Molecular Biology Flashcards
Microscopy: explain dispersion
- The phase velocity of a wave depends on its frequency.
- This causes focused white light going into a lens to disperse into different colors (because wavelengths of light vary by the color) when exiting the lens.
Microscopy: explain chromatic aberration
- White light focused through a lens disperses into the different wavelengths (colors) due to their different phase velocities.
- Chromatic aberration is the failure of the lens to focus all the colors to the same point.
- This can be offset using achromatic lenses.
Microscopy: explain diffraction
Interference of waves at obstacles results in contructive and destructive interference patterns due to the wave nature of light.
Microscopy: explain refraction
Refraction is a change of the angle of light (or other waves) when passing through a boundary between 2 mediums of a different refractive index.
What is the minimum distance resolvable by optical microscopy?
- λ/2NA (or λ/2nsinα, or about 1/2 the wavelength)
- where λ = wavelength of light, n = refractive index, α = opening angle of lens, NA = numerical aperture of lens
- ~ 200 nm (0.2 um) = diffraction barrier
Explain constructive and destructive interference.
- Constructive interference:
- If two coherent waves with phase shift 0 or 2π interfere, a wave with doubled amplitude results
- Destructive interference:
- If two coherent waves with phase shift π interfere, both waves are annihilated
(π = one part of the phase, like half the wavelength)
What is Fourier Analysis and Synthesis?
- Fourier Synthesis:
- Waveforms (signals) can be generated through superposition of different harmonic waves with wavelength λ and frequency ν and different amplitudes
- Fourier Analysis:
- Signals can be disassembled into harmonic waves with different amplitude to describe the signal (waveform) in the frequency domain
- This simplifies computation of waves
What does the Numerical Aperture (NA) of an objective describe?
- NA describes the relationship between the opening angle of that objective’s lens and the refractive index of the medium (air, water, oil) between the objective and the sample being imaged (or its coverslip)
- NA = nsinα, where n = refractive index, α = opening angle of lens (theta can also be used as the symbol instead of alpha)
What are phase objects?
Unstained objects that do not absorb light but slightly alter the phase of the light diffracted by the specimen, usually by retarding such light approximately 1/4 wavelength as compared to the undeviated direct light passing through or around the specimen which is unaffected.
The light is slowed by the specimen because of its thickness, refractive index, or both.
- Amplitude objects absorb light as it passes through them, resulting in differences in amplitude of the wave.
What is fluorescence?
Fluorophores that emit light at another wavelength after irradiation with light
Explain the basics of Time-Correlated Single-.Photon Counting (TCSPC).
A way to measure fluorescence lifetime
- Fluorescence is excited repeatedly by short laser pulses. When a laser pulse hits the sample, time is started. When fluorescence is emitted (and detected by a PMT), time is stopped.
- Time delay between excitation and emission is measured, and the resulting data is a histogram of times to emission, with each line representing one time bin, and the intensity of each line representing a count of how many events took that amount of time (the count of events at each given time t).
- This cycle is repeated many times to generate the histogram.To avoid pile-up (overlap between the cycles/artefacts), <5% of the cycles should result in fluorescence.
- Fluorescence lifetime Tau is then calculated by fitting the histogram to an exponential decay function: N(t) = e-t/T N number of counts as a function of t time = e to the -t/Tau
What is the difference between direct and indirect immunolabeling?
- Direct: using fluorescently labeled primary antibodies to bind directly to your sample of interest
- Indirect: using fluorescently labeled secondary antibodies - these bind to the primary antibody (labeled or unlabeled) bound to the sample of interest
What is photobleaching?
The destruction of a fluorophore/fluorescent protein through irradiation/exposure to light.
Irreversible loss of fluorescence of a fluorophore during irradiation.
What are chemical tags, give examples and explain their mode of action.
The interaction of genetically encoded tags with small molecules allows for fluorescent labeling of proteins in live cells
Covalent
- Substrate is conjugated to the fluorescent probe -> substrate-probe conjugate
- Tag (self-modifying protein/enzyme) is bound to the POI
- Enzyme tag irreversibly transfers the substrate alkyl group to one of its amino acid residues creating a covalent bond of the substrate-probe conjugate to the tag
- Result: The probe tags enzyme and its fusion protein irreversibly
- Example: SNAP-Tag
- Enzyme = mutant hAGT
- Substrate = modified benzylguanine, an O6-benzylguanine (BG) derivative
- Amino acid residue = cysteine
- Example: CLIP-Tag
- Enzyme = a different mutant hAGT
- substrate = benzylcytosine
- Amino acid residue = cysteine
- Example: Halo-Tag
- Enzyme = Mutant dehalogenase enzyme
- substrate = alkylhalide derivatives
- amino acid residue = asparagine, and mutated His289 prevents hydrolysis of this covalent bond
Non-covalent
- TMP-tag
- Tag = eDHFR (E. coli dihydrofolate reductase)
- Substrate = trimethoprim derivatives
- Trimethoprim binds to eDHFR with high affinity as a receptor-ligand pair
- TMP is conjugated to the fluorophore and then binds to eDHFR
What are Quantum Dots? Advantages and limitations of QDs.
- CdSe, for example. These are semi-conducting particles which exhibit stable fluorescence. Wavelength of absorption and emission depends on their size.
- Advantages – they do not photobleach, and they can be labeled with antibodies
- Disadvantages – large size of the QD can affect the molecule of interest
Describe confocal fluorescence microscopy.
Why does confocal fluorescence microscopy achieve improved axial sectioning?
- Confocal fluorescence microscopy uses a pinhole to exclude out of focus light from the image
- One is after the light source
- One is before the detector
- Only light from the plane in focus is imaged at one time, without the additional light scatter from the other planes. Then you only need to change the plane of focus axially in order to image the next portion of the sample in the z direction, acquiring a series of images which can be reconstructed.
- The size of the pinhole determines the thickness of the optical section
Describe two-photon microcopy.
Does two-photon microscopy achieve a higher spatial resolution?
- Longer wavelength light is used at high intensity to excite the fluorophores, so that 2 photons are hitting the fluorophore at the same time. The highest probability of this happening is at the laser focal point, so only fluorophores directly at in the focal point are absorbing 2 photons and getting excited. This eliminates any light coming from the rest of the sample/gives darker background. This reduces phototoxicity and improves light detection.
- No, it does not actually achieve higher spatial resolution because the wavelength being used is longer (spatial resolution = ~ ½ the wavelength), even though fluorescing volume is reduced.
Why does two-photon microscopy achieve a higher imaging depth?
- Because the longer wavelength excitation light can penetrate deeper into the sample due to minimized scattering, and the background signal is strongly suppressed.
Describe how a confocal fluorescence microscope works. Why do you get a better depth of field?
- Confocal fluorescence microscopy uses a pinhole to exclude out of focus light from the image
- One is after the light source
- One is before the detector
- Only light from the plane in focus is imaged at one time, without the additional light scatter from the other planes. Then you only need to change the plane of focus axially in order to image the next portion of the sample in the z direction, acquiring a series of images which can be reconstructed.
- The size of the pinhole determines the thickness of the optical section
Schematically draw the beam path (from the sample to the detector) of a confocal fluorescence microscope.
Explain why electron microscopy has a higher resolution than light microscopy.
What limits the resolution of electron microscopy?
- Because the wavelength of the electrons used in EM are so short, like around 0.004 nm. This is achieved by increasing the velocity of the electrons. The faster the velocity, the shorter the wavelength.
- Current resolutions are ~ 0.1 nm for EM
- Aberrations of optical components limits the resolution of EM
How does EM work?
- A heated cathode/tungsten wire produces electrons. These electrons are accelerated in a vacuum. Magnetic lenses focus and collect the electrons.
- In EM, you see electron density.
Explain FRET.
Which distances can be determined experimentally with FRET?
*
What different functional principles of ion channels do you know?
How to determine the activity of ion channels?
- xxx
- You can use the patch clamp technique, where single ion channels are pulled up into the tip of a micropipette. Voltages can be applied and voltages measured to determine when the ion channel is in the on or off state.
Explain the functional principle of multiphoton fluorescence microscopy.
Why do you achieve a higher depth of field?
- Longer wavelength light is used at high intensity to excite the fluorophores, so that 2 photons are hitting the fluorophore at the same time. The highest probability of this happening is at the laser focal point, so only fluorophores directly at in the focal point are absorbing 2 photons and getting excited. This eliminates any light coming from the rest of the sample/gives darker background. This reduces phototoxicity and improves light detection.
- Because the longer wavelength excitation light can penetrate deeper into the sample due to minimized scattering, and the background signal is strongly suppressed.
What are the 3 phases of transcription?
- initiation
- elongation
- termination
all done by RNA pol
What are the -10 and -35 boxes?
Conserved sequences which act as promotors upstream of a transcription start site, 10 bp upstream (often TATAAT) and 35 bp upstream (often TTGACA).
Promotor sequences lie on the untranscribed coding/sense/+ 5’ to 3’ strand.
Promotors are assymetric and provide a ‘direction’ so that the polymerase knows in which direction to transcribe / which strand is the codeing strand.
The template/antisense/- strand is the 3’ to 5’ strand and transcription (RNA synthesis) takes place 5’ to 3’ for the newly formed RNA molecule.
study the following transcription table for bacteria vs eukaryotes
compare and contrast
