Labs Flashcards
(8 cards)
Microscopy types and components
Widefield: simplest type of fluorescence, whole field view illuminated, microscope focused on single plane but light reaching other planes-> out-of-focus fluorescence, which can obscure image. Easy and cheap.
Confocal: emit light through pinhole, only in-focus fluorescence imaged, out-of-focus light forms disc instead of point at pinhole so doesn’t reach detector.
Excitation filter- selects wavelength to excite fluorophore
Emission filter: selects emission wavelength of fluorophore
Resolution= 0.61lambda (wavelength)/NA (numerical aperture of objective)
EM: more fragile samples mounted on finer mesh, but this results in greater area being overed by grid bars.
Compromise in image acquisition and microscopy
Sample brightness: too bright saturates detector, too weak invisible over background
Dynamic range: (over which values can be measured). 8-bit camera can distinguish 28 different brightness levels, etc. limited by specs of detector and software. Can maximise info on brightness by acquiring raw image over maximum part of dynamic range (controlling exposure/ incident light brightness).
Signal to noise (S/N) ratio: improve (get more photos per pixel per frame) by: using brighter fluorophores, choosing objective with good magnification compromise (magnification dilutes photons) and numerical aperture (admits more photons), brighter excitation light+ longer exposure (more photons per exposure time), larger pixels (to capture more photons), widening pinhole in confocal (let in more photons). Method depends on priorities: sensitivity, spatial resolution, temporal resolution, minimising photodamage, etc
Spatial resolution: depends on magnification, objective numerical aperture, pixel size. If too fine, fewer photons per pixel.
Fluorophore bleaching/ cell photodamage by free radicals: if illumination too bright
Gain/amplification: increase emission by turning gain/amp up on detector (also increases noise)
Binning pixels: merge signal from squares of pixels- doesn’t increase noise, increases signal, decreases res.
Exposure time on camera= brighter signal and better S/N but slower
Numerical aperture: intensity varies with NA2, fluorescence brightness with NA4. Limited by objective availability. High NA requires shorter working distance- tricky with live/ large samples.
Magnification: light varies with 1/Mag2, but more detail may be visible.
Fluorescence microscopy- cameras usually monochrome for greatest sensitivity
Tetrad analysis in yeast
Tetrad analysis in yeast
Wild isolates of cerevisiae usually “homothallic”- haploid cells alternate mating types in mating type switching on budding. Lab strains can be manipulated to be heterothallic- don’t switch.
Ascus walls digested by lyticase/zymolase.
Crossing transformed yeast to parent mutant strain shows that Ura+ phenotype segregates 2Ura+:2Yra-, indicating transforming DNA integrated into genome, undergoing duplication and segregation as part of chromosome using yeast integrative plasmid (YIp)- maintenance over generations depends on integration by recombination.
#single crossovers= TT+ 6 NPD.
Yeast plasmids
Yeast replicating plasmids (YRp): seq for yeast origin of replication (ARS) inserted into YIp, resulting vector can replicate autonomously. Reach high copy number without copy # ctrl system, but transformed phenotype unstable, some transformants lose plasmid every generation even under selection as PRp exhibit bias for segregation to mother
Yeast centromeric plasmids: add yeast centromeric seq (CEN) to YRp-> copy# ctrl, segregation by association w/ mitotic spindle. Centromeres small, well-defined. E.g., plasmid pRS416 (pRS406 derivatie) behaves like small circular chromosome, 1-2 copies per cell, segregates reliably between mother+ daughter.
Yeast Episomal plasmids: multicopy, derive from bacterial vector. Carry yeast selectable marker+ fragment of 2-micron circle plasmid including ARS, copy# ctrl elements-> stable maintenance under selective conditions, ~30 copies/cell. Useful for manipulating gene dosage.
YACs: possible to clone structural components into yeast chromosomes-> construct linear YAC w/ 2 telomeres, centromere, ARS, selectable marker. Stable, acts as small accessory chromosome, tolerates longer inserts, useful for large mammalian DNA (libraries).
Petite mutations and yeast spindles/mitosis
3 types of petite mutations: nuclear (-> mendellian inheritance), neutral (defective mt supressed by wt, no phenotype), suppressive (defective mt-> phenotype)
Yeast spindles/mitosis
Yeast has closed mitosis. SPB (MTOC)= 3-layered structure embedded in nuclear envelope w/ nuclear and cytoplasmic faces for MT nucleation. Spindle orientation req Myo2 (V type myosin). Latrunculin causes spindle misalignment.
Kar9 protein= bridge between astral MTs and Myo2p. Binds astral MTs via MT end-binding protein Bim 1p.
Translocation of spindle across bud neck powered by yeast cytoplasmic dynein- provides pulling force for spindle translocation across bud neck anchored to cell cortex via Num1.
Bioinformatic tools
Bioinformatic tool for comparison, IDing DNA-binding seqs, predict RNA binding sites, etc- Geneious
Used in labs for PPR ID/seq.
Arrays/PPR repeats provide structured interfaces for RNA binding where each PPR binds 1nt in linear array
Immune techniques and golgi imaging
Immune techniques+ Golgi
Blocking solution blocks non-specific binding sites on samples, reduces noise
Lead citrate/ uranyl acetate= heavy metal stains to increase contrast for gold stains- bind diff biocomponents.
Plant Golgi smaller than animal, can have multiple per cell.
Immunogold has better resolution that immunofluorescence, but sample prep much more finnicky.
Nocodazole interferes with MT polymerisation. BFA inhibits Arf1. Both cause golgi proteins to be more diffuse as COPI an MT involved in Golgi function.
calcium indicators
Genetically encoded Calcium indicators- calcium sensitive dyes hard to load into whole organs/ organisms, can’t ctrl where dyes taken up. Ca sensitive proteins introduces as transgenes. GCaMP series widely used- modified GFP connected to CaM and M13 (from myosin light chain kinase, natural binding partner of CaM). As Ca increases, caM binds M13, changing protein conf, GFP fluoresces (non-ratiometric, like the Ca-sensitive dyes). Ratiometric sensors as pairs of GFP variants, commonly cyan+ yellow. Encoded ca indicators can be targeted within cells, e.g., versions targeted to synapses.
