Histology Lecture #1 Flashcards
Histomorphology
Histo = Tissue
Morphology = Structure
Histomorphology = looking at the structure of tissues
- This is what we see on microscope slides
Why is Histology hard to understand?
- Unfamiliarity with tissue structures and microscopicpictures of them
- 2D to 3D view of an object - get a 2D slices form 3D object and you need to translate 2D to 3D
- Problems of Scale (You want to knw the magnification of an image or know if it is zoomed in and without that information it is hard to tell what the image is)
- Complexity
Making Histology Slides
Take sample –> Formalin fix using FFPE –> then remove all the aqueous components using ethonol –> then then embed in wax –> then slice the block
Formalin fixation
Emerge the sample in formadheyhe
- This cross links and stabilizes the tissue allow you to cut it
Thickness of histology slide
4-5 micrometers thick (thinner than seran wrap and the thickness of 2 bacteria)
Being so thin allows you to put the slice on a slide
Why do you stain
Because the slice is so thin without the stain you can’t see anything - color allows you to see better
Most common Stain
Homotoxin and Eosin (H and E; H/E)
Example of unfamiliarity with structures
Skin - the skin has different components but looking a slide means it is hard to know what it looks like in 3D unless you know the structure of skin
- If you know 3D then you can make sense of the shape of the structire
Image - can see that if you know the 3D structure then teh 2D stucture because eaiser to understand
2D to 3D issue
When looking at a slide you have a 3D structure that has a shape
- What you see on the slde depends on the cutting plane (Depending on how it is cut affects the shape hat you get)
Example – see different cuts in cone you get different shapes
Slice fruit exmaple
Shows that depending how how you cut (the plane that you cut in) you will get a different shape/different pattern
ALSO depending on the location that you cut you will get a different image
- Example - different place you cut on orange = get different shape
Tube-like structures in Histology
Many tissues have tube-like structures (Example - have nerves, ducts, and Blood vessels)
- These tubes will live around in 3D space (bend or folds) or can branch
Issue = depending on the cutting plane you can get different images + depending on where you cut you get a different shape/image –> get different shape = don’t know what your looking at
Example image - Circle cut out of bend - if i image the circle is made of cells = this would be circle of cells and you might think its a sphere shape
White space in histology
Often the center of a structure (tube or a duct) - shows an opening
- Means that there was something there that had contents (Ducts or storage structure)
Example in image - see white is a duct and that the duct branches
Issue of complexity
In a textbook you often have a clear cartoon image that is simplified to be less confusing BUT on a slide it is not a simplified cartoon (more complicated)
Issue of problem with scale
In images they often do not tell you the scale = don’t know the magnification or if the image is zoomed in
- You might assume they are all the same magnification and this makes it harder to understand the image
Example - When looking at a map - when all the way zoomed out you can’t see houses -> then zoom in and see a bunch of houses –> then zoom in on one house and see the car in the driveway
Example of Scale issue
Image is of a Kidney –> can see the ducts
- White = interior of ducts
When zoom in = can see things that you didn’t see before (now can see the cells and more ducts)
THEN zoom more on the Glomerulus - now see things that you didn’t see before (see fine details)
Solutions to Issues
- Gain familiarity
- Learn to recognize common elements
- Keep the 3rd dimension in mind
Common elements in histology
- RBCs (very pik/organy red)
- Nucelri/nuc shapes
- White spaces
- Form follows function
Form follows function examples
Top Image - Lungs - oxygenates blood = need many blood vessels + many air spaces = see lots of open space + Lots of RBCs + Lots of capillaries
- See blodd vessels and air
Bottom Image - Kidney - filters blood –> had many blood vessels = in image should see manu blood vessels and ducts
- See many ducts for blood filtration
Using Landmarks
Can use landmarks to determine scale
Example 0 Use RBCs (Fairly uniform - 7 microns) –> if see RBCs in tissue = know the scale + RBCs can give you a sense of what organ you are in
- Know size of RBCs = can know the size of other things (Ex. know the size of nuclei are similar because see that they are similar in size to RBCs)
Histology
The study of the microscopic structure of tissues and ____
- Looks at morphology of cells
- Used for diagnosis of diseases (infections + cancer etc.)
What is often used in histology?
Histology is enhanced using stains –> stains brings out part of the tissue to make it more visible
Types of stains
- H and E
- Special stains (Trichrome, PAS, Gram)
- Imunohistchemistry (IHC)
How do we make a slide
- Place tissue (peice or whole organ) in forlmalin
- Trim the tissue and place in cassetes
- Indictae to technition what tissues are on the slide + often place it in how you want them cut (shows how to orient them)
- Process the Tissue - Remove the formaline and replace with enthnol to remove the water
- Embed in parafin - ethonol in the tisue is replaced with wax
- Section and place on slide - cuts 5-10 microns (thin enough to be translucent)
- Stain slide
Purpose of Formalin
Preserves the tissue - Kills microbes and stops enzymatic activity (Prevents aitosis)
- Prolonged fixation - cross-links the proteins
- This could INTERFERE with IGC = don’t leave it for too long if doing IHC
What does ethonol do in slide preperation
Removes the water - when you replace formalin with ethonol the water is removed
- Need to remove the water for parafin infiltration
Why embed in Parafin
The parafin provides a stable medium that allows you to section the tissue and store the tissue for long periods of time
Why stain slide
Done to visualize the tumor/microbe/cell/protein/nucelic acid of interest
- H/E = can see cells and nucelus
- IHC = can see proteins + nucelc acids
Hematoxylin and Eosin
Hemotaxalin is a blue/purple dye - blue parts = nucleus, RNA-rich parts of the crtoplams + hyaline cartalige matrrix (colors the basophilic structures)
- Basophilic stains purple because of chromatin
Eosin is a pink/red stain - stains the cytoplasm and collogen (Stains eosinphilic parts)
***H/E is hydrophilic and will not stain hydrophobic cells (lipids and fats)
What affects the color of H and E staining
Color is different depending on how the stain is appplied + how long it is applied for
Example H and E Staining
Left image - arrow points to capsid (Satined with eosin)
Bottom right - Nucleus stains purple; cytoplasm stains pink
Peiodic Acid-Schiff (PAS)
Overall: Reaction involved the oxidation of 1,2 glyceral groups of vicinal diols to oxidize to aldehydes
- Schiff reagent reacts with oxidized glycerols to produce deposits of aldeamines
Used to stain glycogens (polysaccrides) + glycoproteins + Basement membranes
When do you use PAS
Use if have a disease that affects basement membranes (because it stains basement membranes) + used for glycogen storgae disorders
PAS example
Staining of basement membrane - doesnt stain the surrounding ___ + see bowman’s capsule in dark purple
*CHECK SLIDE 21
Type of cells that are PAS positive
Globlet cells are PAS positive
- Goblet cells = line the intestine + produce muscus
- Goblet cells = basophilic color = shows PAS positive
Masson’s Trichrome (TRI)
Overall: Uses 3 dyes for different structires
Distictive feature = stans collegen blue
- Stains nuclei brown + cytoplasm red
- Distiguishes cells from extracellular compenents –> allows the pathologst to asses for fibrosis (scarring) of tissues
Use - used t asses fibrosis
Dyes used in TRI
- Iron hematoxylin
- Biebrich scarlet
- Anilline Blue
Example TRI #1
Shows fibrosis in Macaque heart - seen by the adundant abount of collegen (see collegen in the blue fibers)
- Stain difefrentiates collegen fibers
Example TRI #2
Shows collegen fibers in the dermins (shows the collegen stains blue)
- Other cells stain other colors
Verhoef-Van Gieson (VVG)
Overall: Iron hematoxylin interacts with elastic fibers (Elastin) – stains elastin black
- Stains collogen red
- Other structires stain yellow
Use - identifies elastic fibers/bands (Especially within blood vessels)
Example VVG
Shows difference for artiers and veins
- Dfference between artery and vein has to do with the number of elastin fibers = looking at amount alllows you to differentate between the two
Image = can see elastic lamina stains black in artery
Epithelium Definition
One or more layers of cells that together form a barrier betweeen two different envirnments
Location - Lining the outter surface (skin) andinnercavities of the body (ex. lungs + GU tract + reproductive and urinary tract + blood/lymph circulatory systems)
Function = Secretion + absorbtion _+ protection + transprotaion + Sensation
Types of Epithelium
Epithelium Example 1 (blood vessels)
Epithelium lining BV = endothelium
- Type = Simple squamous epithelium
Function = transport + clotting/homeostasis + inflamation + blood pressure control
Epithelium Example 2 (GI tract)
Slide 34 ____ Epithelium lines lumen of the GI tract
- can see goblet cells = PAS positive
- Type = Simple columnar epithelium
Function - Absorptive cells take in nutrients + goblet cells secrete mucus
Connective Tissue
