Slide1 : Electrophysiological techniques applied to vascular smooth muscle Norman Scholfield
Queens University
Basic Medical Sciences (Physiology)
Belfast
UK
http://www.qub.ac.uk/CMSRC
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