exam 1 visualizing cells Flashcards
what are model eukaryotes (+ 4 features of them)
model organisms in which
- the genome has been sequenced
- site-directed and tissue specific mutagenesis is possible
- expression of multiple genes can be tracked across many cells simultaneously
- the developmental sequence is known
what is the main issue with light microscopy
resolution is limited
what is the issue with electron microscopy
it requires special preservation and staining techniques
what does light microscopy image
large-scale cellular structures
what, in light microscopy, provides contrast and sensitivity
chemical stains and fluorescent molecules
which form of microscopy has a higher resolution than light microscopy
electron microscopy
how does flow cytometry work
- uses fluorescent labels to measure levels of biomolecules and ions
- sorts cells into different tubes based on expression levels
what is Cell Theory and who proposed it
that all organisms are made of cells - Schleiden and Schwann
what is resolution
the ability to distinguish two objects that are close to each other
what is the smallest organelle that can be seen clearly by light microscopy
mitochondria
what is the issue with magnification ** on exam or final
we can magnify an image as much as we want, but it may be blurry (due to resolution)
what is detection ** on exam or final
the ability to see something (an item smaller than 0.2 um can be detected if it emits light
what is the limit of resolution for microscopy
the wavelength of the light and the size of the lens
- the bigger the lens, the bigger resolution you can get
why are cells hard to visualize
they are colorless and translucent
what do phase-contrast and differential-interference-contrast microscopy do and what is the result
they use the change in phase of light waves as they pass through a cell
- gets the visualization of live, un-manipulated cells, but have limited detail
what is the pro and con of staining with chemical dyes
pro - chemical dyes can reveal details of cells
con - requires fixation (kills and preserves cells)
how can tissue samples be visualized
they have to be cut into thin sections because they are so large
how do chemical dyes work
by absorbing specific wavelengths of light so the intensity of light goes down
- fluorescent molecules absorb light at one wavelength and emit light of a longer wavelength
what does fluorescence microscopy allow for
detection of very small numbers of fluorescent molecules since they are viewed against a dark background
how can fluorescent stains be introduced
- chemical dyes
- fluorescent molecules attached to antibodies
- intrinsically fluorescent proteins expressed by cell itself (GFP - green fluorescent protein)
how does fluorescent microscopy work
uses barrier filters and beam-splitting mirror to let in certain wavelengths, transmit light, then cut out unwanted fluorescent signals and be seen through eyepiece
how is fluorescence microscopy limited
by fluorescence coming from out-of-focus parts of cells, which reduces practical resolution
how does confocal microscopy work
uses a scanning laser and pinhole apertures to limit detection to the focal plane - creation of an optical section with much better resolution = no need to slice the cell
what type of image of a sample can be reconstructed using confocal microscopy
3D images - formed by taking images from various focal planes (“z sections”)
how does FRET work
fluorescence resonance energy transfer - uses 2 fluorescent proteins where the excitation energy of the 2nd matches the emission of the first. if 2 are very close together, exciting protein 1 will allow protein 2 to fluoresce
- can tell if the molecules are in contact with each other
how does 2-photon microscopy work
2 separate long-wave photons of light excite the fluorophore. this allows for deeper penetration into a sample without sectioning (“vital microscopy”)
what occurs with a long wavelength
the longer the wavelength, the further it can get into the sample before it’s absorbed
what occurs with a shorter wavelength
the shorter the wavelength, the higher the energy
how does electron microscopy work
a focused beam of electrons replaces light - more than 200x increase in resolution than light microscopy
why is a vacuum required in electron microscopy
so electrons don’t bounce. cells must be fixed and desiccated and then sliced into ultra-thin sections
when do you have to use light microscopy specifically
when using live samples
how do scientists solve the issue of cells being mostly transparent to electrons
electron-dense materials are used to stain cells. can also use gold-tagged antibodies to mark specific proteins/structures
what colors do electron microscopy show up in
black and white
what is SEM (scanning electron microscopy)
3D - images the outside surface - uses staining but no sectioning
what is TEM (transmission electron microscopy)
2D - images internal structures - sliced ultra thin
what does an electron beam give more resolution than light
because the wavelength of an electron is much shorter than the wavelength of a photon
what is a simple definition of flow cytometry
single cell analysis - live cells are analyzed in real-time in an aqueous stream as they pass through a laser and a computer collects fluorescence data and laser scatter
