Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Advances in Sub-cellular and Cellular in vivo
Imaging for Systems Biology
#LifeScienceWebinar #ISCxScintica
Peter Delaney ...
Advances in Sub-cellular and Cellular in vivo
Imaging for Systems Biology
Peter Delaney
Chief Technology Officer,
Optiscan...
InsideScientific is an online educational environment designed for life science
researchers. Our goal is to aid in the sha...
To access webinar content, Q&A reports, FAQ document, and
information on lab workshops, we invite you to join our communit...
Overview
• In Vivo Microscopy
• Importance in Systems Biology
• Technologies
• Illustrative Applications Examples
In Vivo Microscopy and Systems Biology
Why is it important?
• Complexity of cell-cell interaction
– e.g. Cancer cells vs m...
What just happened and what will happen next?
What will be the response from the surrounding players and how will they influence the
outcome of the play? While a more c...
Microscope Technologies
• Fluorescence
• Optical Sectioning (Laser Scanning)
• Confocal
• Non-Linear
– Multiphoton
– SHG
•...
Technologies for in vivo Microscopy
Technology Advantages Limitations Cost Complexity
Benchtop Confocal Ability to image u...
Fluorescence – Single Photon Excitation
1. Illumination (blue @ 488nm)
2. Absorption
& excitation
3. Fluorescent emission
...
Confocal Microscopy
• Focal illumination
• Captures via pinhole
• Blur is rejected
• X, Y and Z scanning
• Optical section...
Whole Mount Visceral Adipose Tissue
LYVE-1 AlexaFluor488
3µm steps
183µm volume
Size: 1920px x 1080px
FOV: 475um x 267um
R...
LYVE-1 AlexaFluor488
3µm steps
183µm volume
Size: 1920px x 1080px
FOV: 475um x 267um
3D Image - Imaris
Images courtesy of ...
Fluorescence – Multiphoton Excitation
1. Pulsed Illumination (often NIR eg 800nm)
2. Absorption & Excitation
(the fluoroph...
Multiphoton Microscopy
• Focal Illumination
• Fluorescence only in focus
• Photons must arrive together
• Signal only from...
Benchtop confocal or MP microscope with laser safe
animal chamber for in vivo microscopy
Benchtop confocal or MP microscope with laser safe
animal chamber for in vivo microscopy
Whole Mount Brain Section (800um thick)
• Optical Sectioning – enables in vivo microscopy in thick
specimens
• Single or Multiphoton fluorescence enables this
cap...
Fiber Optic Confocal System
Laser
Objective
lens
Tissue
Detector
Optical fiber
Imaging plane
Scan
mechanism
Confocal microscopy with miniature flexible probes
now allows taking the microscope to the animal
Illustrative Examples
Calcium Imaging
Live Tissue
Fluo-8
1.34sec per frame
Size: 1024px x 1024px
FOV: 475µm x 475µm
Images courtesy of Daniel Po...
Tissue Engineering - Cartilage
Chondrocytes
Contrast Agent:
Fluorescein Sodium 0.5%
in PBS; pH 7.4
fov=500µm
fov=~100µm
Im...
Sheep Cartilage Defect Model
Condyle
Contrast Agent:
Acriflavine 0.05%
Defect area
Normal
condyle
Defect area
post ACI
Nor...
Contrast Agent:
Acriflavine 0.05%
A. Normal trochlear
B. Trochlear post ACI
Trochlear
Wu JP, Kirk TB, Zheng MH.
Study of t...
Contrast Agent:
Acriflavine 0.05%
A. Normal trochlear
B. Trochlear post ACI
Trochlear
Wu JP, Kirk TB, Zheng MH.
Study of t...
In Vivo molecular imaging – Mouse
fov=500um fov=500um
Images courtesy of:
Martin Goetz and Ralf
Kiesslich, Mainz Universit...
fov=~240um
Pancreas:
In Vivo Blood Flow
Images courtesy of:
Martin Goetz and Ralf Kiesslich,
Mainz University Hospital, Ge...
Mouse Liver Necrosis in vivo
A. Contrast Agent:
topical Acriflavine
0.1%, CBDL Mouse
fov=500um
B. Contrast Agent:
i.v. FIT...
cytoplasmic vesicles:
start 15-30 min
nuclear fragmentation:
start 55-80 min
extravasation:
start 10 min
20 6040 150120 18...
Mitotic cell
nucleus
Images courtesy of
Cameron Nowell and
researchers at Monash
University, Melbourne,
Australia
Ejecting...
Images courtesy of:
Ralf Kiesslich, et al,
Mainz University Hospital,
Germany
Images courtesy of:
Ralf Kiesslich, et al,
Mainz University Hospital,
Germany
Images courtesy of:
Ralf Kiesslich, et al,
Mainz University Hospital,
Germany
Technologies for in vivo Microscopy
Technology Advantages Limitations Cost Complexity
Benchtop Confocal Ability to image u...
Bundled Fibre vs. Point Scanning
Bundled Fibre
(Raw Image)
Optiscan Point Scanning
(Raw Image)
Bundled Fibre
(Area of Focu...
Summary
• Advances in fluorophores, imaging systems, and
protocols enable the interrogation of living systems in
dynamic m...
Peter Delaney Chief Technology Officer,
Optiscan Pty. Ltd
For more information on the technology and research applications...
Upcoming SlideShare
Loading in …5
×

Advances in Sub-cellular and Cellular in vivo Imaging for Systems Biology

980 views

Published on

LIVE WEBINAR: Dec 5, 2017
Sponsor: www.scintica.com

Peter Delaney discusses unique combinations of advanced imaging instrumentation, fluorescent probes and key applications in terms of their capabilities, limitations and possibilities. Discussion centers on the importance of cellular and sub-cellular observations in vivo, often producing results counter to in vitro cellular behavior, and therefore streamlining research paths. In addition, he shares a variety of sample images, demonstrating novel capabilities in the world of high-resolution in vivo confocal endomicroscopic imaging.

Background:
In vivo fluorescence microscopy is advancing rapidly with the development of new imaging modalities and fluorescence labeling agents. It is now possible to image morphological, molecular, genetic, pathological and physiological events in the living animal. Laser scanning microscopes have advanced quickly, and even miniaturized confocal microscopes of endoscopic proportions can now create 3D observations at the cellular and sub-cellular level in vivo. This has led to a proliferation of techniques facilitating the transition of therapeutic research, bench-to-bedside, through direct in vivo observation of systems biologic processes.

Published in: Science
  • Be the first to comment

  • Be the first to like this

Advances in Sub-cellular and Cellular in vivo Imaging for Systems Biology

  1. 1. Advances in Sub-cellular and Cellular in vivo Imaging for Systems Biology #LifeScienceWebinar #ISCxScintica Peter Delaney reviews current trends and technological advancements in the field of in vivo fluorescence imaging, with a focus on new applications and capabilities as realized by confocal fluorescence microscopy and 3D automated optical sectioning.
  2. 2. Advances in Sub-cellular and Cellular in vivo Imaging for Systems Biology Peter Delaney Chief Technology Officer, Optiscan Pty. Ltd #LifeScienceWebinar #ISCxScintica
  3. 3. InsideScientific is an online educational environment designed for life science researchers. Our goal is to aid in the sharing and distribution of scientific information regarding innovative technologies, protocols, research tools and laboratory services
  4. 4. To access webinar content, Q&A reports, FAQ document, and information on lab workshops, we invite you to join our community at www.insidescientific.com/register
  5. 5. Overview • In Vivo Microscopy • Importance in Systems Biology • Technologies • Illustrative Applications Examples
  6. 6. In Vivo Microscopy and Systems Biology Why is it important? • Complexity of cell-cell interaction – e.g. Cancer cells vs macrophages • In Vitro studies suggest probable effectiveness to kill the cancer • Adding a third cell type changes the process completely • In vitro models can behave wildly differently to in vivo • Rise in Phase IV (post product approval) FDA withdrawals – Drug development in the post genomic era generates huge lead compound pipelines • Dynamics and context are crucial…
  7. 7. What just happened and what will happen next?
  8. 8. What will be the response from the surrounding players and how will they influence the outcome of the play? While a more complete picture, it is still a snapshot in time!
  9. 9. Microscope Technologies • Fluorescence • Optical Sectioning (Laser Scanning) • Confocal • Non-Linear – Multiphoton – SHG • Endomicroscopy (miniaturized)
  10. 10. Technologies for in vivo Microscopy Technology Advantages Limitations Cost Complexity Benchtop Confocal Ability to image unsectioned tissue (1-300um (tissue dependent). Mature tech with affordable and flexible laser technology. Generally easy to manage laser safety. Cumbersome, only certain tissues can be accessed in vivo, which have to be oriented to suit the optical axis. Several $100K Moderate, depending on configuration. May require significant training although well established. Multiphoton (MP) Fluorescence Deeper tissue imaging up to nearly 1mm in some tissues. Lots of flexibility in configuration, laser sources, and detection techniques. As above. Class 4 laser product requiring in vivo imaging to be performed in a laser tight, interlocked chamber. >$1M High. Normally requires dedicated facility and technician/Operator. Substantial learning curve. Second Harmonic Generation (SHG) As above, Deep imaging of unlabeled collagen, concurrent imaging with MP As above, but also needs collection of light propagating in the direction of illumination (i.e. a detector beyond the subject) >$1M Miniature Confocal Ability to image unsectioned tissue (1-300um; tissue dependent). Flexibility to image tissues from any angle at the operating table (ie. microscope to the animal) Miniaturisation results in a highly integrated system with fewer options than large systems. Several $100K Low. Short learning curve, can be used by anyone that is working with whole animals.
  11. 11. Fluorescence – Single Photon Excitation 1. Illumination (blue @ 488nm) 2. Absorption & excitation 3. Fluorescent emission (green-yellow 505-585nm) The laser illuminates the specimen with a single colour
  12. 12. Confocal Microscopy • Focal illumination • Captures via pinhole • Blur is rejected • X, Y and Z scanning • Optical sectioning • Can collect 3D sets Summary This image only shows a single point. To achieve a field of view the Focal point needs to move in x and y axes.
  13. 13. Whole Mount Visceral Adipose Tissue LYVE-1 AlexaFluor488 3µm steps 183µm volume Size: 1920px x 1080px FOV: 475um x 267um Raw Images Images courtesy of Enyuan Cao; Monash Institute of Pharmaceutical Sciences
  14. 14. LYVE-1 AlexaFluor488 3µm steps 183µm volume Size: 1920px x 1080px FOV: 475um x 267um 3D Image - Imaris Images courtesy of Enyuan Cao; Monash Institute of Pharmaceutical Sciences Whole Mount Visceral Adipose Tissue
  15. 15. Fluorescence – Multiphoton Excitation 1. Pulsed Illumination (often NIR eg 800nm) 2. Absorption & Excitation (the fluorophores in the focal region absorb 2 photons) 3. Fluorescent emission (green-yellow 505-585nm) An ultrafast and powerful pulsed laser delivers burst of light (typically ~100fsec @ 100MHz) over a very narrow wavelength range.
  16. 16. Multiphoton Microscopy • Focal Illumination • Fluorescence only in focus • Photons must arrive together • Signal only from focal plane • Many detector placement options • Deeper imaging than single photon excitation. • Long wavelength ex penetrates • Tissue scattering of emission less important than confocal. Distilled Summary This image only shows a single point. To achieve a field of view the Focal point needs to move in x and y axes.
  17. 17. Benchtop confocal or MP microscope with laser safe animal chamber for in vivo microscopy
  18. 18. Benchtop confocal or MP microscope with laser safe animal chamber for in vivo microscopy
  19. 19. Whole Mount Brain Section (800um thick)
  20. 20. • Optical Sectioning – enables in vivo microscopy in thick specimens • Single or Multiphoton fluorescence enables this capability • A huge catalog of fluorescent markers exist to illuminate specific aspects of microanatomy – Morphological – Protein specific eg mAb – Genetic transfection eg GFP – Process specific eg Ca+ markers Summary of In Vivo Microscope Technology
  21. 21. Fiber Optic Confocal System Laser Objective lens Tissue Detector Optical fiber Imaging plane Scan mechanism
  22. 22. Confocal microscopy with miniature flexible probes now allows taking the microscope to the animal
  23. 23. Illustrative Examples
  24. 24. Calcium Imaging Live Tissue Fluo-8 1.34sec per frame Size: 1024px x 1024px FOV: 475µm x 475µm Images courtesy of Daniel Poole Monash Institute of Pharmaceutical Sciences
  25. 25. Tissue Engineering - Cartilage Chondrocytes Contrast Agent: Fluorescein Sodium 0.5% in PBS; pH 7.4 fov=500µm fov=~100µm Images courtesy of Curtain University, Perth Western Australia Wu JP, Kirk TB, Zheng MH. Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique. J Orpthop Surg 2008; 3: 29.
  26. 26. Sheep Cartilage Defect Model Condyle Contrast Agent: Acriflavine 0.05% Defect area Normal condyle Defect area post ACI Normal condyle Wu JP, Kirk TB, Zheng MH. Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique. J Orpthop Surg 2008; 3: 29. fov=500µm fov=500µm
  27. 27. Contrast Agent: Acriflavine 0.05% A. Normal trochlear B. Trochlear post ACI Trochlear Wu JP, Kirk TB, Zheng MH. Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique. J Orpthop Surg 2008; 3: 29. Sheep Cartilage A B
  28. 28. Contrast Agent: Acriflavine 0.05% A. Normal trochlear B. Trochlear post ACI Trochlear Wu JP, Kirk TB, Zheng MH. Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique. J Orpthop Surg 2008; 3: 29. Sheep Cartilage A B
  29. 29. In Vivo molecular imaging – Mouse fov=500um fov=500um Images courtesy of: Martin Goetz and Ralf Kiesslich, Mainz University Hospital, Germany Goetz M, Thomas S, Heimann A, Delaney P, Schneider C, Relle M, Schwarting A, Galle PR, Kempski O, Neurath MF, Kiesslich R. Dynamic imaging of microvasculature and perfusion by miniaturised confocal laser microscopy. Eur Surg Res 2008; 41: 290–297. A. Before - Low level Fluorescence from a small dose of intravenous dye B. After Octreotate added A B
  30. 30. fov=~240um Pancreas: In Vivo Blood Flow Images courtesy of: Martin Goetz and Ralf Kiesslich, Mainz University Hospital, Germany
  31. 31. Mouse Liver Necrosis in vivo A. Contrast Agent: topical Acriflavine 0.1%, CBDL Mouse fov=500um B. Contrast Agent: i.v. FITC-Dextran 150kD, CBDL Mouse Goetz M, Vieth M, Kanzler S, Galle PR, Delaney P, Neurath MF, Kiesslich R. In vivo confocal laser laparoscopy allows real time subsurface microscopy in animal models of liver disease. J Hepatol 2008; 48(1): 91-97. fov=350um A B
  32. 32. cytoplasmic vesicles: start 15-30 min nuclear fragmentation: start 55-80 min extravasation: start 10 min 20 6040 150120 180 min Morphology of Apoptosis • Untreated Hapatocytes • Administration of “FLIVO” apoptosis markers. • Extravasation • Cytoplasmic vesicles • Nuclear fragmentation • Cell death • Former cellular space filled with diffuse marker fluorophore
  33. 33. Mitotic cell nucleus Images courtesy of Cameron Nowell and researchers at Monash University, Melbourne, Australia Ejecting epithelial cell Brush border & mucus layer
  34. 34. Images courtesy of: Ralf Kiesslich, et al, Mainz University Hospital, Germany
  35. 35. Images courtesy of: Ralf Kiesslich, et al, Mainz University Hospital, Germany
  36. 36. Images courtesy of: Ralf Kiesslich, et al, Mainz University Hospital, Germany
  37. 37. Technologies for in vivo Microscopy Technology Advantages Limitations Cost Complexity Benchtop Confocal Ability to image unsectioned tissue (1-300um (tissue dependent). Mature tech with affordable and flexible laser technology. Generally easy to manage laser safety. Cumbersome, only certain tissues can be accessed in vivo, which have to be oriented to suit the optical axis. Several $100K Moderate, depending on configuration. May require significant training although well established. Multiphoton (MP) Fluorescence Deeper tissue imaging up to nearly 1mm in some tissues. Lots of flexibility in configuration, laser sources, and detection techniques. As above. Class 4 laser product requiring in vivo imaging to be performed in a laser tight, interlocked chamber. >$1M High. Normally requires dedicated facility and technician/Operator. Substantial learning curve. Second Harmonic Generation (SHG) As above, Deep imaging of unlabeled collagen, concurrent imaging with MP As above, but also needs collection of light propagating in the direction of illumination (i.e. a detector beyond the subject) >$1M Miniature Confocal Ability to image unsectioned tissue (1-300um; tissue dependent). Flexibility to image tissues from any angle at the operating table (ie. microscope to the animal) Miniaturisation results in a highly integrated system with fewer options than large systems. Several $100K Low. Short learning curve, can be used by anyone that is working with whole animals.
  38. 38. Bundled Fibre vs. Point Scanning Bundled Fibre (Raw Image) Optiscan Point Scanning (Raw Image) Bundled Fibre (Area of Focus) Optiscan Point Scanning (Area of Focus) Bundled Fibre (Zoomed in Area of Focus) Optiscan Point Scanning (Zoomed in Area of Focus)
  39. 39. Summary • Advances in fluorophores, imaging systems, and protocols enable the interrogation of living systems in dynamic microscopic detail • In Vivo Microscopy enables capture of specific cellular events impossible to recreate in vitro or ex vivo • Longitudinal studies are possible using miniaturized devices • The unexpected nature of some findings suggests an important role in understanding both physiological and pathological cellular and sub-cellular events in vivo
  40. 40. Peter Delaney Chief Technology Officer, Optiscan Pty. Ltd For more information on the technology and research applications discussed in this webinar, please visit www.Scintica.com, www.viewnvivo.com, or email info@Scintica.com Thank You #LifeScienceWebinar #ISCxScintica

×