Transcript Presentazione di PowerPoint

Ratio-imaging :
configuring the
system
Target : obtaining the best from
the experiment
-Why ratio imaging ?
-Is the experiment set correctly ?
-What’s the right set up for me ?
Calcium and Fura-2 as an example
SINGLE WAVELENGHT IMAGING (Calcium
Green) Vs RATIO IMAGING (Fura2)
Single wavelength excitation
imaging
Fluorescence intensity depends
upon
– optical path length through the
sample
– excitation intensity
– fluorophore concentration
– fluorescence quantum yield of
the probe
– calcium concentration
– hidrolysis of loaded dye
Dual wavelength excitation ratio
imaging
Fluorescence ratio depends upon
–calcium concentration
–hidrolysis of the loaded dye
How does excitation ratio imaging work?
Fura-2 changes its spectral characteristics upon
binding Calcium
Fura-2-Ca
340 & 380 nm excitation
Fura-2
Ca – Fura 2
Fura 2
Physiological
range
Fura-2 spectra from Molecular Probes’ website
The Formula for in vivo calcium measurement
[Ca++]=Kd*(R-Rmin)/(Rmax-R)*(F max 380/F min 380)
Kd : dissociation constant for the chelator( Fura ) and ligand ( Calcium )
R : measured ratio (340/380)
R min : 340/380 ratio at zero calcium concentration
Rmax : 340/380 ratio at satured calcium conditions
F : intensity of fluorescence at 380 , Fmax : satured calcium , Fmin :
calcium free
Grynkiewitz, Poenie and Tsien : J. Biol Chem 260, 3440 (1985)
Equilibrium Equation
Kd * [Ca-Fura -- ] = [ Ca ++ ] * [Fura ----]
K dissociation=1/K affinity (high dissociation constants mean
lower affinity between members of the equilibrium)
What is Kd really?
[Ca++] = Kd*(R-Rmin) / (Rmax-R) * (Fmax 380 / Fmin 380)
• Choose denominator (wavelength 2) at
isobestic point (360 nm), we get:
• [Ca++]=Kd*(R-Rmin)/(Rmax-R)
Further, since denominator is the
isobestic value and same for all ratios:
• [Ca++]=Kd*(F-Fmin)/(Fmax-F)
• When F is midway between Fmin and
Fmax , [Ca++]=Kd Half-saturation
point !
Kd = 0,15 um = [Ca++] at half saturation point
In this case the sensitivity to calcium concentrations above 1uM
is very limited.
Fura-4F spectra from Molecular Probes’ website
Choose the right low affinity calcium indicator for your application!
Absolute ratio values of Fura-2 are
equipments dependent
•
•
•
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•
UV transmission of objectives
excitation filters’ transmission maximum
dichroic cube spectral properties
excitation, emission filter bandpasses
relative exposure times
What is the right system ?
•Microscope
•CCD
•Motorized stage xyz
•Filter wheel or
monochromator
•PC
•Control and analysis
software
Which is the right CCD ?
Speed : IPentaMAX
Spatial resolution : MICROMAX 512BFT,
COOLSNAP HQ
Speed + spatial resolution : new CASCADE
I PentaMAX
IPentaMax:RB GenII
IPentaMax:HQ
GenIII
“Red/Blue” Gen II intensifier
Moderate spatial resolution
(45 lp/mm)
“High QE” Gen III intensifier High
spatial resolution
(65 lp/mm)
Sensitive from 475 nm to 900 nm
Ideal for visible-wavelength
imaging
Minimum of 40% QE at 546 nm
Highest available QE on the
market
~20% QE throughout the UV
IPentaMax:HB
GenIII
“High Blue” Gen III intensifier
High spatial resolution
(65 lp/mm)
Sensitive from 350 nm to 900 nm
Ideal for visible-wavelength
imaging
~35% QE at 520 nm
Good QE in the green/blue
region
Fiberoptically coupled to CCD (1.5:1 taper ratio) Highest coupling efficiency available allows full use of 18mm intensifier diameter
512 x 512 imaging array 15 x 15-µm pixels
Frame-transfer readout
5-MHz A/D converter 15 fps at full resolution
1-MHz A/D converter 3 fps at lower noise
I PentaMAX QE
Virtual chip
Virtual chip mode is a special fast acquisition technique that
allows frame rates in excess of 100fps to be obtained
region
ms / frame
fps
160 x 160
7.07
141
98 x 98
3.08
324
89 x 89
2.72
367
68 x 68
1.74
574
56 x 56
1.39
719
51 x 51
1.14
877
41 x 41
0.92
1087
38 x 38
0.78
1282
32 x 32
0.68
1470
32 binned x 5120.52
1923
Virtual Chip speeds determined from scan code
calculator assuming exposure equals readout time, for an
IPentaMax.
MICROMAX 512BFT and CoolSNAP HQ
MicroMax 512BFT
MicroMax
512BFT
CoolSNAP HQ
Backilluminated
CCD
Frame-transfer
readout
Interline, progressivescan CCD
512 x 512
imaging pixels;
13.0 x 13.0-µm
pixels;
1392 x 1040 imaging
pixels;
6.45 x 6.45-µm pixels;
16 bits @ 1
MHz or
16 bits @ 100
kHz
3 fps ( 1MHz)
12 bits @ 20 MHz or 10
MHz; 10 fps ( 20 MHz )
CoolSNAP HQ
New CASCADE
On-chip multiplication
gain
Software selectable; 200x
maximum achievable
gain
Very high sensitivity
Low-noise, impactionization process
653 x 492 imaging array
7.4 x 7.4-µm pixels
Resolves fine detail
Ideally matched to optical
microscope
10-MHz readout
5-MHz readout
Good for high-speed
image visualization
Perfect for high-precision
photometry
ROI size
Wide dynamic range
allows detection of both
bright and dim
signals in the same
image
16-bit digitization
Frames per second
653 x 492
25
256 x 256
48
128 x 128
92
64 x 64
172
32 x 32
307
16 x 16
510
What is
on-chip multiplication gain?
It’s the on-chip multiplication of charge (electrons)
generated by incident photons in each pixel.
Standard CCD architecture
Active
array
Masked
array
Preamplifier
Serial
register
Output
node
(Frame-transfer CCD)
ADC
CCD with on-chip multiplication gain
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
On-chip multiplication gain process
Active
array
Masked
array
Serial
register
Preamplifier
Extended multiplication
register
Output
node
ADC
Why is it so revolutionary?
• Amplifies the signal above the read noise right
on the CCD (without using external
photocathodes)
• Challenges traditional tradeoff between speed
(frame rate) and sensitivity in the world of lowlight imaging
• On-chip multiplication process is easily
controlled by varying the applied voltage
Cascade
I-PentaMAX
Fluorescence from single molecules of perylene diimide in
polymethylmethacrylate gel; 100 msec; 535 nm; 60x; NA 1.3
Experiment required 4x magnifier in front of I-PentaMAX to resolve single molecules.
Cascade
CoolSNAPHQ
Fluorescence from single molecules of perylene diimide in
polymethylmethacrylate gel; 100 msec; 535 nm; 60x; NA 1.3
Excitation wavelenghts devices
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•
•
55 msec shift
High throughput
Fixed bandpass and intensity
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•
•
1,2 msec shift
High throughput
Fixed bandpass and variable intensity
Filter wheel
Galvanometric wavelenght switcher
•
•
•
•
3 msec shift
1 nm resolution
Low throughput
Variable bandpass and intensity
Galvanometric monochromator
Comparison filters/monochromator
Filter wheel
Monochromator
Excitation Intensity
Higher
Lower
Variable Spectral bandwidth
No , discret : 10,15,20 nm
Yes ( 1- 30 nm DeltaRam)
Spectral range
Fixed wavelenghts ( up to
10 ) and no scan
250-700 nm
possible scan
Speed
55msec between adjacent
filters
3 msec
Vibration
Increases with speed , can
be reduced with fiber
delivery
Low/None
Heat effect on the excitation
High : can distort spectral
performance
None
Dynamic range
Medium ( large bandwidth
causes crosstalk , stray light
10-3)
High ( stray light 10-4)
Image quality
Best
Good
Comparison filters/monochromator
RATIO imaging + FISH, Colocalization, GFP
imaging, FRET:
filters and /or fast monochromator
Only RATIO imaging ( BCECF, Fura2.. ) :
fast monochromator
SOFTWARE CONTROL MAIN FEATURES
- # of wavelenghts controlled
-# of ratios controlled
- experiment playback for offline analysis
- macro
RATIO
-Average of ratios Vs Ratio of averages
4 pixel : 1w1 0 2w1 50 3w1 100
4w1 0;
1w2 50
2w2 0
3w2 50
4w2 80
ratio of average (scartando pixel con valore 0 o saturati ) : w1 : 75, w2: 60, R: 75/60 =1.25
average of ratio : ratio 3=2
# wavelenghts and ratio controlled
GFP + Fura 2
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Roberto Becattini
Biology apps specialist