histologia: introduÇÃo regiões s Órgãos moléculas tecidos...
TRANSCRIPT
HISTOLOGIA: INTRODUÇÃO WABeresford
Regiõess
Órgãos
Moléculas
Tecidos
Conecções Células
Partes
Organelas
Desenvolvimento
Funções
Sistemasms
“O estado da arte ”
Junte tudo!
MEDICINA: Alguns aspectos
Regiões
Partes
Conecções
Desenvolvimento
Tecidos
Células
Organelas
MoléculasFunções
Microrganismos
Medicina
IdadePopulações
?
Sexo
Órgãos
Sistemas
Abnormal variants for all the earlier fields of knowledge
Developing judgment - weighing various contributions for relevance & quality of evidence
Foretaste of the ‘pulling it together’ in the PBL experiences, but much omitted, e.g., therapy, follow-up, cost; likewise for clinical correlations
This doubling, plus more fields, e.g. microbes, is why medical training takes several years
Any twit can lay hands on an LCD projector, and push images at you reminds one that the story may be faulty; it is one of many; and there are omissions
?
Feel for the aspects that yield valid risk factors in this particular diagnosis
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 side-by-side)
Images versus REALITY - AnatomyIn Anatomy, the source of the evidence - the essential point of reference - is
the cadaver for Gross &
the microscope slide for Histo
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
Bed-rock
LÂMINA HISTOLÓGICA Vista Lateral
Lâmínula
Lâmina 1”X3”
Fragmento de tecido
Resina (cola)
Resina: é transparente; Índice refração = ao vidro
Etiqueta
Manuseio da lâmina - Cuidados
O vidro é frágil !
Cuidado com as caixas de lâminas
A lâmina vai à mesa com a lâmínula para cima
Lâm. & Microscópio permanecem Lab. didático!
Preparo da Lâmina PassosRemoção & Fixação (preservação do tecido)
Remoção da água & reposição com solvente de parafina
Impregnação em parafina fundida (60oC) e inclusãoPreparação do bloco & microtomia
Montagem dos cortes na lâmina
Adesão dos cortes, & coloração
Desidratação; montagem da lâmínula
Após secagem do meio, microscopia
50 % ethanol 70 %
ethanol95 % ethanol
100 % ethanol
benzene/xylene
Dehydrating series
paraffinwax
Remove the water & replace with wax-solventImbed the oriented specimen in molten wax
Miscible with ethanol; dissolves wax
Fresh tissue
10% Formalin fixative
label
MICRÓTOMO – cortador de presunto sofisticado – prende o bloco de parafina, & corta fatias finas, a mediada que o bloco avança mecanicamente
Block
Knife
Section
Glass slide
Banho - Maria
After it is solid, hold the wax block & cut slices
Montagem das fatias na slides
Lift out floating section on the slide
FREEZING MICROTOME holds the frozen tissue, & cuts off thin slices, as the block is slowly advanced mechanically
Block is the tissue
Knife
Section
Water-bath
Glass slide
For fast biopsy, imbedding is omitted - frozen sections
Mount the thin slices (sections) on slides
Lift out section on the slide
Dissolve paraffin wax
Stain with Hematoxylin - blue
Wash
Stain with eosin - red
Nuclei - blue
Cytoplasm- red
Wash
When dry, remove the wax, & stain the section
Dissolve paraffin wax
Stain with Hematoxylin - blue
Wash
Stain with eosin - red
Nuclei - blue
Cytoplasm- red
Wash
When dry, remove the wax, & stain the section
Potassium+ eosinate- stain + charged amine, etc,
groups on proteins bind -eosin “Acidophilic staining”
“Basophilic”
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 SET number: same as cabinet and Slide number J-7 SET 33 microscope number
PARATHYROID Tissue or organ Source of tissue Human
H & E Stain
Images versus REALITY Artifacts are appearances not true to the original state of the tissue
SLIDE PREPARATION IV Artifacts
Excise & Fix (preserve) the tissue in fixative
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
Knife scores, chatter
Bruising/splitting from cutting; Poor preservation, e.g., gut lining, enzymes, lost fat
Wrinkles, section not flat, splits
Weak/unbalanced staining
Dirt, hair, bubbles
Dirt on lenses, bad illumination
Misleading orientation, Shrinkage & distortion, Mislabeled
.
CLASS LIGHT MICROSCOPE
Max MAGNIFICATION
Eyepiece (10X) times ‘Oil’ Objective
(100X) = 1000XBase
Oculares
MesaLâmina
Fonte de Lux
Corpo
Lentes objetivas
Condensador
CLASS LIGHT MICROSCOPE Controls I
Base
Condenser
Eyepiece/Ocular
Slide
Light
Body
Inter-ocular distance
Moving stage
Iris diaphragm
Field diaphragm
Coarse & Fine focus
Light intensity
On/Off
Objective selection
left rear
CLASS LIGHT MICROSCOPE Controls II
Base
Condenser
Eyepiece/Ocular
Slide
Light
Body
Stage clip for slide Condenser
focusing
Condensercentering
Ocular focusing
left-side
OPERATION I
Base
Condenser
Eyepiece/Ocular
Slide
Light
Body
Inter-ocular distance
Moving stage
Iris diaphragm
Field diaphragm
Coarse & Fine focus
Light intensity
On/Off
Objective selection
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
Field diaphragm
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
Base
Condenser
Eyepiece/Ocular
Slide
Light
Body
Inter-ocular distance
Moving stage
Iris diaphragm
Coarse & Fine focus
Light intensity
On/Off
Objective selection
.
SMEAR - another method of preparationDrop of blood
Slide 1Slide 2
On contact, slide 2 extends the drop along its 1” side
Slide 2
Slide 2
Pushing angled slide 2 along #1 smears the line of blood across slide 1
Lift away slide 2; dry #1 ; stain; coverslip
SmearA few cells are damaged; smear is not evenly thick; & staining is uneven. Same apply to SPREADS
TEASING - a method of preparation
Lumbo-sacral cord
Roots
Terminal thread
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
(Filum terminale)
Cut across BONE shaft twice
Saw out a sector
Lay sector flat & grind thin
Wash ground section
Dry ; place unstained on slide
Coverslip for viewing
GROUND PREPARATION
neuron
collecting duct
osteoclast
CELL DESCRIPTION What is one looking for?
Cell Size?
Cell Shape?Nuclei - #?
Nucleus - size, shape, density?
Nucleoli -prominence , #?
Nucleus -position?Cells’ relations?
Cytoplasm - granular?
Cytoplasm -philia?
Cell membrane - visible?Cell surface
specializations?
Basal lamina
eosinophil
airway lining
GO GRANULAR
Cerebellar Granule layer packed, small neurons- granule cells (& granulosa cells in ovary)
Melanin granules in melanocytes & keratinocytes
BasEosPMN
Blood Granulocytes from their very granular cytoplasm
Layer
Cell
Granule
Some differences between light and electron microscopy I
LIGHT MICROSCOPY ELECTRON MICROSCOPY-----------------------------------------------------------------------------------------------------------------------Section thickness (1-30 m) gives Very thin sections provide noa little depth of focus for depth of focus, but 3-D informationappreciation of the third dimension. can be had from: (a) thicker sectionsSerial sections can be cut, viewed by high-voltage EM; (b) shadowedand used to build a composite image replicas of fractured surfaces; (c)or representation. scanning electron microscopy (SEM).
Most materials and structures cannot Heavy metal staining gives a morebe stained and viewed at the same comprehensive picture of membranes,time; stains are used selectively to granules, filaments, crystals, etc.;give a partial picture, e.g. a stain but this view is incomplete and evenfor mucus counterstained to show visible bodies can be improved bycell nuclei. varying the technique.
Specimen can be large and Specimen is in vacuo. Its small sizeeven alive. 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 Image is in shades of green oneye. Image keeps the colours given the screen; photographically,the specimen by staining. only in black and white.
Modest magnification to X 1500; High magnification,up to X 2,000,000but a wider field of view and easier thus the range of magnificationorientation is greater
Resolving power to 0.25 m. Resolving power to 1 nm (0.001 mm.)
Frozen sections can yield an image Processing of tissue takes a day atwithin 20 minutes. least.
Crude techniques of preparation High resolution and magnificationintroduce many artefacts. demand good fixation (e.g. by(Histochemical methods are better.) 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.
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
SBLC computers have “Histology Lab Assistant”
WebBoard at Course 303 on Anatomy Dept site
****
Did I choose the right medical school?
****Complete Ameba Medicine 10 4 ed. Pp 29
“Please take your zillion+ cells elsewhere. I’m an Ameba doctor.”
.