2.1.1 Microscopy

Question 1

Differential Staining

Answer 1

-Used to differentiate between two types of organisms or different organelles of a single organism.

Question 2

Gram strain technique

Answer 2

-Used to separate gram-positive and gram-negative bacteria.
-Crystol violet is applied, then iodide which fixed the dye and it is then washed with alchohol.
-Gram positive bacteria retain the crystol violet stain and will appear blue or purple. Gram-negative bacteria have thin cell walls that do loose the dye.
-They are then stained with a counter stain of safranin dye, making them red.
-Gram-positive bacteria are susceptible to antibiotic penicillin, which inhibits the formation of cell walls.
-Gram-negative bacteria have much thinner cell walls that are not susceptible to antibiotic penicillin.

Question 3

Acid-fast technique

Answer 3

-Used to differentiate between species of mycobacterium from other bacteria.
-A lipid solvent is used to carry carbolfuchsin dye in the cells being studied.
-The cells are then washed with a dilute acid-alchohol solution, which doesn’t affect mycobacterium, so it retains the carbolfuchsin dye, which is red.
-Other bacteria lose the stain and are exposed to methylene blue stain.

Question 4

Light microscope

Answer 4

-A compound light microscope has 2 lenses-the objective lens, placed near the specimen, and an eyepiece lens through which the specimen is viewed.
-X40, X100, X400, X600 objective lens.
-Image focused with glass lenses.
-Maximum magnification X1500.
-Resolution of 200nm.
-Natural colour, 2D image.
-Staining, e.g, iodide, methylene blue, acetic orcein.

Question 5

Dry mount

Answer 5

-Solid specimens are viewed whole or sectioned, then placed on the centre of the slide with a cover slip over it.

Question 6

Wet mount

Answer 6

-Specimens are suspended in a liquid such as water or an immersion oil, then a cover slip is placed on from an angle.

Question 7

Squash slides

Answer 7

-A wet mount is first prepared, and then a lens tissue is used to gently press doen the cover slip.
-Depending on the material, potential damage to a cover slip can be avoided by squashing the sample between two microscope slides.

Question 8

Smear slides

Answer 8

-The edge of a slide is used to smear the sample, creating a thin even layer coating on another slide, a coverslip is then placed over the sample.

Question 9

Staining

Answer 9

-The images tend to have low contrast as most cells don’t absorb a lot of light. Resolution is limited by the wavelength of light and diffraction of light as it passes through the cell.
-The cytosol(aqueous interior) of cells and other structures are often transparent, and stain increases contrast as different components within a cell take up stain to different degrees.
-To prepare the sample, it’s placed on a slide, left to air dry, and then passed through a flame so it adheres to the slide and takes up the stain.
-Crystol violet and methylene blue=positively charged dyes attracted to negatively charged cell components staining then.
Nigrosin and congo red=negatively charged dyes repelled by cell cytosol, so stay outside of the cells, only dying the background, which mames the cells stand out.

Question 10

Electron microscopy

Answer 10

-In electron microscopy, a beam of electrons with a resolution of 0.2nm is used to illuminate the specimen.
-The smaller resolution allows for separate entities to be seen at high magnifications (over X500000)

Question 11

Transmission electron microscope (TEM)

Answer 11

-A beam of electrons is transmitted through a specimen and focused to produce an image.
-Focused by electromagnets
-Maximum magnification X500000-X3million
-Resolution of 0.2nm
- Black and white, 2D image.
-Stained with heavy metals (lead and uranium)

Question 12

Scanning electron microscope (SEM)

Answer 12

-A beam of electrons is sent across the surface of a specimen, and the reflected electrons are collected.
-Focused with electromagnets.
-Electrons are reflected.
-Maximum magnification X500000.
-Resolution of 2nm-10nm.
-Black and white, 3D images of surfaces.
-Stained with gold and platinum.

Question 13

Sample preparation for electron microscopes.

Answer 13

-Inside an electron microscope is a vaccum to ensure electron beams travel in straight lines so samples have to be processed in a specific way.
-Specimen preparation involves fixation of samples using chemicals or freezing, staining with heavy metals, and dehydration with solvents.

Question 14

Samples for an TEM

Answer 14

-Set in resin then stained again.

Question 15

Samples for an SEM

Answer 15

-May be fractured to expose the inside and then coated with heavy metals.

Question 16

Laser scanning confocal microscopy

Answer 16

-A laser scanning confocal microscope moves a single spot of focused laser light across a specimen (point illumination), causing a fluorescence from the components labelled with dye.
-Only the light radiated from the very close focal plane is filtered through a pinhole aperture to avoid any light emitted from other parts of the specimen being detected and reducing the resolution.
-Laser light source is reflected.
-Focused with glass lenses
-Maximum magnification X1500
-Resolution of 200nm.
-3D image.
-Stained with flourscent tags/antibodies.

Question 17

What is beam splitter in LSCM

Answer 17

-The beamsplitter is a dichroic mirror that only reflects one wavelength (the laser) but allows other wavelengths (produced by the sample) to pass through.
-The position of the two pinholes means the light waves from the laser (illuminating the sample) follow the same path as the flouresced light waves, giving them both the same focal plane.

Question 18

Flourescent tags.

Answer 18

-Using antibodies with flourescent tags specific features can be targetted and therefore studied by confocal microscopy with more precision than staining.
-Green flourescent protein (GFP) is produced by jellyfish Aeqorea Victoria and has been engineered to flouresce different coloursto show where proteins are made and where they go.
-Non-invasive technique to show the productiona dn distribution of proteins within organisms and cells.

Question 19

Creation of artefacts.

Answer 19

-An artefact is a visible structural detail caused by processing specimen and not a feature of them
-They appear in both light and electron microscopy.
-The bubbles get trapped underneath the cover slip in preparation, and the complex preparation in electron microscopy makes it even easier to cause artefacts.
-E.g mesosome.

Question 20

Atomic force microscopy

Answer 20

-The AFM gathers information about a specimen by feeling its surface with a mechanical probe, and they generate 3D images of surfaces.
-It consists of a shapr probe on a cantilever, which, when brought close to the surface of the specimen, causes deflections of the cantilever. -These are measured using a laser beam refelcted from the top of the cantilever into a detector.
-Fixation and staining are not required, and living systems can be exmined at a resolution of 0.1nm.
-Infomation can be gathered at an atomic level, even the bonds without molecules.

Question 21

Atomic force microscopy uses.

Answer 21

-Pharmaceutical insdustry uses AFM in identifying potential drug targets on cellular proteins and DNA.
-Finding and identifying new chemical compounds in the natural, which may have medical applications, can be expensive, but ATM speeds up the process.

Question 22

Super resolved flourescent microscopy

Answer 22

-Electron microscopes can’t be used to examine living cells, and it was always believed that the maximum resolution of light microscopes was 0.2micrometres, which limits detail.
-In 2014, Eric Betzig, Stefan Hell, and William Moerner were awarded a noble prize for achieving resolutions greater than 0.2micrometres using light microscopy.
-Two principles were involved, both forms of SRPM
1. Building a very high-resolution image by combining many images that are very small.
2. Super imposing images with normal resolution to create one very high-resolution image.

Question 23

Stefan Hell’s work

Answer 23

-Developed stimulated emission depletion (STED) which involves the use of two lasers which are slightly offset, the first laser scans a specimen causing flourescence, followed by a second laser which negate the flourescence from all but a moleculer sized area.
-A picture is built up with a high enough resolution to see individual strands of DNA.

Question 24

Eric Betzig and William Moerner’s work

Answer 24

-Developed the second principle, which relies on the ability to control the flourescence of individual molecules. Specimens are scanned multiple times, but each time, a different molecule is allowed to flouresce. The images are then superimposed, and the resolution of the combined image is at the molecular level.

Question 25

Super resolved flourescent microscopy uses.

Answer 25

-Observing proteins involved in Parkinsons and Alzheimers.
-Fertilized eggs dividing into embryo can be studied at a molecular level.

Question 26

Magnification

Answer 26

-How many times larger the image is that than the actual size of the object being viewed. Interchangable objective lenses allow a user to adjsut the magnification.
-Simply magnifying doesn’t increase detail shown, the resolution also needs to be increased.

Question 27

Resolution

Answer 27

-Ability to see individual objects as separate entities. Limited by diffraction of light as is passes through samples (and lenses).

Question 28

Diffraction

Answer 28

-Tendency of light waves to spread as they pass close to physical structures such as those present in the specimen being studied.