How to examine cells and tissues Flashcards
4 types of tissue
- epithelial
- connective
- muscle
- nerve
epithelial tissue
- e.g. skin
- edge of other tissues and surround other tissues
- clusters within tissues (glands)
- polarised at surfaces - apical and basal surface
- basement membrane (basal lamina + reticular layer) on basal surface
- held together by strong anchoring proteins
- communicate through junctions at lateral and basal surfaces
connective tissue
- consist of cells + extracellular proteins/glycoproteins
- fibroblasts, chondrocytes, osteocytes, osteoblasts, osteoclasts, stem cells, progenitor cells, bone marrow, blood, adipocytes
- main products are fibres, groud substance and wax + gel-like materials
muscle tissue
- consists of muscle cells (skeletal, smooth, cardiac)
- major function is to contract - movement, stability, movement of contents
- minor function is to secrete hormones - natriuretic factor, myostatin
nerve tissue
- made of nerve cells and support cells
- nerve tissues can be short or very long
- main fast communication system in the body
- cells congregate into nerve fibres
- fibres congregate into nerves that can be dissected and visualised
what is the standard measurement for a cell
micron (µm)
what is used for sizing of a cell
measurement graticule
what is the limit of resolution
the smallest distance by which two objects can be separated and still be distinguishable as two separate objects (proportional to wavelength of viewing system)
relationship between milli-, micro- and nano- metres
1mm = 1000µm
1µm = 1000nm
1mm = 1000000nm
methods of obtaining a biopsy
- surgery and dissection
- scraping methods (curettes, scalpel scrapes)
- sharp needles (needle biopsy, pipelle, trephine, punch biopsy)
- direct venepuncture (blood smears)
- transvascular (kidney, lung, brain, heart)
what is fixation done for
- protect biopsy from damage as once removed from the body it no longer has the protection of body’s immune defence systems so can be digested by microbes or destroyed by decay
- removes water from sample so stiffens biopsy
why does sample need to be thin (<20µm)
allow light to pass through it so diffraction doesn’t occur producing a blurry image
what is the most common fixation chemical
fixative formalin
- 37% aqueous solution of formaldehyde
- 0.9% sodium chloride solution
why is it important for formalin to be isotonic with intracellular fluid
- allow better penetration of formaldehyde
- reacts with amino groups of amino acids within proteins forming methylated bridges between protein chains
- preserves general structure of cells in tissue
embedding choice for histological studies
paraffin wax
embedding choice for electron microscopy
epoxy resins or plastics
process of paraffin wax embedding
- washed and dehydrated in series of alcohol concentrations
- washed in solvents miscible with alcohol and wax (xylene or toluene)
- immersed in hot paraffin wax overnight
- placed in mould and more hot wax used to completely cover specimen
- allowed to solidify
- gently eased out of mould
microtome process
- block of tissue mounted onto microtome
- thin sections cut using sharp steel blade, sharp edged glass or diamond knife
- fragile sections picked up with paintbrush and floated on warm water bath
- place glass microscope slide under floating section to adhere to specimen
staining process
- dissolve paraffin (toluene or xylene)
- rehydrate with different alcohol concentrations
- stain sections (H&E)
haematoxylin and eosin staining
- immerse section in solution of aqueous haematoxylin
- wash in water and transfer into alcohol
- immerse section in eosin
- wash with alcohol
- specimen mounted in non-aqueous mounting medium
- coverslip applied
- examine specimen once solvents evaporated
haematoxylin
- basic dye so binds to acidic structures like DNA and RNA
- stains blue
eosin
- acidic dye so binds to basic structures like intracellular and extracellular proteins (cytoplasm, collagen, elastic fibres)
- stains pink
preparation for histology
- tissue procurement - biopsy
- fixation/frozen section
- embedding
- microtome
- staining
- examine
Masson’s trichrome
- red = keratin and muscle fibres
- blue/green = collagen and bone
- light red/pink = cytoplasm
- brown + black = nuclei
Periodic acid-schiff stain
identifies anything with a sugar attached - glycocalyx
how can tissue processing lead to formation of shrinkage and other artefacts
leaving biopsy in fixative over 24-48 hours can cause dehydration and fixation artefacts
mechanism of immunohistochemistry and immunofluorescence
both utilise labelled antibodies to localise specific cell and tissue targets
immunofluorescence
- antibodies labelled with fluorescent dye bind to target antigens allowing structure to be visualised directly
- incident lighting often from UV light source
indirect immunohistochemistry
- uses enzyme activated secondary antibody complexes that precipitate a coloured product at site of interaction
- primary antibody binds to target antigen
- secondary antibody with enzyme binds to primary antibody
- dye is added which is converted into coloured precipitate by enzyme
- enzymes routinely used are peroxidases which will produce brown precipitate at reaction site
how is a frozen section prepared
- specimen on metal disc is frozen rapidly
- frozen specimen cut with microtome in cryostat in freezer
- stained with H&E
paraffin wax formalin fixed VS frozen section
paraffin wax formalin fixed
- fixed tissue specimens
- 24-48 hours
- permanent saving time
- clarity in morphology under microscope
- used for pathological diagnosis
frozen section
- fresh tissue specimens
- 10-20 minutes
- months saving time
- opacity in morphology under microscope
- used for intraoperative consultation
difference in tissue preparation for light vs electron microscopy
light microscope
- fix with formalin
- embed in paraffin wax
- stain e.g. H&E, methylene blue
electron microscope
- fix with glutaraldehyde
- embed in epoxy resin
- stain e.g. osmium tetroxide
light vs electron microscope
light microscope
- natural coloured images
- large field of view ~2mm
- cheap and easy
- view living and moving objects
- magnification ~600x
- resolution ~0.25µm
electron microscope
- monochrome images
- limited field of view ~100µm
- difficult and expensive
- view dead and inert objects
- magnification ~500,000x
- resolution ~0.25nm
scanning electron microscopy
- surface of cells coated with electron dense chemicals to reveal 3D aspect of cells and tissues
- good for examining surface of cells
- limit of resolution ~10nm
transmission electron microscopy
- ultra thin sections of cells coated with electron dense chemicals to reveal 2D aspect of cell
- good for examining intracellular structures and organelles, especially membranes
- limit of resolution ~2nm
why are electron microscopes capable of finer resolution than light microscopes
light microscope uses visible light whereas electron microscope uses electrons which has much smaller wavelength therefore much smaller limit of resolution
advantage of phase contrast microscopy
living cells can be examined in their natural state without being killed, fixed, and stained
advantage of confocal light microscopy
- control of depth-of-field
- collect serial optical sections from thick specimens
- create 3D images of the structures within cells
advantage of dark field microscopy
high degree of contrast, making it easy to see samples on difficult backgrounds
how does dark field work
illuminates the sample with light that will not be collected by objective lens so doesn’t form part of the image
plasma membrane functions
- intercellular adhesion and recognition
- signal transduction
- compartmentalisation
- selective permeability
- transport of materials along and across cell surface
- endocytosis
- exocytosis
nucleus
- contains DNA, nucleoproteins and RNA
- inactive cells have small nuclei with condensed heterochromatin
- actively transcribing cells have large nuclei with dispersed euchromatin
- no nuclei in terminally differentiated cells like erythrocytes, stratum corneum, lens fibre cells
nucleolus
- sites of ribosomal RNA synthesis
- disappear during cell division
nuclear envelope
- double layer of membranes bounding nucleus
- perinuclear cisternae between inner and outer nuclear membranes continuous with ER
- contains nuclear pores where macromolecules transported and micromolecules diffuse
endoplasmic reticulum
interconnecting set of membranes, cisternae and vesicles
rough
- ribosomes attached to outer surface
- actively synthesing proteins that destined for cell exterior, lysosomes or cell membrane
- free ribosomes synthesise proteins for cytsol
smooth
- not associated with ribosome
- cisternae not flattened
- lipid biosynthesis and intracellular transport
golgi apparatus
- saucer shaped stacks of cisternae
- sort, concentrate, package and modify proteins synthesised by RER
- vesicles from RER fuse with golgi body
- proteins migrate from convex to concave end of stack
- secretory vesicles condense together and release contents by exocytosis
lysosomes
- contain acid hydrolases
- generated by golgi apparatus
- lysosomal membrane proteins are highly glycosylated for protection from these enzymes
- primary lysosomes phagosomes, endosomes, autophagosomes or excess secretory products to form secondary lysosomes where contents are degraded
- residual bodies = lysosomes which have digested their contents but contain indigestible remnants\
- primarily digest and re-use carbohydrates, lipids and peptides
peroxisomes
- contain granular matrix
- self-replicating - obtain all materials from cytoplasm
- modify toxic molecules before they re-enter bloodstream
- kill bacteria
- major sites of oxygen utilisation and peroxide production
- catalase utilises H2O2 generated to oxidise other substrates
mitochondria
- double membrane with inner membrane folded into cristae
- generation of energy rich ATP molecules by oxidative phosphorylation using glucose and fatty acids
- large numbers in liver and skeletal muscle
- matrix contains enzymes of Krebs and fatty acid cycles, DNA, RNA, ribosomes and calcium granules
- can divide because they have their own genetic information
functions of cytoskeleton
- provide structural support for plasma membrane and cell organelles
- provide way of movement for organelles, plasma membrane and cytosol content around the cell
- provide locomotor mechanisms for amoeboid movements and cilia + flagella
- provides machinery for contractility in cells of specialised tissue like muscle
microfilaments
- 5nm in diameter
- two strings of actin chains twisted together with ability to bind ATP
- contract and change shape of cell
- assemble and dissociate quickly
intermediate filaments
- not dynamic
- 10-12nm in diameter
- common in nerve, neuroglial cells and epithelial cells (cytokeratin)
- form tough supporting network within cytoplasm
- anchored to plasma membrane by desmosomes
- hold cell together and prevent lysis
microtubules
- thirteen α and β subunits polymerise to form wall
- originate from centrosome
- found where structures are moved e.g. mitotic spindle, cilia, flagella
- attachment proteins (dynein and kinesin) bind to organelles and move structures along microtubule
- highways within cell to move molecules and organelles to more distant parts of cell
