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Cell Structure
Organisation of the Body
| Question | Answer |
|---|---|
| Why do we need microscopes to see cells | A nucleus is approx. 5-20 um Cell body approx. 20-30 um Red blood cell approx. 7 um smallest object visible with the naked eye is 2mm or two objects separated by 0.01 mm, which cells are smaller than |
| How do hand lenses work? | This is the simplest way to magnify Uses visible light passed through a glass lens to magnify it |
| Why use microscopes not hand lenses | Hand lenses have limited detail whilst microscopes have multiple lenses and give greater detail |
| Definition of a cell | A single unit or compartment, enclosed by a border, wall or membrane. The smallest metabolically functional unit of life |
| Definition of magnification | The ratio between the apparent size of an object and its try size. This is dimensionless so has no units |
| Definition of resolution | The minimum distance between distinguishable objects in an image. This is different to magnification, magnifying beyond max resolution with make the object blurry |
| What units are used to measure cells | Meters- individuals Millimetre- organs, tumours Micrometre- organ layers, vessels, cells, some organelles Nanometre- membranes, molecular clusters |
| Give some sample types | Sources: post mortem, at operation e.g. biopsy or other e.g. needle Studied by cutting into slices to reduce thickness and all ow light to penetrate through Sections can be a transverse section or a longitudinal section |
| How to prepare a sample | Tissues decay and loose structural detail, so have to be fixed. This uses chemical cross binding (keeps cell stable when dead) or low temperatures. Sample is embedded in wax for support and cut into sections. It can then be stained to increase contrast. |
| Name and describe three routine staining methods | Histochemistry- staining of cells with specific chemicals e.g acidophilic Immunohisto chemistry- localisation of specific antigens with labelled, visible antibodies In situ Hybridisation- localisation of nucleic acid sequences with a labelled probe |
| Name the main types of microscope | Light microscope Scanning electron microscope Transmission electron microscope Fluorescence microscopy Confocal scanning laser microscopy |
| Describe light microscopes | Light from a source below the sample is focused by a condenser lens. This passes through a specimen with the section detail magnified by the objective lens. This is further magnified by eye pieces. Your eyes then see the contrast in the magnified image. |
| How to get images from light microscopes | Images detected by rods and cones in the eye, possibly in colour as visible light is used. Images originally collected by sketching but have recently become digital Resolution = 0.2 um |
| How does phase contrast microscopy work | Converts phase shifts in light passing through specimen to changed in image brightness so we can identify boundaries of certain structures in cells. |
| How does H and E staining work | Section from a wax block placed on a slide and dewaxed by a suitable chemical. Haematoxylin stained nuclei purple as nucleic acids bind with basic dyes (DNA negatively charged- basophilic) Eosin dyes acidophilic structures pink. |
| How does H and E staining help in cancer biology | Normal oesophagus is lined with squamous epithelium, which appears stratified and ordered when stained. Barrett's oesophagus, which occurs when reflux of stomach acid causes squamous to be replaced with columnar epithelium the sample looks less ordered. |
| Trichrome staining | Masson's Trichrome- a three colour staining protocol to distinguish cells from surrounding tissue Blue or green = collagen Pink = cytoplasm Dark red/brown/black = cell nuclei |
| How to interpret 3D stains | What you see depends on the plane of your section e.g. a tube can appear round or oval. You should cut and collect in order serial sections through the specimen to reconstruct 3D relationships |
| Fluorescence microscopy | A lamp produces light for excitation, with is focussed on a section using a mirror. Fluorescence emission light is collected. Different dyes emit visible light of different colours which is visualised as with a light microscope |
| How are samples prepared for fluorescence microscopy | Sample is preserved chemically or by freezing Sample will be labelled via antibodies tagged with fluorescent dyes (immunofluorescent) or the sample may express fluorescent proteins e.g GFP which is exited at 395 nm and emits light at 509 nm |
| Immunofluorescence microscopy | Antibody binds to specific protein region. The antibody can be directly bound to a fluorescent marker or indirectly bound via another antibody. An image is created using excitation and emission wavelengths. Cells have to be leaky to allow antibodies in |
| How does transmission electron microscopy work | Electron beam is used to penetrate a sample with a similar lens arrangement to a light microscope to amplify the image. This must be done in a vacuum as electrons are absorbed by all material and electromagnetic lenses have to be used. |
| How are images recorded by TEM | Resolution is 10nm to 0.5nm. Electrons interact with a phosphorescent screen, which creates an image. Contrast is created with electron dense/heavy metal stains Images are created digitally by interactions of electrons with the photographic plate . |
| How to prepare samples for TEM | Stronger fixation is needed e.g. in strong plastic resins. This must be controlled to preserve fine detail Thinner sections are needed of 50-100nm No colour contrast so heavy metals are used to scatter electrons and prevent reaction with the tissue. |
| How do freeze fracture replicas work | The sample is frozen and fractured at weak points in the cell. This is often at gaps between cells. |
| Confocal Scanning Laser Microscopy | Typically uses fluorescence microscopy and can use a thicker sample than LM so may not need sectioning. A scanned beam is focused at different levels of the section using a pinhole, only light from one focal plane is used. Image is produced on a detector |
| Using confocal images to produce 3D structures | Different images can be obtained by focussing light on different levels on the Z axis. This does optical slicing, so no sectioning is needed. Yu are able to see different layers through the cell |
| How can we view images of the surface of cells | Dissecting light microscope Capsule Endoscope Scanning Electron Microscope |
| What is a dissecting light microscope | Uses visible light reflected from a thick sample. The lenses and eye piece are above the sample. This provides limited resolution but is useful for initial study of specimens and we can view whole tissue surfaces. |
| What is capsule endoscopy | A patient swallows a pill containing a camera, light emitting diode, batteries and a radio transmitter. a data recorder is held on a belt at the waist. The capsule is excreted and recovered. |
| How do scanning electron microscopes work | Use electrons from a probe scattered across the surface of a thick sample. Electrons interact with the sample and the emitted signal goes to detectors. There is a resolution of 2- 0.2 nm depending on the size of the probe and interactions with the sample |
| How to prepare a sample for a scanning electron microscope | Fixed as for light or transmission microscopes. Sample may be glues or unsupported as they can be very large. Due to the movement of electrons a charge could build up so conducting coating is used to minimise this as it would reduce detail seen |
| What are the 4 main tissue types | Epithelium Muscle Connective tissue Nerve |
| What variation can there be in tissues | In position and ratio In cells during cell cycle e.g. between cell division and interphase In cell shape e.g. ratio of nucleus to cytoplasm |
| Nucleus | In LM stained with H and E appears purple and size can vary from 5-10 um. By TEM you can see the nuclear envelope, matrix, chromatin and nucleolus. |
| What two states can chromatin appear in | Euchromatin - lightly stained and finely dispersed Heterochromatin - clumped and course The difference between these is how tightly coiled the DNA is. If it is tightly packed it is not expressed. |
| Nucleolus | Within the nucleus and is responsible for production of rRNA This produces ribosome subunits which are then exported out the nucleus to be involved in protein synthesis |
| Nuclear pores | Responsible for communication between the nucleus and cytoplasm. This is a hole spanning the two nuclear membranes to control what enters and exits the nucleus. |
| Experimental Evidence for Nuclear pores | Unwin and Milligan 1982 Showed more detail to nuclear pores than the eightfold symmetry and flower petals Showed 80nm size with rings, spokes, a central hub, large cytoplasmic particles, cylindrical shape and spoke to attach it to the membrane |
| Function and Structure of cell membranes | This surrounds the cell Its specialisations depend on whether you are viewing apical, lateral or basal aspects of a cell Under higher power TEM membranes are dark/light/dark and are known as unit membranes. EM can show individual lipids |
| Endoplasmic reticulum membrane | Has a network of membrane limited channels called cisterna RER has polyribosomes attached to the outer membrane for protein synthesis for export, SER has specialised functions such as steroid secretion and lipid synthesis |
| Golgi apparatus | A multi layered membranous organelle near the nucleus for packaging of material to be transported out the cell and to endosomes. Cis face receives vesicles from ER Maturing trans face sends vesicles to the cell membrane or endosomal pathway |
| Pathway for proteins through the cell | Created in ribosomes on RER. The nucleus membrane is continuous with the ER. The proteins are carried in vesicles to the Golgi cis face and are released in vesicles from the trans face. They then move to the membrane and endosomes. |
| Lysosomes | Degrade unwanted vesicular material by acid hydrolases. Primary are smaller then secondary. After digesting proteins they become residual bodies. These can be autophagosomes (recycling cells not in use) or heterophagsomes (dealing with new material) |
| What happens is lysosomes are defective | There are many possible consequences depending on enzyme effect e.g neurological impairment, growth disorder. |
| Proteosomes | Present in cytoplasm and nucleus to break down proteins by proteolysis. Misfolded proteins are tagged for breakdown by Ubiquitin. These are not visible on LM or TEM. They have 4 stacked rings around a core where proteases are located. |
| Mitochondria | The ATP generators of the cell. These are spherical or long with different shapes depending on cell type. The have an outer membrane and an inner membrane which folds to form cristae. In LM this is acidophilic. These contain their own small genome. |
| How to interpret a microscope image | Context- what info is available from lab books, labels etc Sample lay out - is it a section of tube or a larger organ Sample size- is it too small to be from an adult human Always consider plane of the section. |
| How to work out magnification | Calculation- from magnifying power or ratios from sample- inbuilt scale markers e.g. nuclei Displayed- numerical e.g. X50 or magnification bar |
| Why should you always find the nucleus when looking at a microscope slide | Helps identify scale- lots of nuclei means low magnification and always have similar diameter Helps identify image type- lots of detail means EM Helps identify cell type - shape/structure/number of nuclei Helps identify cell activity |
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