lecture 1 - Cell Biology and Techniques Flashcards
What is Haematoxylin & Eosin and its uses ?
used in histology sections when cutting slices it binds and allows usto see
Haema-binds to the nucleic acids -nucleas
Eosin -binds to proteins
purple colouring when seen
What is a microtome ?
glorified ham slicer
cuts 1 cell thick samples to get abetter view of the organelles present
as its difficult to handle & manipulate and then analyse tissues this makes it easier as cells are alone
What is important in studies of cell biology ?
We need a source of cellls growing individually and replenished easily
-cells must be homogenous
-A way to see what cells look like
-means to visualise subcellualr structure
-a way to change the activity of the cellular components
What do we need when growing cell ?
- Single layer cells
- indefinitely growing cells
got that from HeLa cells: thus able to build up on other proples work as the cells are the same
What are Fibroblasts ?
they are cells that grow only for several generations
-they divide and occupy till all the space iin the petri is filled the the cells are shaved off using proteases
then move some cells to a diff petri and they then fill it up again
can do this only a few times tho till they just stop
What Tissue Cultures exist now ?
rodent and Human lines
grow on glass & plastic dishes
growth factors are often required and micro-microbials to eliminate contamination
What are some examples of early microscope ?
Robert Hooke
-simple compound microscope
(focused by moving barrel)
One lens near object
Another near the eye
Light focused on specimen by a further lens
Label a modern microscope !
How to manipulate cellls ?
Pharmaceutical -add drugs
also over-express and deplete proteins
2—>:overexpression: extra protein
—–>:depletion:(little to no protein )
eg.) Using CRISPR or RNAi
Targeting both genome and inactivating certain genes using expression vectors
What is Heterologous expression ?
Express something they usually wouldn’t
Taking independent protein and seeing wht happens
How does RNAi work ?
selectively depletes the cell of a specific mRNA
researcher designs the siRNA sequence so that it will bind the mRNA of the protein they are targetting
very effective with more than 90% of protein depleted
can use RT-PCR to determine if levels are reduced
What are Expression Vectors ?
-Eukaryotic promoter
-Polyadenylation signal
-Eukayotic resistance marker
-Bacterial origin and resistance marker
What is contrast ?
see an image were detecting a contrast between object and adjacent backround
due to diff in colour or intensity
-Absorption of light
-Reflection of light
human eye detects changes in amplitude
What to do if object is see through ?
use stains
-use a scope to change amplitude:
1. Brightfield
2.DIC
3.Phase Contrast
How does Phase contrast work ?
- The annular ring creates a hollow cone of light that is focused onto the specimen so its fully illuminated
- light passing through forms hollow cone entering objective lens
- Diffracted light passes takes a diff path
- another ring obstructs light only
- slowed down by a further 1/4 path
- total diff now 1/2 wavelength
- Destructive interference occurs
- gives CONTRAST
How does Digital interference contrast microscopy work ?
very clear and contrast
* Polarised light is split into 2 parallel rays
* the distance is between the 2 the shear is less than the resolution of the objective
* At any boundary one beam is affected differentl to the other
* beams are brought back together
interfere for diff amount of llight
* Pseudo 3D image
Check notebook
How to see in the dark ?
- adaptations to light microscopy are quite limited
We would like to know where substrates even individual molecules are located eithout using EM or biochemical fractionation
Fluoroscence expanded rapidly
What is Flurospar ?
shine green if illuminated with UV light
absorbing UV then reemitting ligh with longe wavelength
-excitation
-emission
-wavelength
What are Fluroscence chemicals ?
staining cellular companents
antibodies are solution
like the one that dyes the eye during opticians
What is Immunoflurocence ?
fluoropheres chemical joined to anitbodies
Ab can be raised to virtually any proteins or cellualr component
bind to antigen
therefore stains can be made for each one as they bind to proteins
high contrast
What if we want to observe live cells ?
- use antibody-fluorophore conjugates to stain cells
- Formaldehyde-alcohols to precipitate or crosslink molecules so they dont move
- some oganisms evolved to prod fluor..#
- molecular cloning-make proteins joined to fluorescent proteins
see where proteins go eithout needing to make antibody
What is GFP ?
green fluorescent protein
put genes in cells
allows them to fully develop allowing cells to make their own fluorescent dye
What is RFP ?
marine organism
makes red fluroesence protein
however tetrameric-causes tons of problems when fused to other proteins
Tsein engineered monomeric protein
Whta us the importance of GFP ?
GFP expression vectors
based on E.coli palsmids so can use the bacterium as a tool tomake DNA construct
Euk promoter upstream of EGFP
Stretch of DNA with many restriction sites so coding sequence to be studied can be spliced in frame with GFP sequence
Poly A tail and marker for selection in euk
