...
Glossary
...
of
...
Light
...
Microscopy
...
Terms
...
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Binning
Binning is a process used to increase the sensitivity of a CCD camera by combining the charge from adjacent pixels to boost the signal-to-noise
...
ratio
...
in
...
the
...
image.
...
If
...
we
...
imagine
...
a
...
hypothetical
...
camera
...
(see
...
below)
...
with
...
a
...
chip
...
consisting
...
of
...
4x4
...
pixels
...
and
...
we
...
apply
...
a
...
binning
...
factor
...
of
...
2x
...
then
...
2x2
...
arrays
...
of
...
adjacent
...
pixels
...
will
...
be
...
combined
...
together
...
to
...
form
...
an
...
array
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of
...
'super
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pixels'
...
that
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are
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four
...
times
...
the
...
area
...
of
...
the
...
originals
...
and
...
contain
...
the
...
combined
...
charges
...
of
...
the
...
adjacent
...
pixels.
...
This
...
improves
...
the
...
signal-to-noise
...
ratio
...
because
...
CCD
...
camera
...
readout
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noise
...
often
...
obscures
...
low
...
signals.
...
If
...
the
...
readout
...
noise
...
of
...
the
...
hypothetical
...
camera
...
below
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is
...
25
...
grey
...
levels
...
then
...
most
...
of
...
the
...
signals
...
in
...
the
...
image
...
on
...
the
...
left
...
will
...
be
...
indistinguishable
...
from
...
noise.
...
By
...
combining
...
the
...
charges
...
from
...
adjacent
...
pixels
...
the
...
readout
...
noise
...
threshold
...
is
...
easily
...
exceeded
...
in
...
most
...
of
...
the
...
pixels
...
in
...
the
...
image
...
on
...
the
...
right.
...
Image acquisition is also faster for binned images because the readout time is shorter and the exposure time is usually shorter too. The drawback of using binning is that the resolution is lower because the specimen is now being sampled by fewer pixels. For example, a specimen imaged with the hypothetical camera above in 2x binning mode would be sampled by four large pixels instead of 16 small ones. The images below broadly illustrate the effect of increasing binning factors on resolution. At high binning factors pronounced pixelation is seen in the images when all are magnified to the same dimensions.
What does binning mean? (Andor Technologies)
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Long Working Distance (LWD) objective lenses
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{float:right}
!collar1.jpg|border=1!
{float}
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Most
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biological
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objective
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lenses
...
are
...
designed
...
to
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focus
...
on
...
specimens
...
mounted
...
under
...
a
...
standard
...
#1.5
...
coverslip,
...
which
...
is
...
about
...
170
...
µm
...
thick.
...
Most
...
of
...
these
...
objectives
...
cannot
...
focus
...
through
...
the
...
plastic
...
in
...
the
...
bottom
...
of
...
a
...
standard
...
microtitre
...
plate
...
because
...
their
...
working
...
distances
...
are
...
only
...
a
...
few
...
hundred
...
µm
...
and
...
the
...
bottom
...
of
...
the
...
plate
...
is
...
in
...
the
...
order
...
of
...
1
...
mm
...
thick.
...
Long
...
working
...
distance
...
objectives
...
are
...
designed
...
to
...
focus
...
on
...
specimens
...
relatively
...
far
...
away
...
from
...
the
...
front
...
lens
...
and
...
can
...
cope
...
with
...
the
...
thickness
...
of
...
a
...
microtitre
...
plate.
...
Many
...
LWD
...
objectives
...
have
...
correction
...
collars
...
that
...
can
...
be
...
adjusted
...
for
...
a
...
variety
...
of
...
specimen
...
thicknesses.
...
If
...
a
...
collar
...
has
...
been
...
adjusted
...
for
...
a
...
1
...
mm
...
thickness
...
(e.g.
...
the
...
bottom
...
of
...
a
...
plastic
...
dish)
...
it
...
will
...
not
...
focus
...
correctly
...
through
...
a
...
glass
...
coverslip
...
and
...
you
...
will
...
need
...
to
...
adjust
...
the
...
collar
...
to
...
a
...
setting
...
appropriate
...
to
...
a
...
170
...
micron
...
thickness.
...
There
...
is
...
no
...
systematic
...
way
...
of
...
adjusting
...
correction
...
collars.
...
The
...
best
...
way
...
is
...
to
...
set
...
the
...
collar
...
to
...
approximately
...
the
...
correct
...
position
...
before
...
observing
...
your
...
sample,
...
then
...
to
...
focus
...
on
...
the
...
sample,
...
and
...
finally
...
to
...
make
...
fine
...
adjustments
...
to
...
the
...
collar
...
position
...
if
...
necessary.
...
The
...
adjacent
...
picture
...
shows
...
the
...
correction
...
collar
...
of
...
a
...
Leica
...
objective
...
lens
...
when
...
adjusted
...
for
...
a
...
1
...
mm
...
sample
...
thickness.
...
The
...
black
...
dot
...
marks
...
the
...
position
...
of
...
the
...
collar
...
and
...
the
...
numbers
...
around
...
the
...
objective
...
barrel
...
indicate
...
the
...
thickness
...
for
...
which
...
the
...
lens
...
will
...
be
...
adjusted.
...
The
...
knurled
...
ring
...
above
...
the
...
dot
...
should
...
be
...
used
...
to
...
rotate
...
the
...
collar.
...
Unfortunately
...
the
...
Leica
...
objectives
...
don't
...
have
...
a
...
mark
...
for
...
the
...
position
...
for
...
a
...
170
...
micron
...
coverslip,
...
so
...
the
...
correct
...
position
...
can
...
only
...
be
...
set
...
approximately.
...
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Numerical Aperture (NA)
...
The
...
numerical
...
aperture
...
is
...
a
...
dimensionless
...
value
...
that
...
characterises
...
the
...
resolution
...
of
...
a
...
lens.
...
It
...
is
...
defined
...
by
...
the
...
following
...
equation:
...
NA
...
=
...
n
...
sinθ
...
Where
...
n
...
is
...
the
...
refractive
...
index
...
of
...
the
...
immersion
...
medium
...
and
...
θ
...
is
...
the
...
half-angle
...
of
...
acceptance
...
of
...
the
...
lens.
...
The
...
higher
...
the
...
NA
...
the
...
higher
...
the
...
resolution
...
of
...
the
...
lens.
...
The
...
NA
...
cannot
...
exceed
...
the
...
refractive
...
index
...
of
...
the
...
immersion
...
medium,
...
so
...
lenses
...
for
...
use
...
in
...
standard
...
immersion
...
oils
...
(
...
n
...
≈
...
1.51)
...
tend
...
to
...
have
...
a
...
maximum
...
NA
...
of
...
1.4
...
to
...
1.45
...
Links:
...
...
...
...
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Working Distance (WD)
The working distance is the distance from the front of the lens to the nearest surface of the coverslip when the specimen is in sharp focus. For objective lenses designed for use without a coverslip the surface of the culture vessel or specimen in used.
Links: