Description: Overview of histology, slide preparation, histological stains, and types of microscopy.

Microscopy & Staining

 I.      Histology

§  Study of Tissues

○      Tissues are made of cells and extracellular matrix (ECM)

§  Why study tissues?

○      We are made of tissues

○      To learn histology is to learn how we are made up and function

○      Injuries & pathologies occur at the atomic & molecular levels manifesting at the cell and tissue levels

○      At present, cell & tissue levels are most easily & economically accessible for diagnosis & to inform treatments

II.    General Tissue Makeup

§  Cells

○      Variety of types

§  ECM

○      Fibers

‒       Collagen

‒       Elastic

‒       Reticular

○      Ground substance

‒       GAGs

‒       Glycoproteins

‒       Proteoglycans

○      Fluids

‒       Plasma-like, mostly water

§  If all tissues are made of the same “ingredients” why do various tissues look & function differently?

○      Types of cells

○      Proportion of ECM components

○      Different tissue types function together à Organ à Organ Systems à Anatomy

§  What technical steps are necessary to study tissues? Histotechnology

○      Tissue acquisition

‒       Cytological swabs

‒       Biopsies

‒       Surgical resections

○      Fixation

‒       Stops tissue degradation

‒       Most common fixative = formaldehyde (penetrates the tissue 1mm/1hr)

‒       Hardens & changes the color of the tissue

○      Tissue processing

‒       Replaces fat and water with hardening agent (paraffin)

○      Embedding

‒       Places the tissue in paraffin block

‒       Thoughtful embedding is important

○      Sectioning

‒       Paraffin block mounted on a microtome

‒       Each slice = 4-10 microns thick

○      Ribbon of thin paraffin slices is placed in water bath

○      Each section is mounted onto a glass slide

○      Staining tissue sections

‒       Remove paraffin

‒       Apply dyes and washing agents

‒       Place a glass coverslip on stained tissue

○      Tissue is ready for microscopic observation

III.      Histotechnology: Dyes and Other Staining Agents

§  H & E: the most common combination of dyes

○      H = Hematoxylin

‒       Positively charged, basic dye à dyes negatively charged, acidic structures (DNA & RNA in nucleus & ribosomes)

‒       Has blue hue

‒       Structures that stain well are said to be basophilic for attracting basic hematoxylin

○      E = Eosin

‒       Negatively charged, acidic dye à dyes positively charged, basic structures (many proteins in cytoplasm)

‒       Has pink hue

‒       Structures that stain well are said to be eosinophilic or acidophic for attracting the acidic eosin

§  Histochemistry

○      Uses natural chemical interactions between the tissues and the chemicals

○      H&E is a type of histochemistry

○      Other histochemical stains:

‒       Trichrome: combination of dyes often used to visualize collagen

‒       Periodic Acid Schiff: reacts with carbohydrates to generate magenta color & is used to visualize carbohydrate-rich regions

Trichome = Tri (three) + chromo (color)

§  Immunohistochemistry

○      Uses specific interactions between antigen and antibody

○      Primary antibody applied to recognize the protein (antigen) of interest

○      Secondary antibody with a chromogen applied to recognize the primary antibody

○      Chemical applied to react with chromogen to turn the tagged region of tissue brown color

§  Immunofluorescence

○      Uses specific interactions between antigen and antibody, just like immunohistochemistry

○      Primary antibody applied to recognize the protein (antigen) of interest

○      Secondary antibody has fluorescent tag instead of a chromogen

○      Excitation by UV light or laser causes the fluorescent tag to emit specific frequency in a color spectrum

○      Can visualize more than one protein of interest

IV.      Microscopy

§  Microscopes allow observation of prepared tissue slides at various levels of magnification

§  Light microscopy

○      Natural light illuminates the tissues for visual inspection

○      Used to observe tissues stained with histochemistry and immunohistochemistry

§  Phase contrast microscopy

○      Natural light illuminates the tissues for visual inspection

○      Leverages different refractory properties of cellular structures

○      Cells or tissues need not be stained à allows observation of live cells and tissues

§  Fluorescence microscopy

○      Works similar to light microscopes but has additional laser or UV light source

○      Used to observe tissues stained with immunofluorescence

§  Transmission Electron Microscopy (TEM)

○      Instead of photons, electrons are used as a source of “illumination”

○      Small size of electrons allow greater magnification and resolution of tissues and cells

○      Electrons pass through the tissue, casting different shades of “shadows”

‒       Darker areas on TEM (electron dense areas): more cellular or tissue materials are concentrated

‒       Lighter areas on TEM (electron light areas): less cellular or tissue materials are present & allowed electrons to pass

§  Scanning Electron Microscopy (SEM)

○      Instead of electrons passing through the tissue, they are scattered by the tissue

○      Able to capture three-dimensional surface contours of the tissue

§  Virtual Microscopy

○      Software or app simulating the use of a microscope on a digital screen

○      Allows navigation of the virtual tissue slide similar to Google Map

V.      Clinical Application of Histotechnology

§  Critical component of pathology practice

○      Biopsies or surgical resections are documented & gross observations noted

○      Large surgical specimens are dissected, photographed and placed in a fixative for appropriate amount of time

○      Fixed specimens are sampled and sampled tissues are processed, embedded, sliced, mounted and stained

○      Diagnosis is made by a pathologist upon visual inspection of the tissues

○      Additional tissue processing, such as immunohistochemistry or immunofluorescence is ordered for ambiguous tissues or to confirm diagnosis

§  During certain surgical procedures, a surgeon may request a rapid tissue observation by a pathologist to ensure that surgical margin is free of malignancy or that a lymph node is free of metastatic cancer cells

○      The tissue is frozen rapidly and sectioned using a cryostat then stained

○      The quality of stained tissue is not as good as routinely processed tissue but can be an effective reference when speed is of importance

○      The frozen tissue is processed for paraffin section for confirmation at a later time

§  Critical component of pathology practice

○      Biopsies, surgical resections, and peripheral blood smears require a pathologist to review a  histopathological slide from the tissue collected to make a diagnosis

○      Additional tissue processing, such as immunohistochemistry or immunofluorescence is ordered for ambiguous tissues or to confirm diagnosis

§  During certain surgical procedures, a surgeon may request a rapid tissue observation by a pathologist to ensure that surgical margin is free of malignancy or that a lymph node is free of metastatic cancer cells

○      The quality of stained tissue is not as good as routinely processed tissue but can be an effective reference when speed is of importance

○      Commonly used by surgical oncologists and dermatologists who specialize in Mohs surgery

VI.      Histology Pro Tips

§  Magnification vs. Field of View

○      Low mag = more field of view but low on details

○      High mag = small field of view but high on details

○      Best practice: Start with low mag then incrementally go to high mag

§  Characteristic landmarks and unique architecture will easily give away its location

Pre-Lecture Quiz

1) What is the subject of study in histology?

(A) Cells

(B) Diseases

(C) Organs

(D) Tissues