what are the 5 control blocks on the instrument front panel?
1) voltage electrode (Vm)
2) bath electrodes (Im)
3) clamp
4) commands
5) current electrode
what’s on the instrument rear panel?
1) BNC connector for GAIN TELEGRAPH INPUT
2) BNC connector for Ve X 10 output
3) an alarm on/off switch
4) binding posts for circuit ground and chassis ground
voltage meter
reports the membrane voltage of the cell, range of +/- 199.9mV
voltage probe connector
7-pin DIN connector for attachment of the voltage probe to the instrument
Vm offset control
10-turn potentiometer providing up to +/- 200mV at the voltage probe input for offset of membrane junction potentials
Vm electrode test control
used to determine the internal resistance of the voltage probe (10mV/M-ohm of probe resistance)
Vm BUZZ push button
facilitates penetration of the voltage electrode by producing a 1kHz square wave at the pipette tip
Vm x10 output BNC
reports the membrane voltage in mV multiplied by 10
current meter
reads the voltage (Ve) of the current electrode when the clamp mode selector switch is off
current meter (clamp mode selector switch set to slow or fast)
current meter displays the current sensed by the bath electrode
gain select controls allows range of gain to be set from:
0.01 to 100
gain selector switch = 0.1 to 10, gain multiplier = 0.1, 1, 10x
I monitor BNC
current outputs at full bandwidth (10 kHz)
I monitor filtered BNC
current outputs filtered at 1 kHz by an integral 4-pole Bessel filter
clamp mode selector: off
the clamp amplifier is disconnected from the current electrode, voltage difference between the current electrode and the bath electrode is read on the meter in the bath electrodes block
clamp mode selector: slow
- useful for screening oocytes or when high clamp speeds are not required
- clamp speed = 0.5ms
- measured currents are displayed on the bath electrodes meter in uA
clamp mode selector: fast
- used for most oocyte clamping
- clamp speed is limited by the resistance of the current electrode and the oocyte membrane capacitance
- fast clamp speed = 350us
gain control
a single turn potentiometer which varies the full bandwidth open-loop gain from 0 to 2000
Ve offset
range of +/- 200mV, used to adjust the offset voltage of the current electrode
Ve x10 output BNC
monitors the voltage of the current electrode (x10) when the clamp mode selector switch is off
gain telegraph output BNC
provides a DC voltage indicating the gain setting of the instrument, output varies from 0.2 to 2.6 volts in 200mV steps
model cell
a simple circuit that mimics the properties of recording from an actual oocyte
follicular layer
connective tissue that is rich in collagen
collagenase
an enzyme that will break the peptide bonds of collagen, thus releasing individual oocytes
denuded oocytes
individualized oocytes
animal pole of oocytes
dark pigmentation, contains the nucleus
vegetal pole of oocytes
lighter pigmentation, pale yellow colour
what defines stage VI oocytes?
light-coloured band separating the animal and vegetal poles
what does it mean if oocytes are covered with blood vessels?
the oocytes are undergoing resorption
stage I oocytes
oocytes are clear and transparent (50-300um)
stage II oocytes
oocytes are white and opaque (300-450um)
stage III oocytes
oocytes are lightly pigmented all over (450-600um)
stage IV oocytes
oocytes are starting to have yolk protein deposited into their cytoplasm and the pigment is primarily concentrated to the upper animal hemisphere (600-1000um)
stage V oocyte
oocytes are accumulating yolk and have a darker pigmented colour (1000-1100um)
stage VI oocyte
oocytes are fully grown and have progesterone receptors on their plasma membrane (1100-1300um)
