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33 Cards in this Set
- Front
- Back
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free vs total intracellular calcium |
- free calcium ion + buffer = bound calcium - bound calcium ~90-99% of all Ca - measure the remaining free Ca in experiments |
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What do you need to measure Ca dye? |
- Calcium dye = fluorescent - Need: calcium indicator, excitation source, detection system (to measure light) |
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What are the two types of calcium indicators? |
1. bioluminescent indicators (photo proteins) 2. fluorescent indicators (fluorophores) |
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What are the two types of bioluminescent indicators and how are they derived? |
1. luminescent - emits from a chemical reaction 2. fluorescence - emits from another light source |
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What are properties of fluorescent indicators? |
- derived from Ca2+ buffers (EGTA) - BAPTA = faster than EGTA, less pH dependent; fluorescence is bound to BAPTA - light excites the molecule, which emits the electron to the excited state; as energy moves back down to the ground state, a longer wavelength (lower energy) is emitted (stoke's shift) |
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What is stoke's shift? |
- light excites the molecule, which emits the electron to the excited state; as energy moves back down to the ground state, a longer wavelength (lower energy) is emitted (stoke's shift) |
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How do you determine the excitation spectrum? |
- change the wavelength of excitation and measure the emitted light at a fixed wavelength |
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how do you determine the emission spectrum? |
- fix the excitation wavelength (440nm on slide) and measure the emission wavelength at various wavelengths |
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Single wavelength dyes |
Single Wavelength dyes: (e.g. fluoro-3) - Calcium concentration is related to the intensity of light emitted - |
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dual wavelength dyes and examples |
Dual Wavelength dyes: (e.g. fluoro-2)- excitation/emission peaks change with bound Ca 1. excitation peaks: measure at the wavelength with Ca binding; then measure at wavelength without Ca binding; ratio of F1/F2=[Ca2+] 2. emission peaks: measure two measurements simultaneously |
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Considerations for Dye Selection |
- high affinity dyes = good for low Kd's (low Ca levels); increase buffering capacity 10-20%, = large effect on transient Ca signaling - low affinity dyes = good for high Ca levels (e.g. ER, mitochondria, etc) - Kd depends on pH, temp, etc - UV light damages cells - autofluorescence (NADH) = background noise |
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Calibration of ratiometric Ca2+ indicator |
- R = fluorescence ratio = F1/F2 - Calibration of single wave Ca2+ indicator is not good for [Ca], but is okay to measure relative changes - in vitro calibration - Sb = intensity of bound Ca - in vivo/in situ calibration: lomomycin (gets rid of Ca in the cell); then load Ca into cell; allows you to calibrate for Ca min and max |
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Kd properties and definition |
- Kd = [Ca2+] at which 1/2 buffer molecules are bound - measured with a single wavelength - log of Kd = x-intercept of graph for calibration purposes |
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Photobleaching definition and effects on single and dual wavelength dyes |
- photobleaching: destruction of the fluorophore because of excitatory illumination - single wavelength: fluorophore with UV light = non-fluorescent species - dual wavelength: ratiometric dye (in theory this cancels out any bleaching effects), but in reality, background fluorescence ratio tends toward 1 as B increases OR ratio tends toward 0 as B increases (fluoresces at one wavelength) |
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Methods of Loading dye into the cell |
- chemical indicators are hydrophilic (they like charge and dislike membranes) Methods: - microinject - wash-in via patch pipette - modify dye into AM esters to add hydrophobicity to cross the cell membrane; esterase inside cell cleaves esters to return to Ca - genetically engineer Ca2+ indicators |
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fast versus slow voltage sensitive dyes |
fast probes = bad signal to noise ratio, but good time resolution; integrated into membrane slow probes = good sensitivity (1% per mV change), but very poor time resolution; distributed across membrane |
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Detection Methods of Fluorescence |
photomultiplier tube (before cameras): - converts light to current - very sensitive, good at low light levels CCD cameras (charge-coupled device): - each pixel absorbs photons and stores electrons - electrons move to positive readout - readout array/time is limiting factor - time resolution (speed) is critical - measured in frames per second (10-30 sec) |
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what can be done to minimize dark current noise from CCD cameras? |
keep it at very low temperatures |
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what comprises the acquisition time for CCD cameras? |
acquisition time = exposure time + read-out time + clearing time + shutter time increase acquisition rate by: reducing exposure time, adding gain to the camera |
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in-vivo calcium imaging options: confocal |
Confocal - blocks out-of-focus light - use PMT to measure focused light - scan point by point (3-30 FPS) - can build a 3-D image |
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in-vivo calcium imaging options: 2-photon |
2-Photon imaging - uses 1/2 of energy, but you need 2 photons simultaneously - light intensity must be increased by ~10^6 to excite two photons, so you can excite with red (2x) and emit green - fluorescence decreases with the 4th power of distance from the focal plane, so there is no bleaching, no out-of-focus light excitation, and minimal scatter - able to conduct deep penetration into tissue |
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Signaling changes in intracellular free Ca2+ concentration |
- sustained elevation (hypoxic-induced) - transient (ATP-induced) - oscillations (glucose-induced) - waves (work its way across the cell) |
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What is Calcium imbalance in a cell? |
- differences in rates of Ca entry and exit from the cytosol |
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Plasma membrane Ca2+ transporters |
- Ca travels inside to outside (usually) 1. Ca-ATPase = active transport; high affinity for Ca (so good at low [Ca]); works slowly (maintains resting [Ca]) 2. Na/Ca transporter via electrochemical diffusion = low affinity binding (for high [Ca]); very fast (good to return [Ca] to basal state) 3. SERCA pump (Sarco ER Calcium ase) = cytosol to ER (2x Ca2+ per ATP); lowers intracellular Ca |
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What does thapsigargin do? |
- inhibits SERCA pump - lowers [Ca] in ER stores |
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How can you tell if there is a Ca influx or intracellular store release of Ca? |
- do experiment with no Ca in extracellular media or with excess buffer to bind all Ca - if Ca signal disappears, Ca was from outside - if Ca signal remains, Ca was from intracellular store NOTE: it could also be co-induced Ca-release |
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What is the ryanodine receptor and what does it do? |
- ion channel on the intracellular store - ubiquitously expressed - 3 isoforms, all ~5000 AA subunits - Ca binds preferentially to open channels - low levels of Ca activate channels - high levels of Ca inhibit channels (>50 uM) |
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what is CICR (calcium induced calcium release)? |
- ryanodine receptors - calcium enters cell, activates ryanodine receptor on ER, causing Ca release from ER |
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what is voltage dependent calcium release? |
- depolarization of Vm causes ryanodine receptor to release Ca through calcium voltage-gated channel |
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what other things can activate ryanodine receptors and what is its main modulator? |
- caffeine activates ryanodine receptors - oxidative stress causes hyper-activity of ryanodine receptors - cytosolic Ca is the main modulator/regulator |
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what is the IP3 receptor? |
- 3 isoforms, ~2700 AA subunits - channel on the ER - IP3 = principle physiologic activator - releases Ca from ER when activated - NOT a CICR; however, Ca modulates the open probability in the presence of IP3 channel - antagonist = heparin - spontaneous Ca spikes/oscillations occur in IP3 - can inhibit upstream pathway through PLC inhibitors (phospholipase C) |
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What do store depletion signals do? |
- sends signals from the ER to the cell membrane to allow more Ca influx to refill the ER calcium stores |
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how do you estimate rmax? |
- measure max peak for lambda 1 (lots of Ca), measure max peak for lambda2 (lowest wavelength peak), |
