CaFluorescenceVascul ar smooth muscle

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Information about CaFluorescenceVascul ar smooth muscle

Published on January 11, 2008

Author: Michelino


Slide1:  Electrophysiological techniques applied to vascular smooth muscle Norman Scholfield Queens University Basic Medical Sciences (Physiology) Belfast UK Slide2:  What does the user need to know? Situations applicable to technique How to use the equipment Functional principals of the equipment Limitations of technique Artefacts created by the technique Analysing results Slide3:  So many cell proteins depend on Ca Cells have many pathways to regulate Ca Easy to measure Why Ca? Slide4:  Chemical assay of total Ca– not very helpful Ion selective electrodes – ionised Ca but disrupts cell Fluorescent compounds Quin Fura -2 dual excitation Indo dual emission Calcium green, red Oregon Greens Fluo 3, and fluo 4 Differ in Kd, excitation-emission wavelengths pH, Na, K, Mg, Cl, heavy metals Some progress on organics Ca measurements Green fluorescent protein:  Green fluorescent protein What is fluorescence ? Green fluorescent protein and many other colours:  Green fluorescent protein and many other colours Marc Zimmer Slide7:  Uses a fluorophore whose excitation/emission changes with free Ca concn (ionised) Assesses Ca in whole cell not Ca imaging Imaging requires massive amount of data analysis Ca microfluorimetry Slide8:  What is fluorescence ? Shifting energy state of atomic electrons in their shells Ground State Higher Energy electronic shell Internal Energy Loss Photon Emitted (longer low energy) Sample absorbs high energy photon (short wavelength) Intermediate State Slide9:  Why use fluorescence? Light easily applied to preparation Light easily measured One fluorophore molecule can repeat many excitation- emission cycles before bleaching -provides a high signal to noise ratio -high cycling rate gives good time resolution Fluorophores are generally small molecules/groups In microscopy, can determine spatial origin of photon Slide10:  Fura 2 Slide11:  Fura 2 FITC Filter Set Ex Filter Dichroic Em Filter Fluorescence Excitation by a shorter wavelength Causing emission of a long wave length Excitation and emission spectra overlap Therefore need mirrors and filters to separate Dichroic mirror Emission filter Excitation filter Slide12:  Fura 2 Filter band width determines magnitude of signal amount of energy passing through filter But a trade off between interference from excitation and signal size Slide13:  Fura 2 FITC Filter Set Ex Filter Dichroic Em Filter Fluorescence Excitation by a shorter wavelength Causing emission of a long wave length Excitation and emission spectra overlap Therefore need mirrors and filters to separate Slide14:  Fura 2 Main change is in excitation spectrum Fura 2 Emission @ 510 nm Ca-free Saturating Ca Excitation wavelength 380 nm 340 nm Slide15:  Fura 2 Main change is in excitation spectrum Fura 2 Slide16:  Fura 2 FITC Filter Set Ex Filter Dichroic Em Filter Fura needs two excitation sources Switching between the two Dichroic mirror Emission filter Excitation filters Slide17:  A m NA=n(sin m) Light cone (n=refractive index) Limit for smallest resolvable distance d between 2 points is (Rayleigh criterion): Slide18:  Resolution and NA 10x NA 0.25 100x NA 1.45 But no there is none Looks like there is co-localisation (yellow parts) Slide19:  Fura 2 Source emits in all directions Microscope objective Microscope objective with high numerical aperture (NA) Best objectives only collect 1/16th of total light Slide21:  Refractive errors in objective lenses Spherical aberration Microfluorimetry – abberations less important than confocal Slide22:  Objective 100x oil fluorescent objective cost 200,000 Baht If damaged, cost 50,000 Baht and lost for 3 months Use only the paddle to apply immersion oil Replace paddle in bottle immediately Cap bottle Clean off excess oil at end of day but NOT from glass cover microscope Cleaning optics Slide23:  Place a drop of suitable solvent on surface Wear surgical gloves (powder free) Distilled water to remove salts Pure absolute ethanol, normally, for greases Use a air canister (air duster) to blow off solvent. Repeat as required. NEVER use any tissue without first a preliminary wash Only use tissue to remove difficult deposits Special lens tissue only Circular movements beginning in middle and work to edge Isopropanol, xylene or chloroform for more difficult dirt Take care because a solvent may dissolve the epoxy glue used to fix the lens in position NEVER use acids, alkalis, bleaches, oxidising agents, etc Multiple compound lenses Cleaning materials may seep in between lenses Difficult to remove Ethanol will evaporate eventually but must be water-free Cleaning optics Slide24:  Shallow well ~80x10 mm Shape and length ensures flow in one direction and streamlined depth of solution 1-2 mm (deep solutions not streamlined flow) Glass bottom and preparation view from under side Thickness of glass bottom ~1mm for patching (glass slide) ~0.12 mm for fluorescence (long glass cover slips) easily and often broken by objective (better if glass breaks!) Some baths have an over-flow avoids flooding microscope Recording bath Slide25:  Repairing bath Bottom of bath of microscope coverslip Easily broken Use a razor blade to cut through and remove old glass and silicon rubber adhesive. Slide26:  Using a single edged razor blade, cut through silicon rubber adhesive Don’t cut yourself on glass Dispose in glass waste container With the razor vertical scrape away remaining silicon rubber from the bottom flat surface from the inner vertical wall of the bath Wash with soap/water Wipe and allow to thoroughly dry Fill a 1 ml disposable syringe with low viscosity silicon rubber (RTV) Done by removing plunger Open end of barrel to opened tube of Si rubber Force the Si rubber into syringe barrel Replace plunger and expel air. Thoroughly clean away all Si rubber from luer end Push on 19 gauge needle with tip broken off leaving ~6 mm. When used, can store in fridge until needed again Replacing glass bottoms Slide27:  Repairing bath Scrape away residual silicon rubber leaving a clean flat surface Slide28:  Repairing bath Fill a 1 ml syringe with clear low viscosity RTV silicon rubber with the plunger removed Replace plunger Break off the end of a 21 gauge needle leaving ~5mm of the metal needle and a clean round opening. Use pliers/hemostats to make a clean break. Place on syringe and remove air. A small bead of Si rubber should be extruded (if Si rubber on needle hub then will not stay in place) Slide29:  Squeeze out an continuous thin bead of Si rubber ~1 mm of the inner edge of bath Seat the glass slips squarely onto the bottom of the bath verify the correct position before dropping the glass on the bath use forceps to press down the glass so that the Si rubber flows to edge of bath do not allow the glass to move sideways Normally coverslips are too short Have one slip in the middle Cut another in 2 Si rubber also needed for the overlapping joints Place each half over the ends of the bath Once in contact with the Si rubber, the glass cannot be moved. Cure at room temperature over night or at 60oC for 1 hr Replacing glass bottoms, cont’d Slide30:  Normal solution providing bulk flow through bath Under gravity or can be pumped Prewarmed by heat exchanger to 37oC before entering bath Nylon tube passes all the way through exchanger Thus water bath electrically isolated from recording bath Sealed at each end with silicon rubber sleeve Very short flexible connection to bath Plumbing – bulk flow Water at ~40oC from water bath Return to water bath Short flexible connection to recording bath Inflow from main reservoir Slide31:  Plumbing – bulk flow Slide32:  Pump for inflow Plumbing – bulk flow Slide33:  Pump to draw off waste -bigger tubing than inflow pump Plumbing – bulk flow Slide34:  Flood control Slide35:  Centred on multiway manifold with n inputs and one outflow The outflow is angled so that drug solution is prewarmed by bath solution Each input is connected to a tap and reservoir (10 ml syringe barrel) via 0.5 mm ID polythene tubing connectors at end are 0.56 mm OD Plumbing – drug delivery Slide36:  Centred on multiway manifold with n inputs and one outflow Each way converges at a single point is 0.3 mm diameter easily blocked keep solutions clean relieve blocks by attaching 0.5 mm ID tube and syringe to outflow and back flush immediately remove back-flushed contaminated solution from reservoir Cleaning End of each day: 2 changes of distilled water in reservoirs Allow to run out through manifold If any line contains air, needs flushing Morning Run through 50% house-hold THIN bleach NEVER, NEVER EVER use ethanol or any organic solvent cracks plastic – becomes useless Plumbing – drug delivery Slide37:  Filling Add main solution to one of the barrels/reservoirs with all the taps open, force solution through Most solution will run back up the other lines to respective reservoirs Air also expelled. Turn off all taps draw off solutions pull out the single plunger (and rubber plunger seal) fill each reservoir and add drugs etc as required. Mix solutions Run through 0.5-1ml to flush out dead space Any air bubbles will prevent flow One of the channels must contain normal solution and be turned on when no other solution is running Drug delivery - filling Slide38:  The second leg of outflow tube should be horizontal The outflow orifice is angled slightly downwards is positioned so that it almost touches bottom of bath under microscope can see contact because outflow tube slides forward along bath bottom also with thin glass, the bath bottom will press down slightly does not normally break glass Position outflow upstream ~200 um from specimen ~half microscope field with 10x objective Drug delivery - positioning Slide39:  On turning on first channel, debris can be seen “blowing” away Check this is in the direction of flow This cleaning effect can be increased by placing finger on top of reservoir and pressing down (increases pressure) On switching solutions, turn old off before turning on new channel. Time for new solution to arrive at cell/vessel ~1 sec Any residual solution from previous channel is marginalised in the streamlined flow Drug delivery - positioning Slide40:  Flow through bath Warmed solution Flowing in Drug delivery manifold Outflow tube bevelled to maintain constant fluid level in bath Inflow orifice must be below water line Keep away from edge of bath because edge meniscus will suck bath dry Slide41:  Flow through bath Glass bottom must be clean put thin layer of detergent in bath every day before starting any beading of solution will create areas with no solution particularly so if inflow ceases - solution continues to be drawn off until meniscus breaks Flow through bath must be a constant rate This maintains a constant temperature important because thermal expansion bows glass bottom down effect small with thick glass in patching bath Slide42:  Air table comprises heavy stand supports a heavy rigid slab via 4 diaphragm pistons compressed air ~2 bar self levelling valves Damps out vibrations through floor Requires little maintenance Verify that no part of the table touches fixed objects, pipes, wires and other services and are flexible Air table Slide43:  Excitation switching Fura needs two excitation sources Switching between the two Here we use a monochromator - not filters diffraction gratings >1200 lines per cm wavelength determined by angle of grating Band-width determines by the angle range controlled by slits in the entry and exit beams The galvanometer mirror directs white light to different angles on grating mirror angle controlled by electronics Slide44:  Excitation switching Monochromator provides flexability But lower throughput Filters waste less light Slide45:  Equipment Xenon lamp Dual monochromator & beam switcher Lamp power supply Specimen Microscope objective Fibre optic Photomultiplier 510 nm filter 670nm long pass filter Video camera VDU Slide46:  Electronics and software Lamp – manual Xenon arc lamp with output extending into ultraviolet High voltage to ignite Useful life of bulb ~1000 hrs cost ~12000 Baht from PTI Become noisy, difficult to ignite and can explode Extending life Do not turn on needlessly Do not switch on and off frequently – turn off if not to be used for >2hr Slide47:  Electronics and software Installing new bulb Cairn instructions Needs re-aligning Slide48:  Electronics and software Monochromator Having lamp on will also cause monochromator to deteriorate Micrometers to control slits manually only Read Cairn web site Do not run program needlessly - causes oscillating mirror to run Optical cable (light guide) Liquid filled Limited life span Do not damage or bend tightly Slide49:  Electronics and software Optoscan Sets up monochromator wavelengths Can have: Single excitation wavelength Wavelength scanning where the monochromator switches over a range in preset steps Can be used to determine the actual excitation spectrum to set up optimum values Switching at variable duty cycles between two wavelengths multiple wavelengths Also specify a “deadtime” during cycle to account for switching delays Monochromator program linked to signal output Slide50:  Electronics and software Photomultiplier and power supply Detects single photons and converts to electrical pulses Normally runs at 400-1000 volts voltage determines gain do not exceed 1200 volts do not switch on high voltage with bright lights Slide51:  Electronics and software Output amplifier Use this to adjust gain High (H) and low (L) ranges Normally use in integration mode (INT) If red on, then signal gain too high or out of range Slide52:  Window adjustment Adjust so that only region of interest viewed and measured Reduces background from areas with no cell Window can be: sized rotated positioned Trial and error learning Slide53:  Camera Use red light to view cells while recording 510 nm emission filter to detector blocks red light Slide54:  Electronics and software Programming Ignore all these Done via the computer (called optoscan) Sometimes the serial link refuses to communicate Try again or ignoring sometimes helps If communication, then the optoscan screens changes Slide55:  Using fura 2 Derivative of EGTA – 4 carboxylic acid groups attached to fluorophore groups - chelates Ca like EGTA - high concentrations reduced free Ca Highly charged molecule -therefore cannot penetrate membranes Use the ester form (fura 2/AM) -membrane permeant -cytosolic esterases release free acid (usually) Usage Stock in anhydrous DMSO (hygroscopic – takes up water) Store DMSO over molecular sieve or over dry nitrogen Water hydrolyses ester Make up 1-2 mM in DMSO Aliquot out as 5-10 ul – enough for one experiment Store under nitrogen or dry air at -20oC 3000 baht /mg (8000 baht Sigma) Slide56:  Using fura 2 Whole tissues need higher concns for loading than isolated cells Avoid high DMSO concentrations (<0.2%) Experiment with different loading times normally 20-100 min some detergent may help (pluronic) overloading reduces free cytosolic Ca need time for esterases to work (not good at 5oC) Slide57:  Calculating [Ca] [Ca] = Kd x b x (R – Rmin)/(Rmax + R) Have to make some assumptions Kd = Ca-fura 2 dissociation constant = 120nM in water = 224 nM in cytosol?? b = viscosity term (=1??) R = F340/F380 Rmin = R in Ca free (value depends on equipment, filters, etc) Rmax = R saturating Ca (value depends on equipment, filters, etc) In practice Rmin and Rmax difficult to attain Gives global Ca concn. Thus too many assumptions best to leave as ratio if [Ca]in changes are small Slide58:  Ca protocols Measurement of Ca levels sometimes unhelpful eg a Ca rise might be due to Ca influx, store release or lower extrusion Influx measured by equilibrating in Ca free adding Ca and measuring initial rate of Ca rise - not good if process is Ca activated Store release comparing effect with full stores and depleted stores using Ca free cyclopiazonic acid thapsigargin or FCCP for mitochondrial stores Use low Na or Na/Ca exchanger carboxyeosin or high affinity extruder Slide59:  Background Photon signal Cytosolic Ca changes Background Fura 2 inaccessable to Ca Tissue autofluorescence Contaminating excitation light Stray light Instrumentation noise Slide60:  Background subtraction Background Fura 2 inaccessable to Ca Tissue autofluorescence Contaminating excitation light Stray light Instrumentation noise Use manganese quenching Mn binds to Fura 2 but prevents fluorescing Mn enters cell through some Ca channels Does not washout do at end of experiment Counts after equilibration is background for each channel Slide61:  Noise Random fluctuations in signal Sources of noise Lamp (both channels affected same) Fluorescence signal noise proportional to 1/(intensity)2 4x signal, 2x better signal to noise ratio Signal/noise ratio More Fura Brighter illumination Longer integration times (amplifier) Longer sampling times Reducing background Slide62:  No signal or cannot see cell fluorescing Lamp no on – green led on power supply Monochromator Slits closed Can you hear it switching (software or Optoscan?) Light from exit – pull out fibre optic from microscope - do NOT look directly into fibre –danger UV flashing feint blue light Is dichroic mirror in light path Slider on right side in middle position Move this and the slight blue haze should disappear Should see a blue spot from objective Slide63:  No signal Nothing on VDU Window at side port closed Bottom front right of microscope Camera power Photomultiplier high voltage not on. Is amplifier red led on? Signal too large and out of range Trace at top of screen Cell not loaded with Fura Move cell out of field of view small drop in signal suggests autofluorescence only Slide64:  Rmin Ratio in zero Ca (after background subtraction) Run Ca free through bath Never completely Ca free Use EGTA Ca buffering May cause fura 2 to leak out of cell Sequestered Ca in ER, etc may take a long time to remove Slide65:  Rmax Ratio in saturating Ca (after background subtraction) Cell has powerful Ca extrusion and binding mechanism to reduce cytosolic Ca Use Ca ionophore (channel molecule) Ionomycin Gramicidine Amphotericin? Saponin (fura 2 leaks out) Slide66:  Artefacts Background Effect of other ions pH K Mg, etc. Fura binding to proteins Assume homogenous cytosol - compartmentalisation - local transients non-linear Buffering effect of fura 2 Fura 2 bleaching No longer fluorescent Greater with high excitation energies Seen as gradual loss of counts Loss stops without illumination Fura leakage continues Slide67:  Isolating cells Normally rely on various proteolytic (usually) enzymes to separate cells Basal lamina Basement membrane Connective tissues Collagenase Proteases Trypsin (or inhibitor) Papain Elastase Hyaluronidase, etc Also some protective agents Taurine, dithiothritol, etc Some work with no real logic – cookery Problems Surface receptors, ion channels lost Cytoskeletal protein attachment to extracellular matrix cells round up Ca intolerance Slide68:  Intact tissues More physiological and stable Difficult to add drugs Responses slow Fura loading difficult Long light path Slide69:  Software Computer runs windows 98 (2nd edition) Limited virus protection No network connection Limited storage (20 GB) but data files small Needs drivers for memory sticks Slide70:  Software Optoscan.exe Programming excitation wavelength settings and switching Often running Optoscan shows a communication error Try switching off/on the terminal or ignoring sometimes helps Or maybe that optoscan already running The communication settings should be correct Com1, 9600 baud If communication, then the optoscan screens changes and message indicating PC control Slide71:  Software - optoscan Optoscan.exe Two types of program Wavelength program – set up one or multiple fixed wavelengths Scanning program – program change wavelength Can be reprogrammed with new settings Tedious but carefully following on screen instructions Save the settings as file xxxxx.hsf Use name which means something to others Previous settings reloaded - fura settings is “fura2.hsf” The last used configuration file is usually loaded next time the “Acquisition Engine” application is loaded Slide72:  Software - optoscan 1 cycle 320 ms 340 nm 250 ms 380 nm 1 ms – no recording 320 ms 340 nm 1 ms – no recording Slide73:  Software – Acquisition engine Acquistion engine 1.1.7 Function Initialise the Optoscan Digitise data Display data Save data Slide74:  Software – Acquisition engine Running the application: Asks for a “user” If new user, then you will need to create new settings But can use existing setting (existing user “CNS7”) OK Blank panel + menu line Slide75:  Software – Acquisition engine Slide76:  Software- acquisition engine Configuring modules Do not change channels Slide77:  Software- acquisition engine Configuring modules Analysis editor on saved files only Slide78:  Software- acquisition engine Configuring modules This sets the screen refresh rate only Slide79:  Software- acquisition engine Configuring modules This sets the screen refresh rate only Slide80:  Software- acquisition engine Configuring modules Because optoscan already running Slide81:  Software- acquisition engine To record, new data file needed Press “Create” Slide82:  Software- acquisition engine To record, new data file needed Press “Create” Slide84:  Software- acquisition engine To record, new data file needed Right click to add a protocol marker Slide85:  Software- acquisition engine To record, new data file needed Can be repositioned and edited later Slide86:  Software- acquisition engine To record, new data file needed Right click to add a protocol marker Slide87:  Software- acquisition engine Recording can be paused Move dichroic mirror out of light path to prevent continued illumination Photodamage and fura 2 bleaching Slide88:  Software- acquisition engine To record, new data file needed Right click to add a protocol marker Slide89:  Software- acquisition engine To record, new data file needed Right click to add a protocol marker Slide90:  Software- acquisition engine To record, new data file needed Right click to add a protocol marker Slide91:  Software- acquisition engine To record, new data file needed Right click to add a protocol marker Slide92:  Software Labcalculator.exe My outdated program to calculate dilutions and concentrations You need the later XP version or XP operating system Slide93:  Solutions Hanks solution convenient HEPES buffer Cells need HCO3 to extrude H+ Therefore need CO2 equilibration

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