for surface topography (by
Scanning Electron Microscope), Element composition and layer arrangement (by X-Ray
diffraction), and nanoscale atomic interactions of microelements (by Atomic Force Microscopy).
Materials and Methods: Confirmed renal stone bearing patients selection was done from post-
COVID subjects: Blood urea, creatinine, GFR lab tests; Stone IVP scans; post-surgery excised
stone examination by SEM, X-Ray Diffraction and Atomic Force Microscope[2].
Results: Six renal calcium oxalate monohydrate stone were grossly examined by optical microscopy
showed distinct rings with radial striations surrounding a stone nucleus. (See Fig 1). AFM and Low
voltage SEM, showed stones having a loosely ordered arrangement of calcium oxalate crystallites
ranging in size from 500Å to 3000 Å. To determine the microstructural origins of the ring structures six
stones were selected after analysis by XRD confirmed their overall composition. The composition
showed abdundance of calcium, nitrogen-rich urate, potassium-oxalate. Both the SEM and AFM images
showed the crystallites of different shapes due to calcification growing patterns in different shapes. Figs
2-4 show the area imaged using AFM and SEM. Both AFM and SEM measured the heights above a
reference surface. AFM showed better resolution while SEM scanned greater depth of field across the
stones.
Rings structures from the box are shown in Fig 1 using both AFM (Fig 3) and SEM (Fig 4). The darker
portions of the ring showed low density of smaller calcium, potassium and oxygen atomes while the
lighter regions showed high density of larger urate and oxalate crystals. In different ring structures from
different areas of stone crystallites showed different irregular alignment of crystallites. These
observations are consistent with the gross optical microscopy.
Outcome: The rings are an intrinsic property of the stone crystallization patterns during progress of
disease in kidneys. The imaging and scanning techniques make them detectable with chemical
determinants. The smaller crystallites in lower density regions are more easily detected. The
physiological cause of different crystallite size distributions is complex. The stone mineralization
analysis and crystallization process may suggest the choice of stone dissolution therapy and cause of re-
occurrence.
References
[1] E.L. Prien and E.L. Prien, Amer. J. Medicine 45 (1968) 654.
[2] H.H. Dorian, P. Rez and G.W. Drach, J. Urol. 156 (1996) 1833.
THANK YOU