level: Microscopy
Questions and Answers List
Flashcards on the case and lecture on Microscopy
level questions: Microscopy
| Question | Answer |
|---|---|
| what are the types of optical microscopy and what are their pros and cons? | brightfield microscopy: specimen is illuminated against bright background, used to observe stained biological samples. pros: simple, inexpensive, widespread use. cons: low contrast, limited resolution. dark field microscopy: specimen is illuminated from the side, used to observe live and stained specimens. pros: enhanced contrast, live specimen observation. cons: limited application, image interpretation. phase contrast microscopy: transparant and colorless objects can be observed. pros: no staining is required, live cell imaging. cons: complex set-up, limited to transparant specimens. confocal microscopy: a laser light is used to create a 3D image of the specimen. pros: optical sectioning, high resolution, 3D reconstruction. cons: cost and complexity, photobleaching and phototoxicity. |
| what are the two main types of electron microscopy and what are their pros and cons? | TEM (transmission electron microscopy): used to study ultra thin sections (< 100 nm) and can visualise the internal structures of cells. only samples with a limited thickness can be used and only 2D images can be generated. SEM (scanning electron microscopy): used to study the surface of solid, thick sections or objects. with this 3D images can be generated, but no internal structures can be visualised. sample preparation is also more complex. |
| what are the main differences between optical and electron microscopy? | optical microscopy creates images in color (depending on the staining), whereas electron microscopy can only generate black/white images. however, electron microscopy can generate much more detailed and magnified images with a better resolution. |
| what are the pros and cons of optical microscopy? | pros: affordable, easy to operate, portable, live samples can be observed, do not require radiation, minor maintenance (costs), preparation is quick and easy, natural color of the sample can be observed. cons: poor resolution at high magnification, low magnifying quality, cannot provide 3D images, staining is often needed, sample thickness is a limiting factor. |
| what are the pros and cons of electron microscopy? | pros: great resolution (up to 0.2nm), great magnification, TEM is able to show the insides of cells clearly, SEM allows surface structures to be seen clearly, high quality images can be generated. cons: expensive, large, affected by magnetic fields, preparation of material is extensive and requires expertise, preparation may distort the sample, only dead specimens can be visualised, only black/white images can be created. |
| what is the difference between specific and non-specific staining? | specific stainings are stainings that make use of (primary and secondary) antibodies coupled to fluorophores or enzymes. non-specific stainings are stainings that bind to all pieces of double stranded DNA and result in fluorescence. an example is SYBR green. |
| what are examples of bacterial, human and biological stains? | bacterial stains: gram staining, endospore staining, Ziehl-Neelsen staining. human stains: haematoxylin and eosin, papanicolaou (PAP), periodic acid Schiff (PAS), silver stain. biological stains: acridine orange, DAPI, Hoechst. |
| what is OCT and what are the pros and cons of using it? | OCT (optimal cutting temperature) compound is used to preserve the structure of a sample when it is frozen and during cryosectioning. OCT is water soluble and does not penetrate the tissue, meaning it can be washed away during sample preparation. pros: preparation is quick, sample shows little autofluorescence and is sensitive to immunohistochemistry, does not require fixed tissue, allows for longitudinal studies. cons: tissue degradation over time, requires rapid processing, loss of cellular viability. |
| what is paraffin embedding and what are the pros and cons of it? | paraffin embedding is done after the tissue is fixed (in formalin) and permeabilized. pros: better preservation of tissue architecture, long-term storage, easily obtainable for research, compatible with IHC, cost-effective, sections are physically stable. cons: preparation time takes about 2 days, shows a lot of autofluorescence, degradation of DNA and RNA, long processing time. |
| what is multiplex immunofluorescent imaging? | in multiplex immunofluorescent imaging, up to 16 different markers can be used to bind to and visualise different molecules in a single section. this can be done if markers with different excitation and emission wavelengths are chosen. |
| what are problems that can occur in (multiplex) immunofluorescent imaging and how can they be solved? | autofluorescence; by placing the tissue in a dark, UV-lit box, which removes the autofluorescence. spectral overlap (the overlap between the emissions of the different fluorophores); this is solved by using multispectral microscopy, in which you stepwise scan the visible spectrum. this generates separate pixels for each fluorophore per scan. these pixels can be separated and you can determine from which fluorophore the pixel comes from. |