The document provides an overview of histology and microscopy techniques. It discusses (1) the process of preparing tissue samples for microscopy, including fixing, staining, and mounting tissue slices on slides; (2) the basic anatomy and operation of light microscopes used to view prepared slides; and (3) sources for further information on histology labs and procedures.

1. HISTOLOGY: INTRODUCTION “ What is going on ?” Pulling it together Regions Organs Molecules Tissues Connections Cells Parts Organelles Development Functions Systems

2. 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 ?

3. MEDICINE: Some aspects What gives with this patient ? Regions Molecules Tissues Connections Cells Parts Organelles Development Functions Regions Systems Systems abnormal Parts Connections Development Tissues Cells Organelles Molecules Functions Microbes Medicines Age Populations Costs ? Gender Organs Organs Abnormal

4. 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 ?

5. PORNOGRAPHY & “THE REAL THING” Images versus REALITY What is the evidence for the real?

6. 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)

7. 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)

8. 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

9. MICROSCOPIC SLIDE Side view of slide Glass coverslip Glass slide 1”X3” Tissue Section Mounting medium Mounting medium: permeates section; fastens coverslip to slide; is clear; has refractive index as for glass Label

10. SLIDE USE - Cautions GLASS IS FRAGILE ! Take care with individual slides & especially with the boxes of slides The slide must go on the stage coverslip up The high-dry & oil objectives cannot focus through the thickness of the slide to the section The label may have been put on the non-coverslip side, as shown ~ Slides & Microscope remain in the teaching Lab, always! Label

11. SLIDE PREPARATION I 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

12. SLIDE PREPARATION I 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

13. 50 % ethanol 70 % ethanol 95 % ethanol 100 % ethanol benzene/xylene Dehydrating series paraffinwax 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

14. MICROTOME - a fancy meat-slicer - holds the wax block, & cuts off thin slices, as the block is slowly advanced mechanically Block Knife Section Glass slide Water-bath After it is solid, hold the wax block & cut slices Mount the thin slices (sections) on slides Lift out floating section on the slide

15. 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

16. 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

17. 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”

18. 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

19. 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

20. 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

21. CLASS LIGHT MICROSCOPE Max MAGNIFICATION Eyepiece (10X) times ‘Oil’ Objective (100X) = 1000X Base Eyepiece/Ocular Stage Slide Light source Body Objective lenses Condenser

22. 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

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

24. OPERATION I 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 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

25. 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

26. 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 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

27. 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)

28. 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 PREPARARTION

29. HISTOLOGY SOURCES 303 Human Structure Syllabus next to last section p.8 Powerpoints - Comments & Standing assignment 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 30 megabytes, and some of the above items could fit on floppies. It is never too soon to attune yourself to examiners’ thinking. Syllabus p. 56 (lower-right #) 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

30. On the 4th floor, straight back from the working elevator, go to the SBLC (Rm 4005) and LEAVE your coat ( in the main lab, there are papers & envelopes on the desks that can get hidden or swept off, if coats are brought in today ) TODAY’s LAB PROCEDURES 1 Take only your book-bag (& computer, if you have it with you) into 4023. Locate your place - labeled in alphabetical order from the far end of the lab. Find the envelope with your name & the set of inventory forms. Open the envelope , remove the slip with the number to your Gross locker in the hallway. Write down this number in two other places. Take the small key out of the envelope and attach it to a key ring or something. If you are worried about your coat, or if this is already too much “structure”, take a break to try your locker combination and put your coat and other surplus stuff away. Once settled back at your place, use the key gently to open the top drawer at your place & take out the two small boxes of slides. Place them near the middle of the bench. Open the locker, and carefully lift out the microscope. Satisfy your curiosity - Find the microscope-use directions p.2 . Follow them with any well-stained slide. Ask us for help. [We will not issue oil for the X100 oil lens] Switch off the scope. Complete the receipt form. Go to p. 1 for slide numbering. Check your slide boxes against the inventory. Place forms in the wall folder .

31. Start the exercise on p.3, but with slide A-1 blood smear LAST of the four on examples of preparation methods. The smear is difficult to focus on. It needs at least the medium-power X10 0bjective; and the glare has to be taken out of the view with the iris diaphragm on the condenser. TODAY’s LAB PROCEDURES 2 The next exercise - Artifacts - should be straightforward. The idea behind the final exercise of this Introductory section is inexhaustible. One can go on and on about cells. Stop when you wish, and come back to it during the next two labs. Skip C-1. The cells are poor examples of neurons. The last part of the Lab on “The General Structure of Cells” is illustrated on the next slide, which indicates some of the variables, but does not show much of the extracellular matrix outside cells, e.g., basal lamina, collagen fibers, reticular fibers. In this and future labs, do not get hung up on a slide. If you cannot get your question answered in a minute or so, go on to the next slide; and come back later, when the question can be answered. Remind yourself with a note by the item. Hand in the inventory. Put your scope & slides away carefully. Lock the drawer & locker. About half of you share with a dental student. Please be considerate

32. 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 neuron eosinophil airway lining

33. GO GRANULAR Cerebellar Granule layer packed, small neurons- granule cells (& granulosa cells in ovary) Melanin granules in melanocytes & keratinocytes Blood Granulocytes from their very granular cytoplasm Layer Cell Granule Bas Eos PMN

34. 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.

35. 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  m.) 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.

36. **** 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.”

37. Test Pattern for the Apocalypse