Microscopy Flashcards

Question

LIGHT MICROSCOPY difference between what is viewed in... - BF - DIC - Ph

Answer 1

BF = no filter DIC = able to contrast background and sample (has some 3 dimensionality) Ph = useful for tissue and cells that are changing shape. Enable observations on whether sample is changing shape.

Answer 2

1. Histology 2. Phase contrast- cell morphology 3. Time-lapse (heart cell differentiation, cell migration)

Answer 3

Histology allows you to view the whole tissue and shows you geographically what is happening in the tissue, HOWEVER, it does NOT tell you what each individual cell is doing

Answer 4

1. immunohistochemistry | 2. laser capture microdissection

Answer 5

You can use antibodies to find where your protein of interest is found (specific antibody will interact with antigen on cell)

Answer 6

remove area of interest using laser that cuts through selected area

Answer 7

You can select which intensity of light goes through the sample – wavelength is not changed, but how much is reflected and how much is refracted is. It's important when looking at where cells or tissue stays

Answer 8

The box life imaging method is used (control CO2 and temperature).

Answer 9

Heart cell differentiation can be observed. Cell migration, e.g. crawling leukocyte chasing bacteria, can also be observed.

Answer 10

An electron source is used instead of light – a beam of electrons go to sample, and gives us a dark image of the areas sampled. If an area is more dense, the electrons have more difficulty passing through the structure meaning it has a darker appearance.

Answer 11

TRANSMISSION EM – this is not 3D; a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through SCANNING EM – the sample is treated with specific reagents, then we scan a beam of electrons through sample at a particular angle, this creates a 3D image

Answer 12

Here, we are controlling the wavelength of light – selecting if light going through is red, green, etc. We are able to modify proteins in the sample to respond to specific wavelengths. ocular: - eyes - camera - PTM

Answer 13

The placement of the light source

Answer 14

Fluorescence is the ability of certain types of molecules to absorb light, become excited and generate energy, and emit energy. The specimen is exposed to light; it then absorbs light (excitation), and releases energy (emission). It emits light at a specific wavelength. Several rounds of this cycle will eventually lead to energy loss and the molecule getting destroyed, leading to no more fluorescence.

Answer 15

Stokes shift is the difference between the wavelength of excitation and the wavelength of the emission Note: the excitation wavelength is ALWAYS smaller than the emission wavelength You'll have 2 peaks: the excitation peak (energy given to molecule), and the emission peak (molecule releases energy) . Due to energy loss, the emitted light is shifted to longer wavelength relative to the excitation light.

Answer 16

High intensity illumination may destroy fluorophores and cause them to permanently lose their ability to emit light. You should work with reduced light intensity, use shorter exposure times or use anti-bleach in mounting media to avoid this.

Answer 17

- using antibodies | - protein fusion (tag the gene)

Answer 18

antibodies can recognise specific antigen proteins and can then attach and give off colour

Answer 19

The advantage: it's specific (can know exactly where fluorescence is, so antibody with specific fluorescent marker can bind to target molecule in wanted area, e.g. protein in nucleus). The disadvantage: it can only be applied to a fixed sample if you want antigen to bind to sub-cellular structures (as the antibody is big and can’t go past cell membrane).

Answer 20

You use plasmids to incorporate a gene that causes fluorescence. It's incorporated into undifferentiated ES (embryonic stem) cells, which can eventually form fibroblasts.

Answer 21

The advantage: it can be used in live cells. The disadvantage: it lacks specificity, and you don’t know if the molecule can be kept (cell can undergo apoptosis if they recognise the inserted gene is exogenous).

Answer 22

If we are able to control the depth that the source of light was going through sample, we’d be able to see through thinner portions of sample. Thus, this involves stopping the beam of light at certain levels, so you can see different levels of sample, e.g. in seeing an epithelial cell (seeing specifically only apical or basolateral side). The light source is a laser – so we are able to control how it goes through sample.

Answer 23

The advantage: better Z resolution, better specifics as we can see through several layers; it also allows live imaging (control temp and CO2). The disadvantage: only a small volume can be visualised by confocal microscopes at once bigger volumes more time consuming, as more sampling and image reassembling is needed (widefield is better for bigger volumes!).

Answer 24

• Developmental dispersal of drosophila haemocytes View the tissue and cellular localisation • Microtubule dynamics using GFP-tubulin • Vesicle transport through microtubules using GFP-kinesin • Look at the cell surface • 3D reconstruction Comparison of neoplastic to non neoplastic