SingleCellMovies

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Unformatted text preview: Bi1x:
Making
movies
of
dividing
cells
 
 As
a
way
to
complement
the
bulk
measurements
you
did
last
week
we’ll
be
measuring
cell
division
at
 the
single
cell
level
under
the
microscope
today.
The
idea
is
to
not
only
compute
the
cell
doubling
time
 based
on
the
movies
you’ll
obtain.
Today’s
experiment
is
also
an
introduction
to
the
kinds
of
 measurements
that
can
be
done
on
single
cells
and
to
the
full
use
of
an
automated
microscope.
 
 
 Note:
There
is
nomenclature
here
involving
petri
dishes,
microscope
slides
and
microscopy
dishes.
Make
 sure
you
know
which
one
is
which
before
starting
the
protocol!
If
in
doubt,
ask
a
TA!
 
 Preparing
the
agar
pads:
 
 1. Stretch
a
piece
of
parafilm
on
a
glass
surface
(or
the
bench).
Place
a
24x50mm
or
24x60mm
 coverslip
on
the
parafilm.
 2. Pipette
2‐3ml
of
LB
with
1.5%
low
melting
temperature
agarose.
 3. Drop
another
coverslip
on
top
in
order
to
“sandwich”
the
agarose.
This
step
might
be
easier
to
 do
with
your
gloves
off.
 4. Let
it
dry
for
30‐60
minutes.
Note
that
in
the
interest
of
time
we’ll
be
giving
you
some
premade
 pads.
 
 Setting
up
the
pads:
 
 1. Cut
two
squares
3‐4mm
squares
of
agarose
and
put
them
on
slide.
Cover
the
pads
while
they’re
 drying
(without
touching
them!).
The
idea
is
to
reduce
pad
drying
before
you
put
in
any
cells.
 We’re
making
two
pads
in
case
something
goes
wrong
with
one!
 2. Spot
2ul
of
cells
on
each
one
of
the
pads.
Remember
that
you
shouldn’t
touch
the
pad
with
the
 pipette
tip!
 3. Close
the
petri
dish
again
and
let
dry
for
a
couple
of
minutes.
Depending
on
how
dry
the
pad
is
 we
might
put
it
at
37C
to
speed
up
the
drying.
 4. Place
the
pads
on
a
microscopy
dish.
This
will
be
either
one
of
the
Wilco
dishes
(the
big
ones)
or
 the
Matek
dishes
(the
smaller
ones).
 5. Place
the
dish
on
the
stage
corresponding
to
your
scope
leaving
the
lid
open.
Make
sure
that
the
 environmental
chambers
are
properly
closed.
The
idea
behind
this
step
is
to
let
the
pad
 equilibrate
with
the
temperature
of
the
scope.
If
you
close
the
lid
and
see
condensation
forming
 then
open
it
again
and
wait
some
more.
 6. After
around
15
minutes
seal
the
dish
using
parafilm.
Make
sure
the
parafilm
doesn’t
interfere
 with
the
placement
of
the
dish
on
the
scope.
This
step
is
meant
to
reduce
evaporation
from
the
 pad
which
would
result
in
drying.
 7. Put
a
drop
of
oil
on
the
objective
and
place
the
dish
on
the
corresponding
holder.
 
 Setting
up
the
movie:
 
 1. Load
“Micro‐Manager
1.3.39”.
NOTE:
It’s
very
important
to
load
this
version
of
Micro‐Manager.
 If
not
your
movie
might
not
work.
 2. Go
to
the
“Multi‐D
Acquisition”
window
and
open
the
XY
list
(next
to
the
“Use
XY
list”
option).
 3. Find
the
cells
and
move
around
the
pad.
Find
5‐10
different
positions
where
you
see
interesting
 things.
For
example,
you
might
want
to
include
some
areas
with
only
one
or
two
cells
in
the
 middle
and
some
other
areas
with
a
lot
more
cells.
Mark
the
positions
on
the
XY
list.
 4. Set
up
the
channels
to
be
used.
You
should
use
the
brightfield
setting
without
binning.
Make
 sure
that
you
have
a
reasonable
exposure!
 5. Set
up
the
autofocus
by
selecting
“Autofocus”
and
open
its
option
dialogue.
Let’s
choose
the
 following
options:
 1st
number
of
steps:
6
 1st
step
size:
1
 2nd
number
of
steps:
6
 2nd
step
size:
0.3
 Threshold:
1
 Crop
ratio:
0.25
 Channel:
Brightfield
 What do these options mean? Check out the Micro‐Manager manual you were given at the beginning of the course. Explain how the search for the optimal focus will go.
 Note
for
the
Nikon
scopes:
The
Nikon
scope
does
not
need
any
software
autofocus.
You’ll
have
 to
set
up
the
Perfect
Focus.
Also,
make
sure
that
the
“Hardware
Autofocus”
options
“Switch
off
 for
XY
move”
and
“Switch
off
for
Z
move”
are
selected.
Ask
you
TA
how
to
work
with
Perfect
 Focus.
 6. Choose
“Save
files
to
acquisition
directory”
and
select
a
folder
where
you
want
to
save
your
 images.
Also,
choose
“Single
Window”
in
the
“Display”
option.
 7. Before
you
start
taking
your
movie
let’s
make
sure
that
everything
is
working.
Let’s
take
only
 one
frame.
If
one
of
the
frames
fails
to
focus
go
back
to
the
position
and
make
sure
it
hasn’t
 drifted
out
of
the
range
of
the
autofocus
search.
If
you’re
having
issues
with
this
ask
you
TA!
 8. Now
you’re
ready
to
take
your
movie.
Think
carefully
about
how
often
you
want
to
take
frames.
 What’s the maximum time resolution you could get? What’s the limiting factor (the bottleneck) in the acquisition at each position?
You’ll
probably
want
to
take
a
frame
every
5
minutes.
 
 
 
 Assignment:
 
 1. Answer
the
questions
in
the
protocol
marked
in
italics
related
to
the
autofocus
and
the
time
 resolution.
 2. Show
the
initial
and
final
frames
of
all
the
positions
you
took
data
from.
Did
all
cells
divide?
 3. Show
a
couple
of
frames
of
one
or
two
of
your
favorite
movies.
You
might
want
to
look
into
the
 Matlab
command
montage
in
order
to
plot
many
frames
right
next
to
each
other.
 4. Estimate
the
cell
doubling
time
by
looking
at
your
movies.
An
easy
and
fast
way
to
look
at
the
 movies
is
with
ImageJ
by
using
the
command
“File

Import

Image
Sequence”.
 5. Make
sure
to
explain
your
reasoning
carefully
by
showing
a
couple
of
division
events
with
their
 respective
time
stamps.
Time
stamps
are
easy
to
add
using
“Plugins

Stacks

Time
Stamper”.
 Also
add
a
scale
bar
by
doing
“Analyze

Tools

Scale
Bar”.
How
do
you
decide
that
a
cell
has
 divided
already?
Do
all
cells
divide
at
the
same
rate?
Do
you
get
the
same
division
rate
in
all
 positions
on
the
pad?

 6. Look
at
the
movies
corresponding
to
one
or
two
positions
and
manually
track
the
different
cell
 division
times.
Draw
a
histogram
of
the
division
times
you
found
and
find
the
mean
division
time
 and
the
standard
deviation.
How
does
the
division
time
compare
to
the
bulk
results
you
got
last
 week?
Is
there
are
any
difference,
why
do
you
think
this
could
be?
 ...
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This document was uploaded on 01/03/2012.

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