HISTOLOGIA: INTRODUÇÃO

1. Regiõess Órgãos Sistemasms Tecidos Partes Células Conecções Organelas Desenvolvimento Moléculas Funções HISTOLOGIA: INTRODUÇÃO “O estado da arte ” Junte tudo!

2. Medicina Microrganismos Sexo Regiões Tecidos Partes Células Órgãos Sistemas Conecções ? Organelas Desenvolvimento Populações Idade Funções Moléculas MEDICINA: Alguns aspectos

3. ? 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 images at you reminds one that the story may be faulty; it is one of many; and there are omissions

4. PORNOGRAPHY & “THE REAL THING” ImagesversusREALITY What is the evidence for the real?

5. 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?

6. ImagesversusREALITY - FunctionalAnatomy REALITY is the living person, often via images Surface anatomyPalpationEndoscopy+RadiologyPET scansUltrasoundDoppler flows Gait & Reflexesetc BiopsiesFine-Needle AspirationCervical, Blood,etc SmearsFlow cytometry & cell sortingCell culture & graftingetc(Bits cut or sucked out for microscopy)

7. REALITY is the dead person DISSECTION[Surface anatomyEndoscopyPalpationRadiologyUltrasoundare 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)

8. Bed-rock ImagesversusREALITY - 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

9. LÂMINA HISTOLÓGICA Vista Lateral Lâmínula Fragmento de tecido Resina (cola) Etiqueta Lâmina 1”X3” Resina: é transparente; Índice refração = ao vidro

10. 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

11. 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

12. Fresh tissue 10% Formalin fixative Remove the water & replace with wax-solvent Imbed the oriented specimen in molten wax 50 % ethanol 70 % ethanol 95 % ethanol label Dehydrating series 100 % ethanol benzene/xylene paraffinwax Miscible with ethanol; dissolves wax

13. After it is solid, hold the wax block & cut slices Knife Section Block Glass slide MICRÓTOMO – cortador de presunto sofisticado – prende o bloco de parafina, & corta fatias finas, a mediada que o bloco avança mecanicamente Banho - Maria Montagem das fatias na slides Lift out floating section on the slide

14. For fast biopsy, imbedding is omitted - frozen sections Knife Section Block is the tissue Glass slide FREEZING MICROTOME holds the frozen tissue, & cuts off thin slices, as the block is slowly advanced mechanically Water-bath Mount the thin slices (sections) on slides Lift out section on the slide

15. Nuclei - blue Cytoplasm- red When dry, remove the wax, & stain the section Dissolve paraffin wax Stain with Hematoxylin - blue Wash Stain with eosin - red Wash

16. When dry, remove the wax, & stain the section Dissolve paraffin wax Stain with Hematoxylin - blue Wash Potassium+eosinate- stain+ charged amine, etc, groupson proteins bind-eosin “Acidophilic staining” Stain with eosin - red Wash Nuclei - blue Cytoplasm- red “Basophilic”

17. 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

18. KEY TO SLIDE LABELING: Slide SET number: same as cabinet and Slide numberJ-7 SET 33 microscope number PARATHYROID Tissue or organ Source of tissue Human H & E Stain

19. SLIDE PREPARATION IV Artifacts ImagesversusREALITYArtifacts 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 Knife scores, chatter Mount the thin slices (sections) on slides Wrinkles, section not flat, splits Weak/unbalanced staining When dry, remove the wax, & stain the section Dirt, hair, bubbles Remove surplus stain & water; mount coverslip When mounting medium has set, do microscopy Dirt on lenses, bad illumination

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23. CLASS LIGHT MICROSCOPE Oculares Lentes objetivas Max MAGNIFICATION Eyepiece (10X)times‘Oil’ Objective (100X)= 1000X Mesa Lâmina Corpo Condensador Base Fonte de Lux

24. CLASS LIGHT MICROSCOPE Controls I 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 left rear

25. Base CLASS LIGHT MICROSCOPE Controls II Ocular focusing Eyepiece/Ocular Stage clip for slide Condenser focusing left-side Slide Body Condenser Condensercentering Light

26. 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 OPERATION I Without looking down the eyepieces, plug in the cord Turn thelight-intensity knob back counterclockwise,Switch on the light,turn the intensity up (about a 90o turn)while observing the lightvia the field openingOpen the field diaphragm wideMove thecondenser 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

27. Eyepiece/Ocular Inter-ocular distance Objective selection Iris diaphragm Slide Body Coarse & Fine focus Condenser Moving stage Light intensity Base Light On/Off OPERATION II Field diaphragm Pull back the clip & place slide, cover-slip up, on the stageUse the stage controls to bring the stained section over the lightFocus, using coarse, then fine adjustmentsClose 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

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30. SMEAR - another method of preparation Drop of blood 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. Same apply to SPREADS Lift away slide 2; dry #1 ; stain; coverslip

31. 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 caudaequina 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

32. 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

33. CELL DESCRIPTION What is one looking for? Cell Shape? eosinophil Nuclei - #? Cell Size? Nucleus - size, shape, density? osteoclast Cytoplasm -philia? Cytoplasm - granular? Cell membrane - visible? Cell surface specializations? collecting duct Nucleus -position? Cells’ relations? neuron Nucleoli -prominence , #? airway lining Basal lamina

34. GO GRANULAR PMN Eos Bas Cerebellar Granule layerpacked, small neurons- granule cells (& granulosa cells in ovary) Layer Blood Granulocytesfrom their very granular cytoplasm Cell Melanin granules in melanocytes & keratinocytes Granule

35. 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 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.

36. 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 mm. 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.

37. 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

41. 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

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