The document provides an overview of various microscopy techniques, including light, electron (SEM and TEM), and NMR, highlighting their functions and applications. It also discusses laboratory instruments like hematology analyzers, differentiating between Coulter counters and flow cytometry, and compares real-time PCR methods using TaqMan and SYBR Green dyes. Additionally, it covers the principles of real-time PCR and the significance of relative and absolute quantification in DNA analysis.

1. Microscope:
 Optical Instrument.
 To view tiny objects that are not visible to the naked eye.
Electron Microscope:
 Uses electrons to illuminate the specimen.
 High resolution.
 Magnify images 2 million times (light = 2000x).
Magnification:
The increase in the size of an object when viewed through the microscope.
Resolution:
 The level of detail that the microscope can reveal in a specimen.
 Measured in nanometers (nm) or micrometers (μm).
Contrast:
 The intensity between adjacent parts of an image or specimen helps to differentiate and
distinguish details.
 degree of difference in brightness between the light and dark areas of the specimen
being viewed.
Methods of enhancing contrast:
 Brightfield illumination: The most common method uses reflected light to provide
essential contrast by creating a bright background and dark features.
 Darkfield illumination: This method uses scattered light to produce a dark background
and brightly illuminated features.
 Phase contrast: This method uses interference of light waves to highlight differences in
refractive index within a specimen, making it possible to see otherwise invisible
features.
 Fluorescence: Fluorescent dyes or stains - making them visible under UV light.
 Differential Interference Contrast (DIC): Polarized light to produce contrast.
Nature of Image:
 Close-up, highly magnified representation.
 Details about the structure, composition, or behavior of the specimen.
 Purpose: Make clearer observations and analyses.
Parts of Microscope: The parts of a microscope typically include:
1. eyepiece lens (10x)
2. objective lenses (4x, 10x, 40x)
3. nosepiece (to hold the objective lenses)
4. coarse focus knob
5. fine focus knob
6. stage (to hold the specimen)
7. stage clips (to secure the specimen)

2. 8. light source (to illuminate the specimen)
9. diaphragm (to control the amount of light)
10. base (to support the microscope)
11. arm (to connect the head and the base)
12. microscope body (to hold all the components together)
Note: The number of lenses and components may vary depending on the type and model of the
microscope.
SEM vs TEM:
 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM).
 Both are electron microscopy techniques to study materials at the nanoscale.
Differences:
 Image formation:
o SEM - scanning the sample surface with a focused electron beam and detecting
electrons that are emitted by the sample.
o TEM - transmitting electrons through a thin sample and detecting electrons that
pass through it.
 Sample preparation:
o SEM – Bulkier sample required.
o TEM – Thin sample required / electron transparent.
 Resolution:
o SEM – Lower Resolution than TEM.
o TEM - Higher resolution than SEM because it detects electrons that have passed
through the sample.
 Information obtained:
o SEM - Surface morphology and elemental composition.
o TEM - Crystal structure, lattice spacings and defects.
 Radiation damage:
o Higher risk of damage with TEM because of the transmission of electrons.
NMR:
Principle:
 By changing the strength of a magnetic field, the nuclei of certain atoms will absorb and
then emit electromagnetiradiationns.
 Change in magnetic field strength causes the spinning nuclei to precess and realign. This
produces an NMR signal.
 The signal can be measured and then used to determine the chemical and structural
properties of the sample under study.
Clinical Application:
NMR - MRI.
MRI:
1. Brain and spinal cord imaging
2. Joint and skeletal imaging
3. Abdominal and pelvic imaging

3. 4. Cardiac and vascular imaging
5. Breast imaging
6. Neurological disorders diagnosis
7. Cancer diagnosis and treatment monitoring
8. Musculoskeletal injuries and disorders
9. Liver, pancreas and gallbladder imaging
10. Evaluation of pelvic floor disorders
11. Diagnosis of sports-related injuries
12. Diagnosis of multiple sclerosis
13. Detection of stroke and brain tumors
14. Evaluation of spinal cord and vertebral column disorders.
Hematology Analyzer:
Definition:
 Laboratory instrument used to count and analyze the different types of cells in a blood
sample, including red blood cells, white blood cells, and platelets.
 Used to diagnose and monitor various medical conditions, such as anemia, infection, and
bleeding disorders.
Types:
There are several types of hematology analyzers, including:
 Three-part hematology analyzer:
o Separates three populations: RBC, WBC, Platelets.
o To study infections, anemia, and bleeding disorders.
 Fiver part hematology analyzer:
o Separate five populations: RBC, WBC, Platelets, immature granulocytres and
lymphocytes.
 Coulter Counter: An electrical impedance-based analyzer that measures cell size and
number.
 Flow Cytometry: A laser-based technology that uses light scattering and fluorescent
labeling to analyze cells.
Coulter counter vs Flow Cytometry:
 Principle:
o Coulter counter -
 Blood cells are suspended in an electrolyte solution and passed through
a small aperture. Cells displace the electrolyte solution and cause a
change in electrical impedance.
 The change in electrical impedance is proportional to the volume of the
cell.
 Helps measure the cell size and number.
o Flow cytometry:
 Blood cells are passed one by one through a laser beam.
 The cells scatter the light.
 The intensity and angle of the scattered light are measured.
 Helps determine the size, shape and internal complexity of the cell.
 Other parameters:

4.  Fluorescence dye as cell marker – Cell characteristic and function,
surface antigen and intracellular proteins.
 Technology:
o Coulter counter - electrical impedance-based analyzer
o Flow cytometry - laser-based technology that uses light scattering and
fluorescence.
 Analysis:
o Coulter counter - Cell size and number.
o Flow cytometry - Detailed information on cell size, shape, and fluorescence
intensity, allowing for the simultaneous analysis of multiple cell parameters
(Cell characteristic and function, surface antigen and intracellular proteins).
 Sample Preparation:
o Coulter counter requires a larger volume of the blood sample.
o Flow cytometry requires a smaller volume of a blood sample. And requires
additional processing steps, such as staining, to enhance the analysis.
 Applications/Uses:
o Coulter counter - Routine complete blood cell count (CBC) analysis.
o Flow cytometry - Specialized applications; immunophenotyping and analysis of
rare cell populations.
Difference between oven and incubator:
 Purpose:
o Oven - Heating, drying, and sterilizing samples.
o Incubator – Growing and maintaining living cells or microorganisms in a
controlled environment.
 Temperature Control:
o Oven: ambient to 300°C
o Incubator: more precise and controlled temperature range, often between 20-
50°C.
 Humidity:
o Oven: No control.
o Incubator: Controlled. Crucial for growing living cells or microorganisms.
 Air Circulation:
o Oven: Poor.
o Incubator: Good to ensure a uniform temperature and prevent contamination.
 CO2 Control:
o Oven: Does not control CO2 level.
o Incubator: Controlled CO2 levels.
Difference between Safety Cabinet and Laminar Airflow:
Lab equipment to protect samples and workers from contamination/ventilation.
Main difference: Type of airflow they use to maintain a clean environment.
Safety Cabinet:
 Protects samples and workers from exposure to hazardous materials by an inward flow
of air to keep contaminants inside the cabinet while clean air is drawn from the room.

5.  The air inside the cabinet is filtered to remove any contaminants before it is exhausted
back into the room.
Laminar Flow Cabinet:
 Used to maintain a clean and contamination-free environment for delicate or sensitive
samples.
 Uses continuous, unidirectional clean air filtered to remove contaminants.
 The airflow is parallel and uniform, creating a "laminar" flow.
 Helps prevent contamination of the samples inside the cabinet.
 Use HEPA filter.
When is a Vertical Laminar Flow Cabinet the Best Choice?
Providing a contamination-free area for non-hazardous products that are safe for an operator to
inhale. Clean and particle free.
Uses:
 Medical, Pharmaceutical, Scientific, and Electronics fields.
 Tasks:
o Preparation, supply and testing of sterile pharmaceutical products
o To handle lab samples, products, and other specimens.
o Cell culture.
o Preparing and pouring bacterial growth media.
o Quality control.
o Electronic and optical assemblies and systems.
Sanger Sequencing vs NGS:
Sanger NGS
Throughput Low Throughput method,
1 Fragment at a time
High Throughput,
Millions of fragments at a time
Other name Di-deoxy chain termination Massive parallel sequencing
Generation First Second
Cost Higher cost More cost effective
Accuracy More accurate Higher error rate
Analogical Digital
Sensitivity Less sensitive High sensitive
Application Gold standard in research,
gene sequencing, mutation
analysis
Larger project, clinical lab
TaqMan and SYBR Green:
TaqMan and SYBR Green are different fluorescent dyes commonly used for real-time PCR
(polymerase chain reaction), a technique used to amplify DNA sequences.
Binding: SYBR Green is a generic dye that binds to double-stranded DNA. Taqman to single-
stranded DNA.
Principle: TaqMan is based on a probe-based system, while SYBR Green is a dye-based method.

6. Specificity: TaqMan is more specific (specific probes - target sequence). SYBR Green can detect
all amplified DNA.
Sensitivity: TaqMan is more sensitive than SYBR Green.
Cost: TaqMan is more expensive than SYBR Green.
Speed: TaqMan is generally slower than SYBR Green as it requires an additional step for the
binding and cleavage of the probe.
Interference: SYBR Green can be prone to interference from non-specific binding to other
sources of double-stranded DNA, while TaqMan is unaffected.
Detection limit: TaqMan has a lower detection limit than SYBR Green, as it can detect lower
levels of target DNA.
Relative vs absolute quantification:
SYBR Green and TaqMan are both commonly used for real-time PCR and can be used for both
relative and absolute quantification of target DNA.
Relative quantification: The goal is to determine the relative amount of target DNA in different
samples, often normalized to an internal control or reference gene. SYBR Green and TaqMan can
be used for relative quantification, where the change in fluorescence over the reaction
determines the relative amount of target DNA in each sample.
Absolute quantification: In absolute quantification, the goal is to determine the exact number of
copies of target DNA in a sample. Absolute quantification typically requires a standard curve,
where a series of known amounts of target DNA is amplified alongside the unknown sample,
allowing the amount of target DNA in the sample to be determined. TaqMan is typically used for
absolute quantification, as the specificity of the probe system allows for a more accurate
determination of the number of target DNA copies in the sample.
It is worth noting that both methods have their limitations, and the choice of method will
depend on the specific requirements and goals of the experiment.
Taqman probe working:
The TaqMan probe is a fluorescent probe used in real-time PCR (polymerase chain reaction) to
detect and quantify specific DNA sequences. The probe consists of a fluorescent dye, a quencher,
and a probe-specific DNA sequence. Here's how the TaqMan probe works:
The TaqMan probe is added to the PCR reaction mixture and the template DNA, primers, and
polymerase.
During the PCR reaction, the primers anneal to the target DNA and the polymerase extends the
primers, creating new DNA strands. If the target sequence is present in the template DNA, the
TaqMan probe will bind to it.
The fluorescence from the dye is quenched by the quencher when the probe is intact.
As the polymerase cleaves the probe during amplification, the fluorescent signal is released and
can be measured. The fluorescence intensity increases proportionally with the amount of
amplified target DNA.

7. The fluorescence intensity can be monitored in real-time during the PCR reaction by measuring
the fluorescence intensity of the reaction mixture.
After the PCR reaction, the fluorescence intensity is plotted against the cycle number to
determine the amount of target DNA present in the sample.
The TaqMan probe is considered a more specific and sensitive method than other real-time PCR
methods, as it relies on a probe-specific sequence to detect only the target DNA and not non-
specific amplified DNA. However, designing TaqMan probes for each target gene is also more
expensive and time-consuming.
SYBR Green technology uses a fluorescent dye that binds to double-stranded DNA and
fluoresces in proportion to the amount of DNA present. The fluorescence intensity can then be
monitored in real-time during the PCR reaction.
Here's how the SYBR Green technology works:
The SYBR Green dye is added to the PCR reaction mixture and the template DNA, primers, and
polymerase.
During the PCR reaction, the primers anneal to the target DNA, and the polymerase extends the
primers, creating new DNA strands. As the DNA strands are amplified, the double-stranded DNA
increases and more SYBR Green dye binds to it.
The fluorescence emitted by the SYBR Green dye can be monitored in real-time during the PCR
reaction by measuring the fluorescence intensity of the reaction mixture. The fluorescence
intensity increases proportionally with the amount of amplified DNA.
After the PCR reaction, the fluorescence intensity is plotted against the cycle number to
generate a melting curve, which can be used to confirm the specificity of the amplification and
the identity of the amplified product.
SYBR Green technology is widely used in real-time PCR due to its simplicity, ease of use, and
relatively low cost compared to probe-based methods such as TaqMan. However, SYBR Green
can also produce non-specific signals and can be affected by interference from other sources of
double-stranded DNA, so it is important to confirm the specificity of the amplification using
methods such as melting curve analysis.
Principle of Real-Time PCR:
The principle of real-time PCR is based on the amplification of target DNA through repeated
cycles of heating and cooling and the measurement of the amplified DNA using fluorescent dyes.
The procedure of Real-Time PCR:
In real-time PCR, the sample DNA is mixed with specific primers, a polymerase enzyme, and
fluorescent dyes (such as SYBR Green or TaqMan probes).
 The reaction mixture is then subjected to a series of temperature cycles in a
thermocycler.
 During each cycle, the temperature is raised to denature the double-stranded DNA into
single strands, then lowered to allow the primers to anneal to the target DNA, and finally
raised again to allow the polymerase to extend the primers and amplify the target DNA.
 As the target DNA is amplified, the fluorescent dye binds to the double-stranded DNA
and emits fluorescence in proportion to the amount of DNA present.

8.  The fluorescence is monitored in real-time during the reaction, allowing for the rapid
and quantitative determination of the target DNA.
 The final result is expressed as the number of copies of target DNA per reaction or per
unit of sample, which can be compared to a standard curve generated from known
amounts of target DNA.