Presentation on theme: "HISTOLOGIA: INTRODUÇÃO Regiões s Órgãos Moléculas Tecidos ConecçõesCélulasPartesOrganelasDesenvolvimentoFunçõesSistemas ms “O estado da arte ” Junte tudo!"— Presentation transcript:
HISTOLOGIA: INTRODUÇÃO Regiões s Órgãos Moléculas Tecidos ConecçõesCélulasPartesOrganelasDesenvolvimentoFunçõesSistemas ms “O estado da arte ” Junte tudo!
MEDICINA: Alguns aspectos Regiões Parte s ConecçõesDesenvolvimento Tecidos CélulasOrganelasMoléculasFunções Microrganismos Medicina IdadePopulações ? SexoÓrgãos 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 - Anatomy In 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 Passos Remoção & Fixação (preservação do tecido) Remoção da água & reposição com solvente de parafina Impregnação em parafina fundida (60 o C) e inclusão Preparação do bloco & microtomia Montagem dos cortes na lâmina Adesão dos cortes, & coloração Desidratação; montagem da lâmínula Após secagem do meio, microscopia
50 % ethanol 70 % ethanol 95 % ethanol 100 % ethanol benzene/ xylene Dehydrating series paraffin wax Remove the water & replace with wax-solvent Imbed 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) = 1000X Base Oculares Mesa Lâmina 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 Eyepiec e/Ocular Slide Light Body Stage clip for slide Condenser focusing Condenser centering 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 90 o 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 preparation Drop of blood Slide 1 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 Lift away slide 2; dry #1 ; stain; coverslip Smear A 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
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 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 Heavy metal staining gives a more be 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 even for mucus counterstained to show visible bodies can be improved by cell nuclei. varying the technique. Specimen can be large and Specimen is in vacuo. Its small size even 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 on eye. 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 2,000,000 but a wider field of view and easier thus the range of magnification orientation 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 at within 20 minutes. least. Crude techniques of preparation High resolution and magnification introduce 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