HUMAN HISTOLOGY – PRELIMS Note: After dehydration, there will be empty spaces

INTRODUCTION TO HISTOLOGY because of the removal of the water. Thus, ethanol must
Made by: Andrea A. Almohallas of BS MLS 2F be removed from the tissue.

HISTOLOGY AND ITS METHOD 3rd STEP

• Clearing – removal of the ethanol from the tissue. A
HISTOLOGY substance called xylene to clear/remove the ethanol
- Study of the tissue of the body and how these tissues from the tissue.
are arranged to constitute organs.
Note: After clearing, holes will be present on the tissue.
- Involves all aspects of tissue biology, with the focus
To have something to support the holes, in the tissue, it
on how cells’ structure and arrangement optimize
needs to be exposed to an infiltrating agent.
functions specific to each organ.
- Two interacting components: cells and extracellular
4th STEP:
matrix (ECM).
• Infiltration – support the holes on the tissue. It takes
place in a high temperature, usually 52°C - 60°C. The
Greek Teminologies:
“histos” - tissue most common infiltrating agent used is paraffin.
“logos” – study
Note: 52°C - 60°C is the melting point of paraffin. Once
the infiltrating agent fills up the holes in the tissue, it needs
Note: The structure of the cells always have something to
to be supported by a medium to help in the process of
do with the function.
cutting (slicing).

5th STEP:
• Embedding – the process of having a medium
supporting the tissue. The most common embedding
medium is paraffin.

Note: Once the tissue is finally embedded, it is now called

a TISSUE BLOCK.

Biopsy – getting tissue sample from someone who is

ALIVE.

Autopsy – getting tissue sample from someone who is

NOT ALIVE.

1st STEP:
• Fixation – use a “fixative”, a chemical that would
preserve the tissue. The goal of fixation is to preserve
the tissue in its life length state as much as possible.
Trimming – is cutting huge portions.
Formalin – most common preservative. Cutting – when tissue ribbons are formed.
In order to remove the formalin, it needs to be removed
together with water. Water is also the main cause for Note: Once ribbons are formed, it will be placed in a water
decay. To avoid that, it is needed to dehydrate, a process bath, and then fished out with a slide.
called DEHYDRATION.
PREPARATIONS OF TISSUES FOR STUDY
2nd
STEP: • STAINING
• Dehydration – expose the tissue to a series of - Acidic structures
increasing alcohol concentration. Usually, some Basic dye = basophilic (e.g., nucleus)
tissues start at 30% Ethanol. Some also starts at
50%, 70%, 90%, 100% (absolute) Ethanol. - Basic structures
Acidic dye = acidophilic (e.g., cytoplasm)
 - Hematoxylin and Eosin is the most common stain project an image to the ocular. The eyepiece will further
used in histology. magnify the image and project this to your retina. The first
part of the microscope that magnifies the object is the
Note: Opposite attracts. objective, not the eye piece.
Basic = alkaline
Acid = acid ✓ Fluorescence Microscopy
- Tissue sections are usually irradiated with
MICROSCOPY ultraviolet (UV) light and the emission is in the
LIGHT MICROSCOPY – uses ordinary light visible portion of the spectrum.
- The fluorescent substances appear brilliant on a
✓ Bright-Field Microscopy dark background.
- Stained preparations are examined by means of
ordinary light that passes through the specimen. o Fluorescence
- It is reliant on resolving power. - Phenomenon wherein certain cellular substances
are irradiated by light of a proper wavelength,
o Resolving Power they emit light with a longer wavelength.
- It is the ability of a microscope lens or optical
system to produce separate images of closely Note: UV light will excite the fluorescent dye. Once it is
positioned objects. already excited, its energy level jumps to a higher energy
level. It becomes unstable. It needs to go back to its
- Defined as the smallest distance between two original energy level. As it goes back to its original energy
particles at which they can be seen as separate level, it will give off/emit excess energy in a form of a
objects. fluorescent light.
- 0.2 µm is the smallest resolving power that bright
field microscope is capable.

Note: If you have objects smaller than 0.2 micrometer (ex.

ribosomes), you might need to use other microscopic
techniques.

Drawbacks:
✓ Expensive
✓ It does not age well. Fluorescence fades within 1 year
or 2 years.

✓ Phase-Contrast Microscopy
- Based on the principle that light changes its
speed when passing through cellular and
extracellular structures with different refractive
indices.
- Uses a lens system that produces visible images
from transparent objects.
The lamp gives of the ordinary light. The light will then - Enables examination of unstained cells and
pass through the condenser. The condenser will the tissues and is especially useful for living cells.
collect and focus the cone of light, illuminating the object. - It is also used if you want to visualize actual
Microscopes uses a dual lens system. cellular processes (e.g., Phagocytosis)

Dual Lens System – lens on the eyepiece and objectives. Note: Once a cell is stained, it is already dead.

Note: The image already illuminated by the condenser will

be enlarged by the objective. The objective will then
o Interference Microscope Advantages:
- Allows quantification of tissue mass ✓ It reduces the stray light to increase contrast with the
image.
o Differential Interference Microscope ✓ You are achieving a higher resolution of the image.
- Using Nomarski optics
- Useful for assessing surface properties of cells
and other biologic objects.

Bright Field Phase Contrast Differential PC

✓ Dark-Field Microscopy
- No direct light from the light source is gathered by
the objective lens.
- Only light that has been scattered or diffracted by
structures in the specimen reached the objective.
- Equipped with a special condenser that
illuminates the specimen with strong, oblique Pinhole Aperture – it is a filter where light only passes by
(redirect) light. this in order to avoid stray lights.
- Field of view appears as a dark background on
which small particles in the specimen that reflect A. Light from a laser source hits the specimen and is
some light into the objective appear bright. reflected.
- The light is refracted (bended). B. A beam splitter directs the reflected light to a pinhole
- Special condenser is used. and a detector.
C. Light from components of the specimen that are
above or below the focused plane is blocked by the
blind.
D. The laser scans the specimen so that a larger area of
the specimen can be observed.

✓ Polarizing Microscopy
- Uses the fact that highly ordered molecules or
arrays of molecules can rotate the angle of the
plane of polarized light.

o Birefringence
✓ Confocal Microscopy - Ability to rotate the direction of vibration or
- Combines components of a light optical polarized light.
microscope with a scanning system to dissect a - It is very common on crystals.
specimen optically.
- Uses (1) a small point of high – intensity light,
often from a laser, and (2) a plate with a pinhole
aperture in front of the image detector.
- Point light source, the focal point of the lens, and
the detector’s pinpoint aperture are all optically
conjugated or aligned to each other in the focal
plane (confocal), and unfocused light does not
pass through the pinhole.
Note: Collagen fibers are birefringent.

ELECTRON MICROSCOPY
- Based on the interaction pf tissue components with
beams of electrons.
- The wavelength in the electron beam is much shorter
than that of light, allowing a 1000-fold increase in
resolution.
- You are able to view higher solution of images. ➢ Scanning Electron Microscopy
- The electron beam does not pass through the
2 Kinds: specimen but is scanned across its surface.
o Transmission Electron Microscope (TEM) - The surface of the specimen is first dried and spray-
o Scanning Electron Microscope (SEM) coated with a very thin layer of heavy metal (often
gold) through which electrons do not pass readily.
➢ Transmission Electron Microscopy (TEM) - When the beam is scanned from point to point across
- An imaging system that permits resolution around 3 the specimen, it interacts with the metal atoms and
nm. produces reflected electrons or secondary electrons
- Magnifications of up to 400,000 times to be viewed in emitted from the metal.
detail.
- Uses the interaction of a beam of electrons with a OTHER METHODS
specimen to produce an image. ENZYME HISTOCHEMISTRY
- A method for localizing cellular structures using a
▪ Cyrofracture and Freeze Etching specific enzymatic activity present in those structures.
- A special method of sample preparation for
transmission electron microscopy.
- It is especially important in the study of membranes.
 IMMUNO HISTOCHEMISTRY
- A highly specific interaction between molecules is that
between an antigen and its antibody.
- You use an antibody against actin filaments.

The antibody carries a fluorescent pad. Once the antibody

and antigen interact, it shines/glows. – DIRECT

You have a primary and a secondary antibody –

INDIRECT

Note: The secondary antibody carries the fluorescent tag.

It is against the primary antibody.

Note: Cutting the tissue is very important because these

DIRECT IMMUNOFLUORESCENCE are 3D structures. How you cut affects the preparation
and it will generate how it will appear.

HYBRIDIZATION TECHNIQUES
- A method of localizing messenger RNA (mRNA) or
DNA by hybridizing the sequence of interest to a
complementary strand of a nucleotide probe.

▪ In situ hybridization
- Binding of the nucleotide probe to the DNA or RNA
sequence of interest is performed within cells or
tissues, such as cultured cells or whole embryos.

INTERPRETATIONS OF STRUCTURES IN TISSUE

SECTIONS