ch1

• Light microscopy

  Uses lenses and refractive glass to focus light
• Magnification

  number of times image is enlarged compared to actual object
• Resolution

  ability to distinguish between two separate points - improve resolution by using radiation with shorter wavelength, e.g. UV light or beam of electrons
• How does light pass through each lens?

  Light passes from bulb under stage through condenser lens and specimen then focused onto objective and eyepiece lens
• Total mag =

  eyepiece mag x objective mag
• Pros of light microscopy

  • observe wide range of specimens including living organisms
  • easy to use and transport
  • relatively inexpensive
  • don't require specialist training
• Con of light microscopy

  • due to limited resolution, most internal structures can't be seen
• Stains

  Stains usually colour particular part of cell, making organelles more visible. Some are added to living cells, others are fixed via alcohol to make proteins and nucleic acids insoluble hence fixing them in position (which kills the cells).
• Cell theory

  • cell is basic unit of all life forms
  • cells are unicellular/multicellular
  • metabolic processes occur inside cells
  • new cells are derived from existing cells
  • cells possess genetic material which passes on to new daughter cells
  • cells are the smallest unit of an organism that can survive independently
• Electron microscopy

  Uses beam of electrons (with shorter wavelength so greater resolving power) as a form of radiation
• Pro of electron microscopy

  Allows finer details to be seen
• Cons of electron microscopy

  • expensive and large
  • requires specialist training
  • specimens must be placed near vacuum as air molecules absorb electrons (so specimens must be dehydrated and dead)
  • also requires more complex staining process
  • artefacts may occur as a result of preparation technique
• As human eye can't detect electrons...

  Electrons are focused onto fluorescent screen which emits visible light when electrons hit. 
• Colouring final image

  Final images are always black, grey and white but can be coloured using specialist computing (false-colour electron micrographs)
• TEM (transmission electron microscopy)

  • requires very thin specimens as electrons can't penetrate materials as well as light rays
  • heavy metals stain specimen as atoms of the heavy metals have large, +vely charged nuclei that scatter the electrons
  • scattered electrons don't hit fluorescent screen so leave dark area in image (2D and black and white)
• SEM (scanning electron microscopy)

  • electrons don't pass through specimen and instead reflect off its surface back and forth in a regular pattern to reflect the contours and produce a 3D image
• CLSM (confocal laser scanning microscopy)

  • obtains high resolution images and 3d reconstructions
  • can produce focused images of thick specimens at various depths via optical sectioning (point source of light directed onto object plane then reflected light is directed through pinhole and enhanced via photomultiplier and appears on screen as a pixel).
  • Blur free due to scanning the object line by line. Fluorescent markers also used to detect biological objects
• Differential stains

  make some structures appear a different colour from other structures
• Temporary slide preparation

  • section tissue before fixation using 70% alcohol
  • place tissue on clean glass slide and add few drops of stain
  • cover specimen using thin glass coverstrip to exclude dust and protect high-power objective lens on microscope
  • specimen mounted in glycerine after staining to avoid specimen from drying out
• Iodine-KI

  for starch, results in blue-black
• Leishmann's

  Blood is dried first then fixed with methanol, flooded then left for 2 minutes then diluted with water and left for another 5-7 minutes. Slide washed until it appears pale pink.
• Wright's

  Used for differential WBC counts and when infections are suspected
• Too thick vs too thin blood smears

  • Too thick means individual cells can't be seen.
  • Too thin means the cells may not be representative.
• Preparing blood smear

  • small drop placed at the end of a dry, sterile microscopic slide and a spreader (another microscopic slide) is held at 30* angle and used to spread blood evenly
  • then the slide is labelled and allowed to dry to allow cells to stick to the slide
  • finally a fixative is added to preserve the cells
• Functions of the blood

  • clotting
  • delivering oxygen and nutrients to tissues
  • removing waste from tissues
  • immunological protection
  • transporting cell signalling molecules
  • acting as buffer to regulate body pH
  • distributing heat to regulate core body temperature
• Erythrocytes (RBCs)

  Deliver oxygen from lungs to rest of body and carry CO2 from tissues to lungs, develop in bone marrow and circulate for approx. 100 days. Contain haemoglobin (Hb), an iron-containing protein that binds reversibly with O2 to form oxyHb.
• RBC adaptations

  Biconcave so flexible and can squeeze through capillaries, large SA:V ratio due to no nucleus.
• Erythropoiesis

  Process of producing new RBC and are stimulated by erythropoietin hormone via kidney
• Thrombocytes (platelets)

  Biconvex discs of cytoplasm fragments surrounded by CSM, produced in bone marrow and have no nucleus, appear dark purple on stained blood smear, involved in blood clotting and clot formation, live for a week.
• Types of WBC

  neutrophil, lymphocyte, monocyte, macrophage
• Neutrophil

  Lobed nucleus, help defend against fungal or bacterial infections, engulf and break down bacteria via phagocytosis, squeeze through capillaries fenestrations due to flexible lobed nucleus
• Lymphocytes

  Large nucleus. B lymphocytes produce antibodies (immunoglobulins) and T lymphocytes have T helper (produce cytokines and help co-ordinate immune response), cytotoxic T cells (bind to antigens on infected cells and destroy them) and T killer cells
• Monocytes

  Largest leucocyte with kidney bean shaped nucleus, carry out phagocytosis and live much longer than neutrophils, eventually leave bloodstream and differentiate into macrophages to remove dead cell debris and attack microorganisms, they can replace lysosomes unlike neutrophil
• Haemocytometer

  Specialised microscopic slide to count cells, centre has platform with grooves. Grid has squares exactly 0.1mm when a cover slip is placed onto platform so possible to count specific volume of cells and calculate concentration
• Importance of haemocytometry

  Thoroughly mix blood sample before taking sample (for representative data), take appropriate dilution so cell number can be accurately counted and repeat to calculate mean
• North-west rule

  Count cells that lie on line on north and west lines
• Flow cytometry

  Electron counting apparatus that uses laserbeam passed over blood stream to count cells, enables analysis of physical and chemical characteristics, counts thousands of particles per second.
• Main parts of flow cytometer

  • Flow cell (liquid stream that carries cells in single file for accurate counting)
  • measuring system
  • detector
  • amplification system
  • computer software.
• Process of flow cytometry

  Specific antibodies 'tagged' (attached) to different fluorochromes. These antibodies recognise and target specific antigens inside cells or on CMS. Each fluorochrome has its own peak wave of excitation and emission wavelength.
• Lamps/lasers in flow cytometry

  Lamps or lasers used to excite fluorochromes so cells fluoresce and are counted.