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HISTOLOGIA: INTRODUÇÃO
WABeresford
“O estado da arte ”
Regiões
s
Sistemas
ms
Órgãos
Tecidos
Partes
Células
Conecções
Organelas
Desenvolvimento
Funções
Junte tudo!
Moléculas
Medicina
MEDICINA: Alguns aspectos
Microrganismos
Sistemas
Sexo
Órgãos
Regiões
Tecidos
Parte
s
Conecções
Células
?
Organelas
Desenvolvimento
Populações
Funções
Moléculas
Idade
Abnormal variants for all the earlier fields of knowledge
This doubling, plus more fields, e.g. microbes, is why medical
training takes several years
Developing judgment - weighing various contributions for
relevance & quality of evidence
Feel for the aspects that yield valid risk factors in this
particular diagnosis
Foretaste of the ‘pulling it together’ in the PBL
experiences, but much omitted, e.g., therapy, follow-up,
cost; likewise for clinical correlations
Any twit can lay hands on an LCD projector, and push
? reminds one that the story may be
images at you
faulty; it is one of many; and there are omissions
PORNOGRAPHY & “THE REAL THING”
Images versus REALITY
What is the evidence for the real?
Noon talks for Internal-Medicine residents’ Board prep
Two recurring themes -Is it what it appears to be ?
Does the treatment/procedure do what is claimed for it ?
What is the evidence?
Images versus REALITY - Functional Anatomy
REALITY is the living person, often via images
Surface anatomy Palpation Endoscopy+
Radiology
PET scans Ultrasound
Doppler flows Gait & Reflexes etc
Biopsies Fine-Needle Aspiration Cervical, Blood,
etc Smears
Flow cytometry & cell sorting
Cell culture & grafting etc
(Bits cut or sucked out for microscopy)
REALITY is the dead person
DISSECTION
[Surface anatomy Endoscopy
Palpation Radiology Ultrasound are sometimes
useful as adjuncts to autopsy & histology
correlations]
Organs and large pieces cut out,
examined, & prepared for
MICROSCOPY- histology &
histopathology (normal & altered sideby-side)
Images versus REALITY - Anatomy
In Anatomy, the source of the
evidence - the essential point of
reference - is
the cadaver for Gross &
the microscope slide for Histo
Bed-rock
As the physician is knowledgeably comfortable with the
patient’s gross & microscopic structure and its
implications, you will become confident at the cadaver &
the microscope, and with the resulting images
TESTS focus on the cadaver, the slides, and interpreting
images - identification, interpretation, & synthesis
LÂMINA HISTOLÓGICA Vista Lateral
Lâmínula
Fragmento de tecido
Resina (cola)
Etiqueta
Lâmina
1”X3”
Resina: é transparente;
Índice refração = ao vidro
Manuseio da lâmina - Cuidados
Lâm. & Microscópio permanecem Lab. didático!
O vidro é frágil !
Cuidado com as caixas de lâminas
A lâmina vai à mesa com a lâmínula para cima
Preparo da Lâmina
Passos
Remoção & Fixação (preservação do tecido)
Remoção da água & reposição com solvente de
parafina
Impregnação em parafina fundida (60oC) e
inclusão
Preparação do bloco & microtomia
Montagem dos cortes na lâmina
Adesão dos cortes, & coloração
Desidratação; montagem da lâmínula
Após secagem do meio, microscopia
Remove the water & replace with wax-solvent
Imbed the oriented specimen in molten wax
50 %
ethanol
70 %
ethanol
95 %
ethanol
Fresh
tissue
10% Formalin
fixative
label
100 %
ethanol
benzene/
xylene
Miscible with ethanol; paraffin
dissolves wax
wax
After it is solid, hold the wax block & cut slices
Knife
Section
Block
MICRÓTOMO – cortador de
presunto sofisticado – prende
o bloco de parafina, & corta
fatias finas, a mediada que o
bloco avança mecanicamente
Glass slide
Banho - Maria
Montagem das fatias na slides
Lift out floating section on the slide
For fast biopsy, imbedding is omitted - frozen sections
Knife
Section
Block is
the tissue
FREEZING MICROTOME
holds the frozen tissue, & cuts
off thin slices, as the block is
slowly advanced mechanically
Glass slide
Water-bath
Mount the thin slices (sections) on slides
Lift out section on the slide
When dry, remove the wax, & stain the section
Dissolve paraffin wax
Stain with Hematoxylin - blue
Wash
Stain with eosin - red
Wash
Nuclei - blue
Cytoplasm- red
When dry, remove the wax, & stain the
Dissolvesection
paraffin wax
Stain with Hematoxylin - blue
Wash
-
Potassium+ eosinate stain
+ charged amine, etc, groups on
proteins bind -eosin
“Acidophilic staining”
Nuclei - blue
“Basophilic”
Cytoplasm- red
Stain with eosin - red
Wash
SLIDE PREPARATION III Steps
Excise & Fix (preserve) the tissue in fixative
Remove the water & replace with wax-solvent
Imbed the oriented specimen in molten wax
After it is solid, hold the wax block & cut slices
Mount the thin slices (sections) on slides
When dry, remove the wax, & stain the section
Remove surplus stain & water; mount
coverslip
When mounting medium has set, do microscopy
KEY TO SLIDE LABELING:
Slide number
Source of tissue
J-7
SET 33
PARATHYROID
Human
H&E
Slide SET number:
same as cabinet and
microscope number
Tissue or organ
Stain
SLIDE PREPARATION IV Artifacts
Images versus REALITY
Artifacts are appearances not true to the
original state of the tissue
Excise & Fix (preserve) the tissue in fixative Bruising/splitting from cutting; Poor
preservation, e.g., gut lining, enzymes, lost fat
Imbed the oriented specimen in molten wax
Misleading orientation, Shrinkage &
distortion, Mislabeled
After it is solid, hold the wax block & cut slices
Mount the thin slices (sections) on slides
Knife scores, chatter
Wrinkles, section not flat, splits
When dry, remove the wax, & stain the section
Remove surplus stain & water; mount coverslip
When mounting medium has set, do microscopy
Weak/unbalanced staining
Dirt, hair, bubbles
Dirt on lenses, bad illumination
.
CLASS LIGHT MICROSCOPE
Oculares
Lentes objetivas
Max MAGNIFICATION
Mesa
Lâmina
Corpo
Condensador
Eyepiece (10X)
times
‘Oil’ Objective (100X)
= 1000X
Fonte
de Lux
Base
CLASS LIGHT MICROSCOPE Controls I
Eyepiece/
Ocular
Inter-ocular distance
Objective selection
Iris diaphragm
Slide
Body
Coarse & Fine focus
Condenser
Moving stage
Field
diaphragm
Light intensity
Base
Light
On/Off
left rear
CLASS LIGHT MICROSCOPE Controls II
Ocular focusing
Eyepiec
e/Ocular
Stage clip
for slide
Slide
Body
Condenser
Condenser
centering
Base
Light
Condenser
focusing
leftside
OPERATION I
Eyepiece/
Ocular
Inter-ocular distance
Objective selection
Iris diaphragm
Slide
Body
Coarse & Fine focus
Condenser
Moving stage
Field
diaphragm
Light intensity
Base
Light
On/Off
Without looking down the eyepieces, plug in the cord
Turn the light-intensity knob back counterclockwise,
Switch on the light, turn the intensity up (about a 90o turn)
while observing the light via the field opening
Open the field diaphragm wide
Move the condenser assembly to its top position
Switch the shortest objective lens (X4) into the working position
Open the iris diaphragm wide
Select any well-stained slide
OPERATION II
Eyepiece/
Ocular
Inter-ocular distance
Objective selection
Iris diaphragm
Slide
Body
Coarse & Fine focus
Condenser
Moving stage
Light intensity
Base
Field
diaphragm
Light
On/Off
Pull back the clip & place slide, cover-slip up, on the stage
Use the stage controls to bring the stained section over the
light Focus, using coarse, then fine adjustments
Close the iris diaphragm to take the glare out of the view
Push (pull) the eyepieces together to match your eye spacing
Shut one eye, focus with the fine focus; then shut that eye,
open the other, and focus for it with the ocular focus (turning
the eyepiece knurled ring)
Switch in the next higher objective, and focus, using the main
focusing controls & testing for binocular fusion
.
Drop of
blood
SMEAR - another method of preparation
Slide 1
Slide 2
Slide 2
On contact, slide 2 extends
the drop along its 1” side
Slide 2
Pushing angled slide 2
along #1 smears the line
of blood across slide 1
Smear
A few cells are damaged;
smear is not evenly thick;
& staining is uneven.
Lift away slide 2; dry #1 ; stain; coverslip
Same apply to SPREADS
TEASING - a method of preparation
Terminal thread
(Filum terminale)
Roots
Lumbo-sacral cord
A technique you know from using a
needle to separate out the
connective-tissue filum terminale
from the nervous cauda equina of
dorsal & ventral roots
On the MICROSCOPE SLIDE, with a
needle point one can tease apart
individual nerve or muscle fibers from
their bundles in nerve or muscle
When tissue is already
thin, it can be draped SPREAD - over the
slide like a tablecloth
GROUND PREPARATION
Lay sector flat & grind thin
Cut across BONE
shaft twice
Saw out a sector
Wash ground section
Dry ; place
unstained on slide
Coverslip for viewing
CELL DESCRIPTION What is one looking for?
Cell Shape?
eosinophil
Nuclei - #?
Cell Size?
Nucleus - size,
shape, density?
Cytoplasm granular?
Cytoplasm philia?
Cell surface
specializations?
neuron
Cell membrane visible?
collecting duct
Nucleus position?
Cells’ relations?
Nucleoli prominence , #?
osteoclast
airway lining
Basal lamina
GO GRANULAR
Cerebellar Granule layer
packed, small neurons- granule
cells (& granulosa cells in ovary)
Layer
Blood Granulocytes from
their very granular cytoplasm
PMN
Eos
Bas
Cell
Melanin granules in melanocytes
& keratinocytes
Granule
Some differences between light and electron microscopy I
LIGHT MICROSCOPY
ELECTRON MICROSCOPY
----------------------------------------------------------------------------------------------------------------------Section thickness (1-30 mm) gives
Very thin sections provide no
a little depth of focus for
depth of focus, but 3-D information
appreciation of the third dimension.
can be had from: (a) thicker sections
Serial sections can be cut, viewed
by high-voltage EM; (b) shadowed
and used to build a composite image
replicas of fractured surfaces; (c)
or representation.
scanning electron microscopy (SEM).
Most materials and structures cannot
be stained and viewed at the same
time; stains are used selectively to
give a partial picture, e.g. a stain
for mucus counterstained to show
cell nuclei.
Heavy metal staining gives a more
comprehensive picture of membranes,
granules, filaments, crystals, etc.;
but this view is incomplete and even
visible bodies can be improved by
varying the technique.
Specimen can be large and
even alive.
Specimen is in vacuo. Its small size
creates more problems with sampling
and orientation.
Some differences between light and electron microscopy II
LIGHT MICROSCOPY
ELECTRON MICROSCOPY
-------------------------------------------------------------------------------------------------------------------Image is presented directly to the
eye. Image keeps the colours given
the specimen by staining.
Image is in shades of green on
the screen; photographically,
only in black and white.
Modest magnification to X 1500;
but a wider field of view and easier
orientation
High magnification,up to X 2,000,000
thus the range of magnification
is greater
Resolving power to 0.25 mm.
Resolving power to 1 nm (0.001 mm.)
Frozen sections can yield an image
within 20 minutes.
Processing of tissue takes a day at
least.
Crude techniques of preparation
introduce many artefacts.
(Histochemical methods are better.)
High resolution and magnification
demand good fixation (e.g. by
vascular perfusion), cleanliness
and careful cutting, adding up to
fewer artefacts.
HISTOLOGIA - FONTES
BIOMANIA.COM.BR
http://www.bris.ac.uk/Depts/PathAndMicro/CPL/he.html
Histo Powerpoints
Histology Full-text* & Histology Lab Guide
http://wberesford.hsc.wvu.edu
http://www.geocities.com/Athens/Academy/1575
Recommendation - catch it while you can: download the above this
week. We’re talking about 50 megabytes, and some of the above items
could fit on floppies.
WebBoard at Course 303 on Anatomy Dept site
SBLC computers have “Histology Lab Assistant”
It is never too soon to attune yourself to examiners’ thinking.
Syllabus p. # presents the formats in which Histo lab exam questions
will be framed
Did I choose the right medical school?
“Please take your
zillion+ cells
elsewhere. I’m an
Ameba doctor.”
Complete Ameba Medicine
10 4 ed. Pp 29
.