SCAS0106E UB

CELL SIZE
6.5
f. 02
PIXEL ARRAY
f. 03
2048 X 2048
EFFECTIVE AREA
13.312
IMAGES COURTESY OF:
PEAK QUANTUM EFFICIENCY_
f. 05
QE @ 560 nm 82%
QE @ 500 nm 77%
Robert Harvey,
INSTITUTO GULBENKIAN DE CIÊNCIA
UNIVERSITY OF NEVADA
SCHOOL OF MEDICINE
Qi Zhang,
MAXIMUM EXPOSURE TIME 10 s
A B S O L U T E M A X I M U M F R A M E R AT E
25,655
NAGOYA INSTITUTE OF TECHNOLOGY
Philipp Keller,
Kenji Kanai and Kenta Saito,
HHMI
f. 41
HOKKAIDO UNIVERSITY
Michael Davidson,
Joerg Bewersdorf,
FLORIDA STATE UNIVERSITY
FRAME
YALE UNIVERSITY
Bruce Gonzaga,
41
Wang Haibo,
MOLECULAR DEVICES
HUAZHONG AGRICULTURAL UNIVERSITY
Sato Honma,
Tian Tian,
HOKKAIDO UNIVERSITY
NANJING MEDICAL UNIVERSITY
Hamamatsu Photonics Deutschland GmbH
(49) 8152 375 0 EMAIL: [email protected]
Hamamatsu Photonics France S.A.R.L.
(33) 1 69 53 71 00 EMAIL: [email protected]
Hamamatsu Photonics Norden AB
(46) 8 509 031 00 EMAIL: [email protected]
Hamamatsu Photonics Italia S.R.L.
(39) 02 93581733 EMAIL: [email protected]
Hamamatsu Photonics UK Limited
(44) 1707 294888 EMAIL: [email protected]
Hamamatsu Photonics (CHINA) Co., Ltd.
(86) 10 6586 6006 EMAIL: [email protected]
Hamamatsu Photonics K.K., Systems Division
(81) 53 431 0124 EMAIL: [email protected]
hamamatsucameras.com
SCAS0106E02 Created in the U.S.A.
SPRING 2016
Hamamatsu Corporation, U.S.A.
(908) 231 0960 EMAIL: [email protected]
SCIENTIFIC CAMERAS
UTHSCSA
X2
2016 V2
Jun Hee Kim,
QE @ 750 nm 61%
MINIMUM EXPOSURE TIME 1 ms
Kazuaki Nagayama,
VANDERBILT UNIVERSITY
QE @ 670 nm 76%
f. 06
S PAC E
Emilio Gualda,
LT
TIME
SPRING 2016
SCIENTIFIC CAMERAS
When the history of this decade is
written, what will be the memorable
scientific achievements?
Usually it’s the big discoveries or culminating achievements that are celebrated. But
every day, every scientist takes small steps toward a potential breakthrough. In this
regard, scientific research is the embodiment of Kaizen, the Japanese philosophy of
continuous, incremental improvement rooted in empirical investigation and relying
on teamwork and best of practice methods. Within Hamamatsu engineering, Kaizen
V2
is never discussed, but it is always practiced. Since the launch of the ORCA-Flash4.0
sCMOS camera, we have stood side by side with researchers to learn what works and
2016
what can be done better. As of October 2015, every ORCA-Flash4.0 V2 has exactly
LT
X2
what you asked for...increased quantum efficiency. We understand that by capturing
more of the light emitted from you samples, you are now free to go a little faster, to
resolve those extremely dim structures, to achieve greater localization precision in
super-resolution experiments and to more efficiently image developing organisms with
light sheets. We believe a small change can have a big impact.
+
2016
+
2 016
+
+
+
FRAME
+
01
+
CELL SIZE
ORCA-Flash4.0 V2
D I S C O V E R
T H E
B R E A K T H R O U G H
From its introduction the Flash4.0 has challenged the status quo of imaging and undergone
a series of useful enhancements. The most recent is perhaps the most exciting; a notable
increase in the ability to detect photons. This enhanced QE means that you have a possibility
of detecting the faintest of signal or for brighter samples that ideal image quality is achieved
with shorter exposure times, perhaps saving your cells from phototoxicity or bleaching. With
the ORCA-Flash4.0 V2 already delivering wide field of view, large dynamic range, and fast
frame rates, this QE enhancement only makes it more versatile and powerful. If you have
not yet discovered the performance of the ORCA-Flash4.0 V2 sCMOS now is the time. What
biological breakthrough will you make with your extra photons?
FRAME RATE
100 FPS max @ full resolution
DYNAMIC RANGE
37,000:1
READ NOISE
1.4 electrons rms minimum
100
V2 (2016)
80
QUANTUM EFFICIENCY (%)
.015
13.312 X 13.312
0.5
2048 X 2048
6.5
PIXEL ARRAY
EFFECTIVE AREA
AIR COOLED
W AT E R C O O L E D
DARK CURRENT_
+
V2 (original)
60
40
20
0
400
500
600
700
W A V E L E N G T H ( nm )
800
900
1000
04
05
FRAME
01
CELL SIZE
E X P LO R E
M O R E
W I T H
C M O S
Ready to make the jump from a traditional CCD to the increased flexibility and sensitivity of a
scientific CMOS (sCMOS) camera? With established performance and an affordable price, the
ORCA-Flash4.0 LT fits into any experiment that needs simple connectivity, moderately fast frame
rates, and great sensitivity. The ORCA-Flash4.0 LT is designed to bring all the advantages of
sCMOS technology—wide field of view, low-light sensitivity, and large dynamic range—to every
research lab. Easy connectivity and powerful performance help you explore your pressing biological
questions. And when your green channel is screaming but your red is ho-hum horrible, the LT’s
“W-VIEW Mode” in combination with the W-View Gemini (p.12) ensures more balanced exposure
for reliably quantitative dual wavelength imaging. Think of all that LT can help you discover.
FRAME RATE
30 FPS max @ full resolution
DYNAMIC RANGE
33,000:1
READ NOISE
1.5 electrons rms minimum
80
QUANTUM EFFICIENCY (%)
.015
13.312 X 13.312
0.5
2048 X 2048
6.5
PIXEL ARRAY
EFFECTIVE AREA
AIR COOLED
W AT E R C O O L E D
DARK CURRENT_
ORCA-Flash4.0 LT
60
40
20
0
400
500
600
700
800
900
1000
W A V E L E N G T H ( nm )
06
07
ImagEM X2 512
ImagEM X2
T H E
L I M I T S
O F
SERIES
ImagEM X2 512
Frame Rate: 1076 FPS MAX
LO W
What do you do when your light levels are truly low? How low? As in the dimmest of dim,
just a few photons-of-signal-per-pixel dim with very little background. In these demanding
situations EM-CCDs still shine. Why? It’s all in the EM-CCD’s larger pixel size and specialized
architecture, which multiplies the weak input signal before it reaches the amplifier and the
unavoidable addition of readout noise. The ImagEM X2 (512 x 512 pixels) and the ImagEM
X2-1K (1024 x 1024 pixels), are ideal for applications like luminescence that require long
exposures, big pixels and/or ability to analog bin.
ImagEM X2-1K
Frame Rate: 314 FPS MAX
100
QUANTUM EFFICIENCY (%)
P U S H
ImagEM X2 -1K
75
50
25
0
300
400
500
600
700
800
900
1000
1100
W A V E L E N G T H ( nm )
CELL SIZE
6.5
08
09
2 016
1.3
2 016
The Living Image
C E L E B R AT I N G T H E L I T T L E T H I N G S I N L I F E
Science is both beautiful and awe-inspiring. The images that are created in
the process of scientific discovery astound us because of their significance
and because of the raw beauty of life at the microscopic level. Although
the images can and do stand on their own as art, there is a story behind
every image. That story starts with a question. And, in the search for
the solution, researchers encounter barriers at the bench that must be
overcome. Eventually, the quest for answers is achieved leading to even
greater possibilities.
The Living Image is an evolving collection of these Bench Stories from
some of the best researchers in the world, on topics from light-sheet of
whole embryos to implementing in vitro-like diagnostics in vivo. We invite
you to read their stories and hope you can experience the same thrill that
we do at seeing discovery in action. And through the library of articles and
interactive tools in the Resources section explaining camera technology,
we hope to make it easier to apply your camera as part of the solution. And,
by all means, contact us if you’d like to see your work as a Bench Story—
we’d love to help you share the story behind your work of art.
10
11
M I S H A
M I C H A E L
D R E W
SEEING THE
LIVING BRAIN
N.
B.
B.
A H R E N S
W I L L I A M
O R G E R
S T E F A N
R.
C.
L E M O N
P U L V E R
R O B S O N
B U R K H A R D
J E N N I F E R
M.
K A T I E
L I
P H I L I P P
H Ö C K E N D O R F
J.
K R I S T I N
K E L L E R
J E R E M Y
M C D O L E
B R A N S O N
F R E E M A N
P H I L I P P
J.
K E L L E R
01 THE QUESTION
02 THE BARRIERS
03 THE SOLUTION
04 THE POSSIBILITIES
How does the brain work?
Light-sheet technology
Redesigning the imaging strategy
Next generation light-sheet microscopy
Scientists have been searching for answers to this question for decades. What’s been
To acquire these groundbreaking images of the brain, Ahrens and Keller had to extend
Advancing light-sheet microscopy technology required a redesign of the existing imaging
The promise of light-sheet is real-time imaging of neuronal ensembles, allowing scientists
missing is an actual image of the complete picture—the ability to directly view neurons
the capabilities of existing light-sheet technology to speed up volumetric acquisition
strategy. The team optimized the hardware components and configuration to streamline
to probe functional patterns associated with sensory, motor and homeostatic behaviors.
firing in a whole, living brain in real time. This view is exactly what the Keller Lab
time. Fast frame rates are important for imaging moving, living systems, whether you
communications throughout the microscope control system.
First demonstrated in 2013, the Keller lab radically advances the possibilities in 2015 with
delivers as they push the limits of light-sheet microscopy.
are looking at an entire brain or watching microtubules dynamics.
a multi-view hs-SiMView. In both systems, the speed of the ORCA-Flash4.0 V2 is essential.
T E C H N O LO GY B A R R I E R S
ILLUMINATION OBJECTIVE 2
DETECTION OBJECTIVE 1
SAMPLE
Above: Ahrens et al. use wide field of view at single cell resolution,
and imaging every 1.3 seconds to capture projections of whole-brain,
neuron-level functional activity (reported by the genetically encoded
calcium indicator GCaMP5G in an elavl3:GCaMP5G fish via changes in
fluorescence intensity (ΔF/F), superimposed on the reference anatomy).
Above: The single view
hs-SimView set-up.
DE
TE
LIGHT SHEET 1 AND 2
(405-1,080 nm)
CT
IO
N
SAMPLE
HOLDER
DE
TE
ILLUMINATION
OBJECTIVE 2
O
N
I
AT
IN
M
LU
Z
X
CT
IO
N
DETECTION OBJECTIVE 2
Communication speeds between
existing hardware components of the
laser illumination system limited image
acquisition rate.
Camera acquisition time of first gen
sCMOS technology limited the rate of
image capture.
ILLUMINATION OBJECTIVE
DETECTION OBJECTIVE
SYNCHRONIZED PIEZO SCANNING
DE
TE
FOCUSED LASER BEAM
CT
IO
N
Y
Find details of this 2013 work by Ahrens et al. at DOI:10.1038/NMETH.2434
SAMPLE
HOLDER
DE
TE
SECOND
ILLUMINATION
OBJECTIVE
OPTIONAL
O
N
I
AT
IN
IL
M
LU
Z
X
Y
New camera technology was needed to keep
up with the faster image acquisition times.
Camera:
(2) Two Orca Flash 4.0
cameras were used
SAMPLE
The imaging strategy of physically
moving the sample extended the total
acquisition time of each plane to allow
for setting.
IL
All images courtesy of Phillip Keller, Lab Head, HHMI Janelia Research Campus
REVISED IMAGING
CT
IO
N
Faster image acquisition required the
development of a more robust computational
pipeline.
Acquisition rate:
0.8 Hz (for a volume of the
size 800 × 600 × 200 μm3)
Changing to an imaging strategy that keeps
the sample immobile reduced the need for
extended settle times.
For more information on this and other Bench Stories visit http://thelivingimage.hamamatsu.com/
Above: Functional imaging of the entire, isolated central nervous system
of a Drosophila larva.
Above: The multi-view hs-SiM
view set-up.
By taking a multi-directional approach and revising the optical, mechanical
and computational imaging strategy, the new hs-SiMView achieves a 25x speed
improvement over the 2013 version. Because of these enhancements, it’s now
possible to perform functional imaging of non-transparent biological samples
such as the Drosophila larva above.
Find details of this 2015 work by Lemon et al. at DOI:10.1038/ncomms8924
MATCHED TO THE PERFORMANCE
OF GEN II SCMOS CAMERAS
CHROMATICALLY CORRECTED
USER-DEFINED FILTER COMBINATIONS
EASILY ALIGNED AND STABLE
HIGH TRANSMITTANCE
W-View Gemini
G E T
C LO S E R
T O
T H E
B I O LO G Y
Have you ever thought, “Optical splitters—great idea but so hard to use!” They hold the
promise of more efficient multi-wavelength imaging, but need careful alignment. The slightest
bump of the microscope table or stomp in the neighboring lab can disrupt the setup. The team
at Hamamatsu understands how important it is for tools to get out of your way and just work.
They’ve taken on the optical splitter problem and crafted the W-VIEW GEMINI. Optically and
mechanically stable, chromatically corrected, with simple software-assisted alignment and
flexible configurations, the W-VIEW GEMINI lives up to the promise of what an optical splitter
should be. And when you don’t need a splitter, just switch to “bypass mode”—it’s as though
there’s nothing between your camera and your microscope. Simplifying multi-wavelength
experiments like FRET, the W-VIEW GEMINI gets out of your way to bring you closer to the
biology. What biological processes will the W-VIEW GEMINI bring closer to you?
Simultaneous Dual Wavelength Imaging
12
13
Relative SNR
Relative SNR vs Signal @ 580nm
1.0
How can I easily compare cameras?
Calculating SNR is a simple ratio of the total signal to the total noise.
For microscope cameras, the equation looks like this:
QE * S
Fn 2 * QE * (S + lb ) + (Nr /M) 2
TYPICAL INTENSITY OF BIOLOGICAL SAMPLES
0.5
Perfect
Flash4.0 V2
Flash4.0 LT
CCD
0.25
Gen I sCMOS
EM-CCD
0
1
10
100
1000
10000
INPUT SIGNAL PHOTON
Relative SNR at 580nm. EM-CCD gain is 500x. Read noise: ORCA Flash4.0s = 1.4 e- rms, CCD = 6 e- rms,
Gen I sCMOS = 2.4 e- rms. At 500x gain, EM-CCDs saturate around 1000 photons/pixel.
Thinking in Photons
Calculating SNR
SNR =
RELATIVE SNR
0.75
No single technical specification can provide all the necessary information to match a camera to an
application. But when the quantum efficiency and noise characteristics of a camera are considered
in light of the signal and signal noise, we can understand the theoretical limits of a camera under
the full range of light conditions. These signal to noise (SNR) curves provide tremendous value in
predicting which camera performs best for certain applications, assuming the light levels for that
application are known (more on this later). To make SNR data even more approachable, a useful
variation is to look at relative SNR (rSNR), where all data is normalized to an imaginary “perfect”
camera that has 100% QE and zero noise. With this transformation, it’s easy to see that at the
lowest light levels (less than 4 photons per pixel with 0 background), EM-CCDs achieve the highest
possible SNR. And yet, above 4 photons per pixel, the 2016 ORCA-Flash4.0 V2 surpasses the
SNR performance of the EM-CCD and exceeds all other technology, including CCD and previous
generations of sCMOS. This SNR performance, combined with fast frame rates and large field of
view make the ORCA-Flash4.0 V2 an excellent choice for most every biological application.
How many photons do I have?
QE=Quantum efficiency
S= Signal (Photons/pixel/frame)
Ib = Background (Photons/pixel/frame)
Nr =Readout noise (e- rms)
M =EM gain
Fn =Excess noise factor
(1.4 for EM-CCD; 1 for CCD & sCMOS)
We capture images of light but rarely have any sense of the absolute amount of light that was incident
on the camera. To get a useful estimate of your light levels, it’s possible to make a quick calculation.
Gather this information: the pixel full well capacity and bit depth of your current camera in the mode you
normally use to collect images and without gain. Divide the full well by the bit depth (e.g. 30,000/65536
= 0.46). This number is the conversion from grey level (gl) to electrons. Then, from one of your typical
images (collected without any EM or analog gain), identify a pixel or small region of pixels that
represents typical brightness. Measure the intensity of this area in grey levels and subtract the camera
offset (average grey level of a dark image; e.g. 1100 gl -100 gl = 1000 gl). Then convert the grey levels to
electrons by multiplying the intensity in grey levels by the conversion factor (e. g. 1000 gl x 0.46 e-/gl =
460 e-). Now you have your intensity in electron and can convert to photons by dividing by the QE at the
emitted wavelength (e.g. 460 e-/.82 = 560 photons). This is a rough but reasonable approximation of
how many photons per pixel were captured from your sample in your imaging system.
14
15
SPECS
ORCA-Flash4.0 V2
USB 3.0
ORCA-Flash4.0 V2
With Camera Link Option
ORCA-Flash4.0 LT
USB 3.0
ImagEM X2
512
Product Number
Imaging Device
Cell (pixel) Size (µm)
Pixel Array (horizontal by vertical)
Effective Area (horizontal by vertical in mm)
C11440-22CU
sCMOS
6.5
2048 x 2048
13.312 x 13.312
C11440-22CU
sCMOS
6.5
2048 x 2048
13.312 x 13.312
C11440-42U
sCMOS
6.5
2048 x 2048
13.312 x 13.312
C9100-23B
Back-Thinned EM-CCD
16
512 x 512
8.19 x 8.19
C9100-24B
Back-Thinned EM-CCD
13
1024 x 1024
13.3 x 13.3
Dark Current (electrons/pixel/sec.) – Air Cooled
Dark Current (electrons/pixel/sec.) – Water Cooled
Full Well Capacity in electrons (typ.)
Readout Noise (Nr) median in electrons (typ.) slow scan
Readout Noise (Nr) rms in electrons (typ.) slow scan
Readout Noise (Nr) median in electrons (typ.) standard scan
Readout Noise (Nr) rms in electrons (typ.) standard scan1
Dynamic Range (typ.)
0.06
0.006
30,000
0.8 @ 30 fps
1.4 @ 30 fps
1.0 @ 30 fps
1.6 @ 30 fps
37,000:1
0.06
0.006
30,000
0.8 @ 30 fps
1.4 @ 30 fps
1.0 @ 100 fps
1.6 @ 100 fps
37,000:1
0.6
N/A
30,000
0.9 @ 30 fps
1.5 @ 30 fps
1.3 @ 30 fps
1.9 @ 30 fps
33,000:1
0.005
0.0005
370,000 7
8 @ 4x gain
<1 @ 1200x gain
Gain Dependent
0.01
0.001
400,000 8
3 @ 10x gain
<1 @ 1200x gain
Gain Dependent
Peak Quantum Efficiency (QE)
Quantum Efficiency (QE) @ 500 nm
Quantum Efficiency (QE) @ 670 nm
Quantum Efficiency (QE) @ 750 nm
Noise Factor (Fn)2
(QE) 82% @ 560 nm
(QE) 82% @ 560 nm
(QE) 73% @ 580 nm
(QE) 92% @ 580 nm
(QE) 92% @ 580 nm
(QE) 77% @ 500 nm
(QE) 77% @ 500 nm
(QE) 67% @ 500 nm
(QE) 91% @ 500 nm
(QE) 91% @ 500 nm
(QE) 76% @ 670 nm
(QE) 76% @ 670 nm
(QE) 68% @ 670 nm
(QE) 83% @ 670 nm
(QE) 83% @ 670 nm
(QE) 61% @ 750 nm
(QE) 61% @ 750 nm
(QE) 53% @ 750 nm
(QE) 66% @ 750 nm
(QE) 66% @ 750 nm
1
1
1
1.4
1.4
Minimum Exposure Time
Maximum Exposure Time
In-Camera Binning
Subarray
Maximum Full Resolution Frame Rate (fps)
Absolute Maximum Frame Rate (fps)3
1 ms4
10 s
2 x 2, 4 x 4 (digital)
Yes
30
25,655
1 ms4
10 s
2 x 2, 4 x 4 (digital)
Yes
100
25,655
1 ms 4
10 s
2 x 2, 4 x 4 (digital)
Yes
30
25,000
13.85 ms 5
2 hours
2 x 2, 4 x 4 6
Yes
70.4
1076
52.7 ms 5
2 hours
2 x 2, 4 x 4
Yes
18.5
314
Electron Multiplying Gain
Analog Gain
A/D Converter
Interface Type
Lens Mount
N/A
No
16 bit
USB 3.0
C-mount
N/A
No
16 bit
CameraLink
C-mount
N/A
No
16 bit
USB 3.0
C-mount
4 - 1200x
Yes
16 bit
IEEE 1394b
C-mount
10 - 1200x
Yes
16 bit
IEEE 1394b
C-mount
1
2.0 electrons rms, guaranteed.
2
If this value is greater than 1,
multiplicative noise is present.
3
Using maximum binning
and/or smallest subarray.
4
40 µs using internal trigger
and subarray.
5
10 µs using external trigger.
6
8 x 8, 16 x 16 binning optional.
7
FWC in EM-CCD Mode.
FWC for normal CCD mode
is 140,000 e-
ImagEM X2
1K
8
FWC in EM-CCD Mode. FWC for
normal CCD mode is 50,000 e-
16
17
L I G H T- S H E E T
CALCIUM IMAGING
SINGLE MOLECULE
FLUORESCENCE MICROSCOPY
SINGLE MOLECULE FLUORESCENCE
WIDEFIELD FLUORESCENCE
L
L I G H T- S H E E T
FLOW
A T
T
I
C
E
L
I
G
H
T
S
H
E
E
GFP
T
LIVE CELL FLUORESCENCE
LIVE CELL
FIXED CELL FLUORESCENCE
R AT I O I M A G I N G
LIVE CELL
IMAGING
FLUORESCENCE MICROSCOPY
TIRF
L
BRET
TIRF
U
M
I
N
E
S
C
E
N
C
CELL SIZE
BRET
6.5
L I G H T- S H E E T
S T R U C T U R E D I L L U M I N AT I O N
E
PIXEL ARRAY
2048 X 2048
SUPER RESOLUTION
IRDIC
FRAME
BLOOD FLOW
EFFECTIVE AREA
13.312 X 13.312
SPINNING DISC CONFOCAL
DARK CURRENT_
01
AIR COOLED
0.5
W AT E R C O O L E D
.015
THERE’S A STORY
F U L L W E L L CA PAC I T Y
PALM
R A T II ON EILM
NSG_
E CAT G
R OI N
TIRF
f. 01
30,000
L AT T I C E L I G H T S H E E T
SUPER-RESOLUTION
S
U
P
E
R
R
E
S
O
L
U
T
I
O
CELL SIZE
N
6.5
BEHIND EACH OF THESE IMAGES
f. 02
PIXEL ARRAY
f. 03
L O C A L I Z AT I O N M I C R O S C O P Y
EFFECTIVE AREA
BLOOD FLOW
X
E
D
C
E
L
L
QE @ 560 nm 82%
QE @ 500 nm 77%
F
L
QE @ 670 nm 76%
f. 06
U
O
R
E
WHAT’S YOUR STORY?
QE @ 750 nm 61%
S
C
E
N
C
E
MINIMUM EXPOSURE TIME 1 ms
MAXIMUM EXPOSURE TIME 10 s
HIGH SPEED BRIGHTFIELD
S PAC E
I
PEAK QUANTUM EFFICIENCY_
f. 05
LUMINESCENCE
F
13.312
TIME
SINGLE MOLECULE
CELL
2048 X 2048
L AT T I C E L I G H T S H E E T
A B S O L U T E M A X I M U M F R A M E R AT E
25,655
LIVE CELL
SUPER RESOLUTION
f. 41
FRAME
41
IRDIC
L O C A L I Z AT I O N M I C R O S C O P Y
L
I
G
H
T
S
H
E
E
T
M
I
C
R
O
S
C
O
P
Y
18
19