HISTOLOGY LAB BASIC MICROSCOPY 1/024/2021

- Illumination system
MICROSCOPE - Substage condenser
- Comes from the word: - Filter holder and filter
- Mikros = small - Light intensity adjustment
- Skopein = look knob/rheostat
- It is an instrument used to see objects that are too
small for the naked eye. MECHANICAL SYSTEM
1. Support system
Compound Microscope - BODY
- Is used to view minute details of cells and primarily - Base – Must rest on a firm, flat bench or
tissues. table
- Compound = set of lenses (ocular and objective) - Arm - Supports the main tube and the
used simultaneously to magnify the image. mechanical stage
- Light = necessity to use a light source for better - Revolving nosepiece (turret) – Holds
viewing of the object. the objectives

HANDLING and STORAGE PRECAUTIONS

1. When carrying the microscope, use both hands.
One holding its arm and the other is supporting
base with the cord in secure position.
2. Clean all glass components by wiping gently with
lens paper. In the absence of such, use tissue
paper moistened with a mixture of ether 70% and
alcohol 30% to remove fingerprints and oil
smudges.
3. Don’t disassemble any part of the microscope as A= Proper way of carrying the microscope –
this could result in malfunction or reduced Base = Dominant hand ; Arm = Non-dominant hand
performance.
4. Store microscopes in a cabinet with cover to avoid
subjecting it to direct sunlight, high temperature,
humidity, dust or vibrations.

MICROSCOPE
- Binocular microscope = 2 oculars
- Monocular microscope = 1 ocular
- Aid in examining all the specimens and their
contents that are invisible in the naked eye
Components of the microscope:
1. Mechanical system
- Support system
- Body - Mechanical stage – Hold the slide secure
- Mechanical stage and allows the specimen moved smoothly
- Observation tube backwards, forwards and sideways
- Adjustment ▪ Stage clip – keeping the specimen in
- Coarse adjustment knob place
- Fine adjustment knob ▪ Vernier Scale – can be used to trace
2. Optical system part of the blood film you need to re-
- Magnification system examine
- Objectives ▪ Feedknob (x and y axis)
- Eyepiece
- Prism
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OPTICAL SYSTEM
• Magnification System
o Objectives

- Observation tube/head – composed of main

tube which houses the prism
o Scanner = x4
▪ Diopter – used to compensate the
o LPO = x10
difference in eyesight between the left
o HPO = x40
and the right eye
o OIO = x100
▪ Interpupillary distance – used to
o Eyepiece (oculars) – where we view the
adjust the eyepieces in relation to the
specimen; has x10 magnification
distance between the left and right eye
o Prism – refracts rays, splitting the image
directing them towards the two eyepieces
2. Adjustment System

• Illumination System
o Condenser

- Tension ring – adjustment of the focusing

mechanism that is made at the factory;
already set so that it is easy to focus but also
tight enough to avoid stage drifting - Made up number of lenses
Stage drifting – out of control - Centers the light from the mirror or
slipping of the mechanical stage, electric light source to central on
which is cause by the adjustment the microscopic field
of the tension ring or weight of the - Can be raised or lowered to give
stage maximum/minimum illumination
- Coarse adjustment knob – control the - Near light source = madilim
upward movement of the stage preventing - Far light source = maliwanag
the collision of the objective; movement from Iris diaphragm – has a corresponding
scanner to lower power objective number (kung x10, dapat x10 din)
- Fine adjustment knob – movement from - If lower ang magnification, lower
low power objective to high power objective din ang light na kailangan and vice
and to oil immersion objective versa.
- Tend to have eye strains bc
examining lower obj ang ginagamit
kay kusog na light
Height adjustment knob
Security knob
 HISTOLOGY LAB BASIC MICROSCOPY 1/024/2021

• Electron microscopy
o Mirror/light source – Provides light in o Transmission electron microscope
viewing the specimens; white or yellow light o Scanning electron microscope
o Filter holder and filter – Blue filter make • Autoradiography
microscopic field white rather than yellow • Cell tissue and culture
o Light intensity adjustment knob – • Enzyme histochemistry
controls the intensity of the light ; found at • Visualizing specific molecules
the base o Immunohistochemistry
PROBLEM CAUSES ACTION o Hybridization
The light is poor • The lamp bulb • Replace the
the field is is busted lamp bulb with LIGHT MICROSCOPE
completely dark • The a new one 1. Bright-field microscope
condenser is • Raise the
too low condenser to
• The iris correct
diaphragm is position
closed • Open the
diaphragm

Poor image • The slide is • Turn over the

quality upside down slide
• There is an air • Move the oil - Stained specimens are examined by
bubble in the immersion means of ordinary light that passes
oil (class A) objective through the specimen
• There is dirt quickly from - Conventional microscope that we use in
on the side to side the laboratory
objective • Clean the lens
• The oil is too • Use specified
sticky immersion oil
No image or • Revolving • Revolve to
image is dark on nosepiece is click stop
one side not in click position
stop position • A new
• Objective is objective
out of order maybe needed Left = (stained by H and E) cannot stain carbohydrates
There are dark • The surface of • A new Right = (stained by periodic acid Schiff)
shadows in the the eyepiece eyepiece
field of view has scratches maybe needed 2. Fluorescence microscope
which move as • The eye piece • Clean the
you turn around is dirty eyepiece
the eyepiece

METHODS OF STUDY
• Light Microscopy
- Bright field microscope
- Fluorescence microscope
- Phase-contrast microscope
- Confocal microscope
- Polarizing microscope - The sample can either be fluorescing in its
natural form or treated with fluorescing
chemicals
 HISTOLOGY LAB BASIC MICROSCOPY 1/024/2021

4. Confocal microscope
- Tissues sections are irritated with UV
light
- Fluorescent substances appear brilliant
on a dark background

- Scanning the specimen at successive

Left = applied with Acridine orange (bind w/ nucleic acid focal planes with a focused light beam,
and cause the nuclei emit yellow light while the cytoplasm often from a laser, and produces a 3D
will appear orange because of the RNA) reconstruction from the images
Right = stained w/ DAPI (4′,6-diamidino-2-phenylindole) - Employ a pair of pinhole apertures to limit
bind w/ DNA and stain blue while cytoplasm is green the specimen focal plane to a confined
because it was stained with another fluorescent chemical volume
called fluorescent phalloidin high affinity to the filaments in - Going to produce images from cell/tissue
cytoplasm. with the use of image sectioning
Stray light from other microscope is going to reduce the
3. Phase contrast microscope contrast within the image and compromises the resolving
power of the objective lens – a problem being avoided
when using confocal microscope

- Lens system that produces visible images

from transparent objects
- Used for observing unstained living cells
and tissue cultures
- MUST use for living tissues

Image are by section. All of them are going to fused

together in order to form a 3D image.

A= Bright-field microscope (transparent) (hard to identify)

B = Phase contrast microscope (able to see nucleus &
nucleolus)
C= Differential interference microscopy – modification of
phase contrast microscope; better image bcs it is going to
give a 3D like aspect of the cells. Uses Nomarski optics.
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5. Polarizing microscopy

(Urine specimen) Hexagonal shape= Cystine crystal

A= Bright-field microscope
B = Dark-field microscope
- Uses polarized light producing images
appearing as bright structures against a
dark background
- Often used to analyze tissue components
that contains highly oriented molecules
such as cellulose, collagen, microtubules
and actin filaments
- Dependent on birefringence – ability of
tissue components to rotate the direction
of the vibration of the polarized slide
(Skins scrapings of a patient w/ syphilis)
Dark-field microscopy is also utilized as a diagnostic
method to detect the presence of Treponema pallidum,
which is the causative agent of your syphilis

ELECTRON MICROSCOPE
1. Transmission electron microscopy

A = Bright-field microscope (can see nucleus)

B = Polarizing microscope (can only see tissue components
that are highly oriented molecules thus, u cannot see the
nucleus)

6. Dark-field microscopy

- High resolution images magnified as much

as 4000,000 times to be viewed in detail
- A bean of electrons focused using
electromagnetic lenses producing an image
with black, white, and gray regions
- Makes use of heavy metal ions added to
the fixative or dehydrating solutions used
for tissue preparation (Osmium tetroxide,
lead citrate, uranyl compounds) in which
they are going to bind smaller cellular
- Has a dark background wherein small
macromolecules to increase their electron
particles in the specimen that reflect
density and visibility leading to the
some light appear bright
production of back, gray or white images
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AUTORADIOGRAPHY

(very detailed compared to bright-field)

2. Scanning electron microscopy

- Makes use of photographic emulsion placed over a

tissue section to localize radioactive material within
tissues
- Able to detect newly synthesized macromolecules
in cells or tissue sections

- High-resolution view of cells, tissues, and

organs
- Does not pass through the specimen
- Specimen is spray-dried and spray-coated
with a very thin layer of heavy metals
(Gold) which reflects electrons in a bean
scanning the specimen
- Gives a 3D image
CELL AND TISSUE CULTURE
- Direct observation of cellular behavior in-vitro under
a phase contrast microscope
- To study molecular changes that occur in:
- Cancer
- Viral infections
 HISTOLOGY LAB BASIC MICROSCOPY 1/024/2021

1. Immunohistochemistry
ENZYME HISTOCHEMISTRY - Visualization od specific molecules can be
- Method for localizing cellular structures using a done with the aid of immunohistochemistry
specific enzymatic activity present in those – manly for the detection of specific
structures compounds with the use of antibodies.
- Phosphates (remove phosphate group)
- Dehydrogenases (transfer hydrogen ions) 2 types of immunohistochemistry:
- Peroxidases (promotes oxidation of 1. Antibodies binds to specific antigens
substrates w/ a transfer of hydrogen to form 2. Direct and indirect
hydrogen peroxide) a. Direct – use one type of antibody and its
going to be labeled
b. Indirect – use two antibodies.
1st antibody = not labeled
2nd antibody = labeled
The label could eb a form of enzyme or fluorescein dye.

Dark areas = presence of alkaline phosphatases

VISUALIZING SPECIFIC MOLECULES

- Detection of specific macromolecule using
compounds that bind specifically to molecule of
interest
- Detect and localize specific sugars, proteins, and
nucleic acids
- Phalloidin
- extracted from mushrooms which
interacts strongly with the actin
protein of microfilaments
(Fluorescein chemical)
- Protein A
- Purified substance taken from
staphylococcus aureus bacteria
that binds specifically to the fc SLE = serum lupus erythematosus
region of antibody molecules and Oragnelle contains DSDNA antigen (yun ang i-identify
can therefore be used to localize - If may fluorescence, ang serum ni patient may
naturally occurring or applied antibody against DSDNA antigen kasi nag attach
antibodies that are bound to siya.
cellular structures - Then sa second antibody which is attracted sa fc
- Lectins portion of the primary antibody which carries a
- Glycoproteins that is derived from label.
plants in order to specifically
identify the presence of
carbohydrates
 HISTOLOGY LAB BASIC MICROSCOPY 1/024/2021

Applied a probe that is going to identify the following

chromosomes:
- Green = chromosome 13
- Red = chromosome 21

(treated with an antibody against the enzyme lysozyme)

Find out the presence of lysozyme enzyme. They applied
an antibody against the lysozyme and the 2nd antibody is
labeled with peroxidase. The label of the 2nd antibody is not
necessarily a fluorescein dye but could also be an enzyme.
Since nag attach si antibody kay lysozyme, pag add ng
secondary antibody, nag attach pud siya and actually
labeled with the peroxidase enzyme. In order to detect the
presence of the lysozyme, add substrate sa peroxidase
which will lead to the production of gold color indicating the
presence of lysozyme enzyme.

3. Hybridization techniques
- Localizing mRNA or DNA by hybridizing the
sequence of interest to a complementary
strand o nucleotide probe
- Also known as In-situ hybridization – the
hybridization is conducted inside the cell

FISH – Florescence In Situ Hybridization

Right = Normal cell
Left = (3 red cell) = not normal
Trisomy 21 = indicated that the baby will suffer with down
syndrome