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Presentation

What’s up with EM-CCDs II?
EM-CCDs and Fluorescence microscopy.
To answer the question of the hour
What do microscopists really want?
Sensitivity!!
...but what kind?
presented by Jim Pawley, [email protected]
2nd International EMCCD Symposium
1
/55
This kind?
or this kind?
2 /55
What everyone needs to know about photodetectors
• Some of the electrons in the sensitive volume of
the photodetector are capable of absorbing one
photon and using its energy to produce an excited
(photo)electron.
e-
•
e•
e•
e•
e•
sensitive
volume
of detector
• So measuring light intensity comes down to simply
measuring these photoelectrons.
The problem is that you can’t quite do this...
3 /55
Two problems:
Problem 1.
Only a fraction of photons produce photoelectrons.
(This fraction is called the Quantum Efficiency or QE)
QE varies
w/wavelength
Signal lost to
low QE must
be “replaced”
by making
more signal:
i.e., more
excitation
4 /55
Problem 2.
The few-electron signal is very weak and must be
amplified. All amplifiers add “read” noise to the
signal.
e•
e•
e•
e•
e•
sensitive
volume
of detector
electronic
noise
FET
amplifier
amplified signal +
electronic noise
5 /55
Everyone wants it but no one
knows what it is.
What is “sensitivity” anyway?
It must be a mixture of QE
and read noise...
6 /55
Let’s settle for:
• High-QE,
• Low Noise,
• Fast readout!
Surely, the EMCCD has provided all this...
What do microscopists really want NOW?
7 /55
Menu...
Old problems
• Interline transfer would solve Moiré problems.
• Software: compatibility! Build in deconvolution/filtering.
• How much cooling for @ 106 FPS read out?
• Know/control the EM gain, store data as electrons.
• Need a way to measure CCD performance.
New applications
• Applications for linear EM-CCDs
• “Proportional color” CCDs?
• Can an EM-CCD replace the PMT in the LSCM?
But first, a bit of microscopy...
8 /55
9
OBEY
THE RI
RULES!
Use a
water
lens!
OBEY NYQUIST
>4 PIXELS/BLOB!
7µm pixels better
than 16µm pixels. OB
TH
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Of course, “proper
SA correction” will
not solve all your
optical problems:
Cells are still lumpy
and will scatter and
distort the nice
laser beam so it
doesn’t go where it
should.
nucleus
Backscattered light image
of a cheek cell. 488 nm.
Nuclear “Shadow” distorts
image of smooth glass
surface.
10 /55
This leaves diffraction and statistics...
The problem of live-cell microscopy:
• How to get a good, 3D image of
a living cell without killing it.
• More photons make better images
but also cause more damage.
In confocal fluorescence imaging, “too
few photons” is usually a more severe
limit on resolution than diffraction!
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11
This is really true...
12 /55
13
Signal
is
lost
at
every
stage.
Signal is lost at every stage
.
detector
Input:
1015 photons/mW or
~109 phot./pixel/mW
(assume 512x512 in 1 sec)
Detected output:
107 photons/mW or
~100 phot./pixel/mW
(assume 512x512 in 1
sec)
The Specimen interacts with the incoming light in some way that produces contrast (i.e.,
some pixels brighter than others): commonly epi-fluorescence.
Though it depends on specimen stain level, the in-focus fluorescence signal is commonly
~10 6 times less intense than the excitation.
Because of geometrical and reflection losses, and the low effective QE of the PMT, only
about 1% of in-focus, signal photons are detected.
/55
The weak-signal problem:
• The rate at which signal is produced is always
proportional to the intensity of the input light.
• Although the input light level can be made
almost arbitrarily high, the fluorescent dye
response saturates at about 1 mW in a halfmicrometer spot.
• Even 1 mW causes considerable damage.
Much lower excitations are preferable.
14 /55
15
All methods of 3D microscopy are not equal...
High power
density not
good for dyes
or cells.
Damage/excitation:
2-photon
> 1-beam confocal
> disk/line confocal
> widefield (?)
Need 2D detector
Peak power
x
Widefield
Illuminates entire field (1 million
points!) at one time
5
10 x
Confocal
Illuminates only one point at a time
(Ok, really about 12 points)
10
10 x
2-photon
Illuminates about 12 points, and it
is turned off 99.999% of the time)
/55
Disk-scanners are fast and spread the light load.
Single-beam Confocal
Multi-beam Confocal
PMT
CCD
Using many beams, each one can have less photon/sec,
without decreasing the data rate.
BUT, until recently, they lacked a photodetector that
operated as well as a PMT in the 0-10 photon range. 16
/55
ENTER: The EMCCD!
When φ1 goes to ground, the high positive voltage on φ2 pulls the charge
packet past the “DC” electrode, through the high-field region, where charge
amplification may* occur
*Only ~1% of single-PEs multiply to become 2.
17 /55
First, the bad news:
Multiplicative noise in the EM amplifier doubles
“Poisson Noise”: effectively halving QE.
FOM EM-CCDs, March 2005
18
19
Then, the good news:
Intrinsic QE is pretty high!!
/55
What does EM-CCD offer?
• Unique ability to produce
useable images with signals in
the 0 - 50 photon range.
• Rapid (10 - 35 MHz) readout
with no increase in read noise.
20 /55
21
We understand EM-CCDs
Simulated/actual results for early E2V EM-CCD camera: 0.8 and 10.4 electrons/pixel.
They have no read noise but the multiplicative noise
effectively cuts the measured QE in half.
/55
And it works pretty well!
Ixon 512x512, EM-CCD
Intensified-CCD
“Normal” dye loading, No wave
propagation.
Wave propagates!
7x less dye, EM-CCD
Images kindly provided by Mark Hollywood,
Queens University, Belfast
22/55
Because the EM-CCD measures zero so well, it can
detect 4 grey levels at the bottom of the signal
range that are lost in noise with a conventional CCD.
and it does this @35MHz!
output,
photons/pixel
Fortunately, this is just
where the disk-scanning
confocal microscope operates!
However, nothing is perfect!
input, photons/pixel
If the lowest signal is >50 photons and speed isn’t
important, then the conventional, cooled, slow-scan
CCD wins because its effective QE is 2x higher.
23
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Perkin Elmer/Yokogawa disk-scanning confocal:
Micro-lenses and
lasers provide enough
light for fluorescence.
20,000 micro-lenses
cover surface of upper
disk, concentrates laser
excitation into pinholes
fiber from
laser: 2λ
• Each lens of the micro-lens array is
located so as to focus the light striking it
onto a specific pinhole in the lower disk.
collimating
lens
axis of
double
disk
CCD
camera
• The lenses and pinholes are laid out in a
constant-pitch helical pattern such that
every part of the illuminated area is
scanned equally about 360x per second.
• The micro-lens pattern repeats 12
times/turn. Twelve hundred 50µm pinholes
cover 3%of the disk surface.
disk spacing
= fmicro-lens = 1 cm
1:1phototransfer
lens
doubledichroic
beamsplitter
cube
laser illuminates
7 x 10 mm area in
intermediate plane
• As the disk rotates, the CCD
accumulates data from an entire optical
section.
Overall performance depends
strongly on that of the CCD camera.
objective
pinhole pattern on disk
Pinhole disk
located in
intermediate
image plane.
Band of pinholes
in lower disk,
50µm diam. on
250 µm centers,
focus plane
24
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So what is the problem?
Son of Moiré lives!
Frame transfer effects:
Get the orientation right!
EMCCD Rotation
A better solution would be an interline transfer EM-CCD,
with microlenses!!
25
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Old problems
• Interline transfer would solve Moiré problems
• Software, software software compatibility!
/55
But to make this clever
camera work on an diskscanning confocal,
?
it needs to talk to the software!
Integrating the camera drivers into the
microscope software has been slow.
27/55
Microscopy is degenerate!
• The accuracy of the
measurement of the number
photons recorded in each of
these 64 voxels is limited by
√n or Poisson Noise.
• The resulting √n S/N
ratio will be greatly
improved if a way can be
found to average all these
photons together.
Deconvolution is the way!
28/55
These results generously provided by Erik Manders
Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam
What else should software do?
Deconvolve everything!
Projected, raw, 3D confocal data
Same data, deconvolved.
The cells moving from late anaphase to interphase. DNA stained with GFP makes cells extremely
sensitive for oxidative stress since 1) the GFP is very close to the DNA and 2) the cells have to pass
the very sensitive checkpoints around mitosis. Standard Zeiss LSM510, 488 Argon,10 mW output, AOTF @
0.2 %, 488 beamsplitter and 505 LP emission filter. Power measured at specimen, 150nW.
29/55
Cooling is good!
Dark “noise”
and temp:
-30 0C
-50 0C
...so, cool it!
-80 0C
7 /55
30
Lowest temperatures = lowest noise
Lowest temperatures need UH vacuum.
Permanent
Hermetic
seal
Vacuum
Window
(anti-reflection coated)
TE Cooler
CCD
• Less heat conduction.
• Window stays warmer: no condensation.
• Only one glass window needed.
But higher cost and complexity.
Do you need it at 1,000,000 FPS?
31 /55
Old problems
• Interline transfer would solve Moiré problems
• Software, software software compatibility!
32
/55
The EM gain is nice to get the signal
away from the noise BUT it makes it very
hard to know how big your signal is.
sense
sense
read
read
digitize
memory
digitize
memory
Or the level of statistical noise.
What we need is a system to...
33/55
monitor its gain on-the-fly, and divide
the digitized number by the appropriate
factor to make the number stored, equal
(approx.) to the number of electrons in
the original packet.
Display LUTs can be used to make
the image visible on the screen.
These LUTs might be linear or SqRt.
34/55
So we have a neat photodetector.
How reliable is it?
We need a method of measuring
photodetector performance that is:
• Foolproof
• Reproducible
• Intuitive
• Informative
I have a suggestion...
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36
Measuring the Intensity Spread Function (ISF) of a CCD
2. Extract the values for a
particular pixel from each
image.
1. Obtain ~100 CCD images,
identical but for noise.
.
3. Plot histogram from set of
intensities obtained from these
pixels (This plot shows data from
~ 600images).
What could
be simpler?
# trials
100
4. For no-light,
halfwidth at half
max. is approx.
equal to RMS
noise, when
expressed in
electrons.
50
0
0
8
16
photons detected = ne
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37
OTHER VARIABLES
Stray light that varies during data collection will skew the ISF results
(as it will also skew “real” results).
Protect the camera from stray light by working in a dark room and placing
shields to prevent stray light entering via the objective or the oculars.
Light source instability over time will produce “fixed-pattern” noise that
will confuse ISF calculations. Ensure that the transmission source is
powered by a regulated DC source. Stray signal from fluorescent room
lights can be a particular problem on inverrted scopes. Turn them off!!
The software can be programmed to warn of, and even partially correct
for, “fixed-pattern” noise in the time domain.
/55
What does EM-CCD offer?
• Unique ability to produce
useable images with signals in
the 0 - 50 photon range.
• Rapid (10 - 50 MHz) readout
with no increase in read noise,
with no increase in read noise,
with no increase in read noise!
38 /55
New applications
39
/55
EMCCD sensors so far…..
E2V – ‘L3’
CCD65 – 576x288, 20x30µ pixels
FI only
TI – ‘Impactron’
Now:
FI and BI
TC285 – 1Kx1K, 8x8µ pixels
FI only - virtual phase
FI and BI
Here soon: 128x32,
10x10µm pixels, 35MHz
40 /55
41
Proposed Gain-register Linear CCDiode
• The 270µm x 15 mm sensitive area would be divided into 9 x 512 pixels, with the linear signal
light centered on row 3 (center).
• At the end of each 52 µs line scan period, charge is rapidly transferred from the sensor to the read
register.
• This signal is then read out @ 70 nm via the Horizontal and Gain registers to the Read amplifier
and the Digitizer.
Read
amp
512 pixel, 4-phase gain register
3-phase horizontal
register
Storage
register
Sense
register
Array length, 512, 30 µm pixels or about 15 mm
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42
To mimic the effect of changing the slit width, the digitized signal is
then “decoded” into 3 data streams, representing signal from the
center, the adjoining and the outside rows of the sensor.
Signal decoder:
small slit signal
- separates signals
from pixels representing
different “slit widths.”
medium slit signal
clock
512 pixel, 4-phase gain register
Read
amp
large slit signal
Digitizer
/55
The new Zeiss
“Live-5,” linescanner almost
cries out for a
512 x 1 linear
EM-CCD!
Actually, 2
of them!!
1
2
43/55
TX309
32
pixels
128 pixels
Frame transfer
 Virtual Phase QE ~ 65% max
10 µm2 pixels
35 MHz pixel readout (possible extension to 50 or 60 MHz)
 0.16 µs/row parallel transfer
44/55
TX309
(Texas Instruments)
Dark Events ~ 1 electron every 70 pixels.
–70 C
Exposure 10ms
EM Gain ~ x1000
45/55
ent
mes 46
ety
s,
Spectral detectors
Fluorescent
light comes in
a variety of
colors,
reflection
losses?
prism
pinhole
collimating
lens
PMT 4
PMT 3
and these colors carry
useful information
PMT 1
Leica SP
spectral detector system:
Truly ornate
detector
PMT 2
getting away from interference filters, method 1.
systems have been
designed to make this
• When pinhole is small,
collimation is excellent and spectral accuracy superb.
possible!
• When pinhole is larger, or specimen thick/heavily stained,
collimation is less good and spectral accuracy decreases.
/55
47
Linear
photodetector
arrays are
already in use.
diffraction
grating
pinhole
collimating
lens
higher
order
diffraction
losses?
• The signal from each PMT can
be stored individually or added
to that from others.
• Up to 8 separate grouped or
individual signals can be stored.
• Spectral precision slightly
reduced by large pinhole size
or thick stain.
Zeiss META spectral detector:
getting away from interference filters,
method 2.
32 PMT array
could be replaced
by a 32x128 EMCCD, binned 32x4
photocathodes
glass
envelope
“dead zone”
en-face view of PMT array
but, as binning wastes time, 1x32 is preferred.
/55
48
Is there an easier way?
If the eye can discriminate a million colors with
only 3 sensors,
Green channel
why not try it
in fluorescence
microscopy!
All you need
is the filter
arrangement
used in 3-chip
color CCDs.
Blue channel
Red channel
Channel 1
Channel 2
Channel 3
Signal wavelength is coded as proportional signals in 3 (or more) channels
/55
49
Dichroic 1
Dichroic 2
Light from
microscope
laser-line filter,
to remove stray
reflected light.
Proportional
color coding
Detector 2
Detector 1
to digitizer
100% transmission
Dichroic 1
Wavelength of light
Dichroic 2
/55
New applications
50/55
51
3 things PMT specs don’t say
Effective QEQE
1. Not all PEs are amplified equally.
Poisson statistics affects the gain of the multiplication
process, as it applies to each PE. Even a very high first
dynode gain of 25x, will produce a muliplicative noise of
about 20%*.
1/1.4
to less
= 0.71
than 70%
2. Lost signal
PMT QE curves describe the fraction of photons striking the
photocathode that produce photoelectrons (PE), About 30%
of these either miss dynode 1, or they produce no SE.
0.7
3. Optimistic “statistics”: The published curves tend to
describe the very best tubes produced. Actual QE of tube in
your scope, probably ~30% lower.
0.7
The Bottom Line: QE = (0.7 x 0.7 x 0.7)QE
= ~34% of the quoted QE !
(effective)
(published)
/55
52
200
µm
Proposed Gain-register CCDiode
• The 200 x 200 µm sensitive area would be divided into
25 pixels, with the signal light centered on the center.
• At the end of each 2 µs pixel period, charge is transferred
from the sensor to the read register.
•
This
signal
is then transferred via the Horizontal and
5x5
Gain registers to the readout amplifier.
sensor
•
The
digitized
signal is then “decoded” into 3 data
register
streams, each representing one of the concentric
areas of the sensor.
small pinhole signal
medium pinhole signal
read
register
large pinhole signal
signal decoder:
- separates
signals from the
different “rings”
digitizer
read
amplifier
horizontal register
gain register, 512 to 768 stages
gain = 1kx to 2kx
/55
53 /55
What if we made a Dedicated Electronic Pinhole EMCCD…
Theoretical frame rate calculation of
a dedicated 5x5 interline EMCCD sensor…..
@ 50 MHz pixel readout
~ 1 µs to shift under interline mask
0.16 µs/row parallel shift
~ 400,000 frames/sec
5 pixels
~ 2.5 µs/frame
5 pixels
54/55
Step LED brightness (6.86%)
1x1 ROI
LED on
~1,500 x 20µs
exposures
from one
pixel over ~
one second
Step LED brightness (0.27%)
When the signal level
drops below one
photon/measurement,
the Poisson Noise is
pretty high.
LED on
1x1 ROI
LED on
55/55
39.5%
6x6 pixels,
binned
29.3%
22.2%
17.6%
14.1%
11.7%
Step LED brightness (97-90)
39.5%
9.9%
8.2%
6x6 ROI
29.3%
Step LED
brightness (97-90)
6x6 ROI
Step LED brightness
(39.5 to 8.2%)
1x1 ROI
22.2%
17.6%
14.1%
11.7%
9.9%
8.2%
Single pixel signal
56/55
Comparison to CCD97
LED 1.9%
(E2V)
1x1 pixel ROI
1000
1100
Expanded
1050
1000
57/55
TX309 for rapid spectral imaging
1 ms exposure time – ROI plot from one spectral peak
Switch source on and off during 10,000 frame kinetic series
 Very weak signal, but still easy distinction from instrument detection limit
58/55
Acknowledgements
Andor Technology
Queen’s University Belfast
Colin Coates
Donal Denvir
Mark Hollywood
University of Vienna
Markus Aspelmeyer
US Genomics
Sandia National Labs
Mike Sinclair
Ray Meyer
59/55

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