Solutions to Imaging for Extended Depth and Time Prof John Girkin

Solutions to Imaging for Extended
Depth and Time
Prof John Girkin
Biophysical Science Institute
and
Department of Physics
University of Durham, UK
j.m.girkin@durham.ac.uk
© University of Durham 2013
Outline
• Background
• High Speed Imaging
• Cell Tracking in vivo
• 3D Imaging in Tissue
© University of Durham 2013
Complications in Biology: Scale
Time Scale:- Grain of salt to distance of sun from earth
Length Scale:- Grain of salt to distance round earth
© University of Durham 2013
What the Life Scientist Desires
•  Sub-cellular resolution images
•  Three dimensional imaging
•  Minimally invasive imaging
•  In depth imaging
•  In vivo imaging
© University of Durham 2013
Mitochondrial Movement
•  Previous work “slow” (1s to 1/10th ish)
•  Novel high speed camera
– Camera analysis images
– Send only positions
– Track position 100 particles 20kHz
– ~ 2-5 nm relative position movement
•  Specialized data analysis
© University of Durham 2013
nm Camera Imaging
•  Take camera image
•  Image into on board FPGA
•  Find centre of mass of object
•  Centre of mass then forms that pixel
in image
•  or image correlation for movement
Saunter et al FEBS Letters 483, 1267, 2009
© University of Durham 2013
SPIM: 3D optical sectioning
Side illuminate with
excitation
laser
Side
illuminate
with
excitation
laser
Side
illuminate
with
excitation
laser
Side illuminate with
excitation
laser
Side
illuminate
with
excitation
laser
Side
illuminate
with
excitation
laser
Side illuminate with
excitation
laser
Side
illuminate
with
excitation laser
Standard
SPIM: only
imaging:
one
can’t
layer discriminate
illuminated
between
at a time layers
• 
• 
• 
• 
• 
• 
Depth discrimination (3D reconstruction)
Reduced phototoxicity
Reduced bleaching
Degrades more gracefully than confocal
High speed imaging
Fraction of the price of confocal!
© University of Durham 2013
SPIM: Dual white light/
fluorescence imaging
Red/IR image
(transmission)
SPIM sheet
Biological sample
Imaging objective
Illumination beam
launch objective
Fluorescence
image
© University of Durham 2013
© University of Durham 2013
Method overview
•  Fast imaging/image processing
-> ok
•  Accurate timing/triggering mechanism
-> hard! (dedicated FPGA electronics)
(ECG is very impractical in tiny embryos!)
© University of Durham 2013
Heart period/phase recovery
•  Peak-finding heuristic used to identify matching
frames from previous video frames
•  Sub-frame fitting used to obtain exact period/
phase
"Real-time optical gating for three-dimensional beating heart imaging” J. Taylor, C. Saunter, G.
Love, and J. Girkin, D. Henderson, B. Chaudhry, J. Biomed. Opt. 16, 116021 (2011)
© University of Durham 2013
Heart synchronization systems
Frame data
Free running
imaging
camera
Trigger
Science
camera
Timing
controller
Microscope
PC
Standard camera images
Laser fire signal
Laser fire signal
UV Laser Ablation
PALM/STORM
bolt-on
Science
camera
Monitoring
camera
Microscope
Timing controller
(could be completely
separate system)
PC
"Real-time optical gating for three-dimensional beating
heart imaging” J. Taylor, C. Saunter, G. Love, and J.
Girkin, D. Henderson, B. Chaudhry, J. Biomed. Opt. 16,
116021 (2011)
© University of Durham 2013
A single laser pulse to the mid-cavity of the ventricle (Fig. 1) resulted
in instantaneous cardiac injury confirmed optically by a 2 to 4 s pause
followed by marked bradycardia and a small amount of bleeding into
the pericardial space. There then followed a gradual and progressive increase in heart rate (HR) over the following 2 to 3 min with a significantly
reduced HR compared with controls by 2 h post-laser (117 ± 11 vs
157 ± 9 bpm, p ≤ 0.001, Fig. 2B). Laser injury also resulted in temporary
Haematoxylin & Eosin (H&E) staining of whole embryos was performed at 2, 24
and 48 h post-laser injury and serial 4 μm sagittal sections were stained according to
standard protocols [21].
Heart Recovery
2.8. Whole-mount TUNEL assay
Apoptotic cell death in whole-mount zebrafish was detected according to a modification of the ApopTag rhodamine In Situ Apoptosis Detection kit (Chemicon,Temecula, CA)
Before laser
A
2 h post-laser
24 h post-laser
50 m
B
C
180
Heart rate (bpm)
Cardinal vein blood flow
( m s-1)
***
160
140
120
100
80
60
40
20
0
25
***
20
15
10
5
0
Before laser
72hpf
2h
24h
350
250
200
150
100
50
14
12
***
10
8
6
4
2
0
48h
***
300
0
E
30
2
Diastolic area (103 m )
Ejection fraction (%)
D
400
Before laser
72hpf
Post-laser injury
control
2h
24h
48h
Post-laser injury
injury
Fig. 2. Effects of a laser pulse to the ventricle on cardiovascular function. Panel A. Images showing an in vivo zebrafish embryo heart ventricle before laser (72 hpf), at 2 and 24 h
post-laser injury. Panels B-E Heart rate, cardinal vein blood velocity, ejection fraction and ventricle diastolic area assessed before laser and 2, 24, 48 h post-laser injury
(Mean ± sem, N = 30 per group, 5 experiments, *** = p b 0.001, ANOVA test).
“Laser-targeted ablation of the zebrafish embryonic ventricle: A novel model of cardiac injury and
repair” Gianfranco Matrone, Jonathan M. Taylor, Kathryn S. Wilson, James Baily, Gordon D.
Love,
John
M. Girkin,
John J. Mullins,
Carl
S. Tucker,
Martin
A. Denvir,
International
Journal
of
Please cite this
article
as: Matrone
G, et al, Laser-targeted
ablation of
the zebrafish
embryonic
ventricle:
A novel model
of cardiac injury
and repair,
Int J CardiolCardiology,
(2013), http://dx.doi.org/10.1016/j.ijcard.2013.06.063
July 2013
© University of Durham 2013
Skin Model Requirements
•  In vitro skin reconstitution
model
•  Compatible with long-term
imaging
•  Ability to mix cell types
•  Induce chemical and physical
stress
•  Induce wounding
Means extended depth and time imaging
© University of Durham 2013
The Imaging Problem
Images of Philadelphia Cream Cheese
taken using two-photon microscopy
10µm
25µm
50µm
90µm
“Adaptive Optics for Deeper Imaging of Biological Samples”
J M Girkin, S Poland, A J Wright Current Opinion in Biotechnology 20 106-110 (2009)
© University of Durham 2013
Astronomy Solution
Galaxy NGC 7469
Imager
Wavefront
sensor
© University of Durham 2013
Multiphoton:- Post Scanning
“Practical implementation of adaptive optics in multiphoton microscopy”
P N Marsh, D Burns, J M Girkin, Optics Express 11 1123-1130 (2003)
© University of Durham 2013
Cells imaged for 24 hours
Basal
© University of Durham 2013
Suprabasal
Analysis by Moises Santos
Challenges
•  Image at the correct speed
•  Vast quantities of data
•  Data analysis in 4D with spectral and
temporal information complex
•  Extended imaging periods required
•  Ensuring minimal perturbation
© University of Durham 2013
Acknowledgements
•  Durham University
Prof Gordon Love, Dr Jonny Taylor (Glasgow),
Dr Chris Saunter (Durham), Dr Cyril Bourgenot,
Dr Carrie Ambler
•  University of Edinburgh
–  Prof John Mullins, Dr Martin Denvir
Funding
British Heart Foundation, EPSRC, EU, Durham University
© University of Durham 2013