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  • 08/12/11
  • We chose a transgenic mouse line developed in the Sanes lab at Harvard University. Expressing fluorescent protein under the control of the neuron specific promoter, Thy1, multiple transgenic mice lines were developed with neuronal expression of fluorescent protein. Transgene insertion site and the number of transgene copies results in unique expression patterns across the multiple mouse lines. In one strain, YFP-H, expression can be seen throughout the cerebrum and brainstem, but, importantly in the neocortex expression is limited to Layer V pyramidal neurons, a population of neurons our lab has previously shown is particularly vulnerable to diffuse axotomy.
  • As we will discuss in further detail later, diffuse axotomy following cFPI can easily be observed in the axons of Layer V pyramidal neurons. You can clearly see both the proximal and distal processes and further can correlate the damaged axon with its related somata of origin.
  • The locus of injury was consistent at about 1 mm proximal to the chiasm. diffusely injured axons exhibiting the development of focal axonal swellings proximally and distally over time. And progressive dieback of the proximal and distal axonal segments over time. reflected in the finding that the number of axonal swellings per unit area decreased proximally while increasing distally from the injury. Figure 3. Spatial and temporal progression of diffuse axonal injury within the optic nerve following fluid percussion brain injury visualized via confocal microscopy of the intraaxonal YFP fluorescence. The top panel shows, in a sham injured animal, the intense YFP fluorescence that can be visualized within the individual axons coursing within the optic nerve. The insert is provided to illustrate the points within the optic nerve assessed via confocal microscopy, with the rostral segment originating from the globe and the caudal segment terminating in the optic chiasm. Note that at 1h postinjury, little overt damage can be seen within the optic nerve. The majority of fibers manifest normal morphology, with only scattered, small punctate axonal swellings. Although the center of the optic nerve (for detail see insert) gives the impression of decreased immunofluorescence, this is related to the development of local brain edema, which exerts a quenching effect upon local immunofluorescence (also see Figure 7). At 3h to 12h postinjury, this immunofluorescence quenching continues, although there is now evidence of axonal disconnection and dramatic swelling on the proximal axonal segments in continuity with the retina. In contrast, over the next 24h and 48 h periods, these proximal swellings become less prominent, with a concomitant increase in caudal axonal swellings (Figure 4). We interpret this decline in the rostral swellings and their apparent continued separation from the caudal swellings to reflect the dieback of the rostral segment (Figure 5), with the implication that long-range evaluation of the caudal axonal swellings may serve as a more valuable tool in assessing the overall burden of axonal damage.
  • Confocal microscopic evidence of axonal swellings within the optic nerve of Thy1-YFP-16 mice at 12 hours post TBI. The axonal swellings now manifest further maturation (A). Note that the distal axonal swellings expand to form spheroids (B and D), while the proximal axonal swellings now assume a more truncated form (C and E). Scale bar, 200 μm (A), 20μm (B, C, D and E).
  • After 2 days survival, the optic nerves, optic chiasm, as well as the relevant target tissues were well labeled with CTB fluorescence (Fig. 7). At this time point, the optic nerve was labeled fully by CTB-fluorescence. The decussation of retinal projections could be seen in the optic chiasm. The controlateral, ipsilateral as well as overlapping (Fig. 7 yellow) retinal projections to the SCN, LGN, SC and OPN were well delineated. The LGN, SC and OPN were innervated by optic nerve fibers, with most originating from the contralateral side. Only a small part of the LGN, OPN and the rostral SC was innervated by the ipsilateral optic nerve. While we appreciate that these studies were only conducted in normal rats, they constitute important proof of concept studies, suggesting the feasibility of this approach planned in the studies of downstream consequences of the mice optic nerve DAI.
  • An important point to emphasize is that using this model we can identify and study axonal pathology in great detail from minutes to weeks post injury. Here, at 30 minutes post-injury, one can clearly see the disconnection along the length of the axon, with beading and swelling of both the proximal and distal processes. One important question is how does this approach compare with known methods for assessment of axonal injury.
  • 3d PI
  • GFC – electren lucent center devoid of ribonucleic acids contains protein involved in SUMOylation pathway – SUMO-1 and Ubc9 (and not Ubiquitination pathway) UBF – IHC marker of GFC’s
  • 08/12/11
  • 08/12/11
  • In normal brain BAD and Bcl-xL are separate The Distance is greater than 100 Angstroms
  • In traumatically injured brain, if the apoptotic cascade is active: BAD and Bcl-xL Heterodimerize and the Distance is less than 100 Angstroms
  • As we will discuss in further detail later, diffuse axotomy following cFPI can easily be observed in the axons of Layer V pyramidal neurons. You can clearly see both the proximal and distal processes and further can correlate the damaged axon with its related somata of origin.
  • Brian Kelley has further described the pathology related to perisomatic axotomy Perisomatic axotomy abundant in the thalamus ultra-rapid: 5-15 min after injury within 100  m of the soma, prior to the first myelinated segment detectable up to 72 hours after injury These observations bring us to the question…
  • 08/12/11
  • While this approach allows for the study of proximal processes at later time points, it also may help in understanding how early, punctate swelling evolve into these highly complex processes later on. Here, 3d post injury, reactive change suggestive of reactive sprouting can be see following image deconvolution. Here note the multiple fibers projecting from the proximal stump.
  • With increased knowledge concerning the fate of the related somata, questions remained about the fate of the proximal processes at later time points. At later time points post-injury the majority of proximal processes are much more complex than those simple swellings seen at earlier time points, as they have multiple brances and many have a more tortuous course through the neocortical gray matter. When measured, these proximal processes show a significant increase in length at later time points, all suggestive of reactive and perhaps regenerative changes occuring in these processes at later time points.
  • SLIDE

Povlishock, John Povlishock, John Presentation Transcript

  • NNTS 2011 J T Povlishock
  • Acknowledgements
    • Jonny Liftshiz- Ph.D.
    • John Greer-MD-Ph.D.-student
    • NINDS-P30
    • Scott Henderson-Ph.D.
    • My goal is to discuss DAI and its upstream and downstream consequences through the use of traditional as well as modern imaging approaches, with emphasis on the unique capabilities of modern imaging for unmasking this complex pathobiology.
    Retrograde Anterograde
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  • Darkfield Microscopy
    • highly refractile structures
    • autoradiography
    • silver / gold labelling
    • Can detect but not resolve structures
    • below the resolution limit
    Molecular Expressions - http://www.microscopy.fsu.edu/
  • Phase Contrast Microscopy
    • differences in refractive index cause phase shifts
    • requires phase objective & condenser
    • less compatible with fluorescence microscopy
    • compatible with plastic substrates
    • both sides of sample must be clean
    Molecular Expressions - http://www.microscopy.fsu.edu/
  • Differential Interference Contrast Microscopy (DIC) - Nomarski
    • requires polarizer, analyzer & prisms
    • compatible with fluorescence microscopy
    • incompatible with plastic substrates
    • both sides of sample must be clean
    Molecular Expressions - http://www.microscopy.fsu.edu/
  • Fluorescence Microscopy Molecular Expressions - http://www.microscopy.fsu.edu/
  • Electron Microscopy
  • ModernTransmission Electron Microscopy
    • Provides unprecentended subcellular and synaptic detail
    • Further detail has been provided through the use of 3D image reconstruction employing SSTEM,SBFSEM & SSET.
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  • However, these TEM approaches provide major challenges as they require:
    • Accurate image alignment
    • Contour recognition
    • Automated segmentation
    • Error checking
    • Importantly, does not allow dynamic imaging
  • Microscopy Physics Mathematics Biology Chemistry Computer Sciences
    • Hardware development
    • optics
    • illumination sources
    • detectors
    Probe development Image Processing & Analysis
    • Clinical Applications
    • Diagnostics
    • Therapy
    Basic Research
  • Click to edit Master title style Human Diffuse DAI
  • Using multiple imaging tools the study of traumatic axonal injury as well as TBI in general, has been aided:
    • By the development of a wide-array of commercially available antibodies, together with multiple immunocytochemical approaches using chromagen based or fluorescent labeling.
    • By the use transgenic mice expressing fluorescent reporters in specific populations of neurons/injured neurons.
    • By the use of knockout mice breed with these endogenously fluorescent mouse strains.
  • Fluorescence Microscopy Molecular Expressions - http://www.microscopy.fsu.edu/
  • Buki et al. J. Neurosci. 20:2825,2000
  • Confocal Microscopy
  • Laser Scanning Confocal Microscope Molecular Expressions - http://www.microscopy.fsu.edu/
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  • Point Spread Function widefield confocal
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  • YFP Expression in YFP-H strain
    • YFP under the control of Thy1 promoter (neuron specific)
    • Transgene insertion location and/or copy number results in unique expression patterns
    • YFP-H expresses YFP throughout cerebrum, but..
    • In neocortex expression is limited to Layer V pyramidal neurons
    Feng et al., 2000
  • Mouse Central Fluid Percussion Injury Model
    • A circular craniotomy made along sagittal suture midway between bregma and lambda
    • Leur-Loc syringe hub affixed to craniotomy site using cyanoacrylate
    • Moderate severity (1.65-1.75)
    • No contusion
    • No intraparenchymal hemorrhage
  • Utilization of YFP-H Mice Allows the Continued Visualization of Axonal Damage and its Sequelae Over Time 24H 24H
  • cFPI in Thy1-YFP-H Mice Results in TAI in Layer V Greer et al. J. Neurosci. 31:5089, 2011
  • Sham 1h 3h 12h 24h 48h Wang et al. J. Neurotrauma, 8: 2011 P321
  • 12 hour 200 μm 20μm 20μm
  • 14d post-injury
  • Scale Bar: 500µm
  • Technique Extends the Temporal Window for Studying Axotomized Processes and Sustaining Somata 30m
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  • Phospho-c-Jun Expression is Induced Preferentially within Layer V Pyramidal Neurons Sustaining TAI Greer et al. J. Neurosci 30: 5089,2011
  • Phospho-c-Jun Expression is Induced Preferentially within Layer V Pyramidal Neurons Sustaining TAI
  • TAI Induces Persistent Neuronal Atrophy
  • Importantly, in some cases, these Fluorophore-containing elements can be targeted for TEM analysis.
    • Specifically, antibodies to the Alexa 488 Fluorophore can be used with subsequent use of avidin/biotin/peroxidase strategies to generate an electron dense reaction product
    • Alternatively, antibodies with the appropriate chromagen targeted to the protein of interest can be used to yield an electron dense reaction product
  • Ultrastructural Evidence of Reactive Change
  • The use of antibodies to fluorophores with their subsequent conversion to an electron dense reaction product Farkas et el. J. Neurosci 26:3130, 2006 VCU
  • Alternatively, photoconversion using confocal laser scanning microscopy can be employed for subsequent TEM analysis.
    • Specifically, using region of interest scanning capabilities of the confocal microscope, together with an acousto-optical filter a controlled and regional specific reaction product can be generated in the presence of diaminobenzidine.
    Tozer et al. J. Neurosci. Methods 164:240, 2006
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  • Bioimaging in determining drug targets
  • Ca 2+ Na + Na + Ca 2+ Procaspase 9 Procaspase 3 Calpain Calcineurin Cyto c Apaf-1
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  • Illuminating brain tissue protein interactions using confocal and two-photon excitation fluorescent resonance energy transfer microscopy-FRET Mills et al. J. Biomed Optics 8:347-356
  • Multi-photon laser scanning microscopy MPLSM 2-photon laser scanning microscopy 2P-LSM
  • Multi-photon (2-photon) laser scanning microscope
    • fixed stage upright (in situ / physiology)
    • 3 confocal detectors
    • (2 descanned + META)
    • 2 direct (non-descanned) detectors
    • transmitted light detector
    • 4 lasers:
      • Argon (458, 476, 488, 514 nm)
      • yellow diode (561 nm)
      • red HeNe (633 nm)
      • Ti:sapphire (710 - 990 nm) (MP)
    • high NA (oil & water) IR objective lenses
    • + dipping IR objective lenses
    • 3 stages
      • standard translating stage
      • slice incubation
      • stereotatic
    • Luigs & Neumann 380FM workstation
    • micromanipulators
    • AMPI Master 8 VP timing unit
    • stimulators
    • Multiclamp 700 amplifier
    • pre-amplifier - A/D board
    • peristaltic perfusion pump
    • in-line heater
    Zeiss LSM 510 META NLO
  • Bcl-xL BAD 520 nm 488 nm
  • Bcl-xL BAD 580nm 488 nm
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  • In Vivo and In Vitro Imaging
    • Multiphoton Approaches
    • Spinning Disk Confocal Microscopy
  • Multi-photon (2-photon) laser scanning microscope
    • fixed stage upright (in situ / physiology)
    • 3 confocal detectors
    • (2 descanned + META)
    • 2 direct (non-descanned) detectors
    • transmitted light detector
    • 4 lasers:
      • Argon (458, 476, 488, 514 nm)
      • yellow diode (561 nm)
      • red HeNe (633 nm)
      • Ti:sapphire (710 - 990 nm) (MP)
    • high NA (oil & water) IR objective lenses
    • + dipping IR objective lenses
    • 3 stages
      • standard translating stage
      • slice incubation
      • stereotatic
    • Luigs & Neumann 380FM workstation
    • micromanipulators
    • AMPI Master 8 VP timing unit
    • stimulators
    • Multiclamp 700 amplifier
    • pre-amplifier - A/D board
    • peristaltic perfusion pump
    • in-line heater
    Zeiss LSM 510 META NLO
  • Multi-photon (2-photon) microscopy
    • Reduced photo-toxicity
    • (IR light produces fewer free radicals)
    • Reduced photo-bleaching
    • (only volume around focal point is excited)
    • Greater depth of imaging
    • (IR is less scattered than shorter wavelengths)
    • Prolonged live cell studies
    • Thick sample imaging
    • In situ imaging
  • Ultrafast Pulsed Lasers for MPLSM High peak power Low average power Short pulse width
    • average power laser = 10 mW
    • pulse rate = 100 MHz
    • pulsewidth = 100 fsec.
    • energy per pulse = average power/pulse rate = 0.1 nJ
    • peak power = energy per pulse/pulsewidth = 1000W
    • duty cycle = pulsewidth x pulse rate = 0.001%
    • period (time between pulses) = 1/pulse rate = 100 msec
  • Multi-photon (2-photon) laser scanning microscope
    • fixed stage upright (in situ / physiology)
    • 3 confocal detectors
    • (2 descanned + META)
    • 2 direct (non-descanned) detectors
    • transmitted light detector
    • 4 lasers:
      • Argon (458, 476, 488, 514 nm)
      • yellow diode (561 nm)
      • red HeNe (633 nm)
      • Ti:sapphire (710 - 990 nm) (MP)
    • high NA (oil & water) IR objective lenses
    • + dipping IR objective lenses
    • 3 stages
      • standard translating stage
      • slice incubation
      • stereotatic
    • Luigs & Neumann 380FM workstation
    • micromanipulators
    • AMPI Master 8 VP timing unit
    • stimulators
    • Multiclamp 700 amplifier
    • pre-amplifier - A/D board
    • peristaltic perfusion pump
    • in-line heater
    Zeiss LSM 510 META NLO
  • Spinning Disc confocal microscope
    • High-speed multi-dimensional confocal imaging
    • Ca 2+ imaging
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  • Axotomized and intact neurons are easily identified in the living slice and labeled with biocytin during whole cell patch clamp recordings Axotomy at both short (A) and long (B, C) distances from the soma could be easily identified by the bulb on the axon in the live slice prior to patch clamping recordings. Two focus levels of same image are seen in B. Greer & Jacobs, Poster 320
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  • Both intact and axotomized neurons are healthy Parameters shown are typically measured when assessing the physiological health of recorded neurons. For these ‘health’ parameters, injured neurons were not different from controls. Although there was a trend towards an increase in input resistance for some injured neurons, suggesting possible cell shrinkage, this was not significant. Data shown are from the following number of neurons: 15 control, 9 axotomized and 6 intact one day after injury, and 6 axotomized and 6 intact two days after injury.
  • Axotomized neurons show a decrease in intrinsic excitability at 1 day that recovers by 2 days after injury. Intact neurons show an increase in intrinsic excitability at 2 days post injury Membrane properties reflecting intrinsic excitability show a decrease for axotomized neurons one day after injury and an increase for other groups relative to controls. During depolarizing current steps, intact neurons from injured brains tended to fire at increased frequencies relative to control cells (A, B). A shows examples from individual neurons. The slope of the plot of frequency versus injected current was significantly different between experimental groups (B, 1-way ANOVA, p<0.05, LSD post-hoc). The rheobase (lowest current to produce action potentials) was significantly greater in axotomized neurons one day after injury (C, 1-way ANOVA, p<0.05, LSD post-hoc). The primary instantaneous frequency = 1/(interspike interval for first 2 action potentials responding to injected current). Only non-doublet RS neurons were used to calculate this measure. There was a trend toward a lower frequency for axotomized neurons one day after injury (D-F), while there was a significant increase in this measure for intact neurons two days after injury (D-F, 1-way ANOVA, p<0.05, LSD post-hoc). D shows examples of the response to 400 (upper) and 200 (lower) pA current injections. E shows examples of the plot of primary frequency versus current injection for individual neurons. Data shown are from the following number of neurons: 15 control, 9 axotomized and 6 intact one day after injury, and 6 axotomized and 6 intact two days after injury.
  • 24H 24H
  • Acknowledgements
    • Andras Buki, M.D. Ph.D.
    • Daniel Erb, Ph.D.
    • Orsi Farkas, M.D. Ph.D.
    • Guoyi Gao, M.D
    • John Greer, M.D. Ph.D. student
    • Brian Kelley, M.D. Ph.D.
    • Hiro Koizumi, M.D. Ph.D.
    • Jonathan Lifshitz, Ph.D.
    • Tina Marmarou, Ph.D.
    • Melisa McGinn, Ph.D.
    • Yasukata Oda, M.D. Ph.D.
    • David Okonkwo, M.D. Ph.D.
    • Ted Pettus, Ph.D.
    • Linda Phillips, Ph.D
    • Tom Reeves, Ph.D.
    • Rich Singleton, M.D. Ph.D.
    • James Stone, M.D. Ph.D.
    • Eiichi Suehiro, M.D. Ph.D.
    • Yugi Ueda, M.D. Ph.D.
    • Joan Wang, M.D.
    • Enoch Wei, Ph.D.
  • Image formation in TEM
    • column under vacuum
    • electron beam focused by magnetic lenses
    • electrons interact with stained sample
    • elastic (2) & inelastic (3) scattering of electrons
    • scattered electrons blocked by aperture
    • non-scattered electrons (1) strike phosphorescent screen or film or CCD
  • DAI Axotomy
    • APP positive swelling and disconnection
    Kelley et al., Poster Number 64
  • Proximal YFP Swellings, Not Distal Contain  -APP
  • CALPAIN MEDIATED SPECTRIN PROTEOLYSIS (CMSP) AND NFC CO-LOCALIZE AT FOCAL SITES OF TAI Calpain has been shown to target spectrin a major structural link of the actin cytoskeleton to the axolemma. AB38 (CMSP) and NFC show a co-localization. Buki et al., 1999
  • Early Reactive Sprouting in Phospho-c-Jun+ Neurons
  • Evidence of Reactive Sprouting Early Post Injury * * * ** ** * ** * * C A 2D
  • Technique Extends the Temporal Window for Studying Axotomized Processes and Sustaining Somata 14D 14D 7D 7D * *
  • Early Morphological Evidence for Regeneration at Later Time Points Post-Injury
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  • DIFFERENTIAL RESPONSE OF AXONS TO TBI Stone, J.R., Singleton, R.H., and Povlishock, J.T. Experimental Neurology 172, 320 – 331 (2001) C-APP RMO14
  • YFP-positive neurons show a variety of action potential firing patterns. Two days after injury, intact neurons are less likely to show bursts of action potentials Most YFP recorded neurons were non-adapting, non-doublet regular-spiking (RS) pyramidal neurons. A small percentage of YFP-labeled neurons were of the previously described intrinsically-bursting (IB) type (A, B, Connors, 1990). Some RS neurons had an initial doublet of action potentials, followed by non-adapting, single action potentials (C). These were similar to those previously reported for YFP-H neurons (Yu, 2008). Non-doublet RS neurons were also similar to those previously described for layer V somatosensory neurons in YFP-H mice (D, Sugino). A series of hyperpolarizing currents produced rectification and a prominent sag of the voltage response that is likely due to hyperpolarization-activated current (E arrow). A transient calcium (T) current also appeared to be present in some cells as indicated by the action potentials produced after return to rest from hyperpolarization (E). All examples shown (A-E) are from controls. Intact neurons recorded two days after injury, only produced single action potentials, thus this population had a significantly different percentage of the three cell types compared to the control population (F, z-test, p<0.05). The other injured groups showed these three neuronal types in percentages similar to controls. Data shown are from the following number of neurons: 20 control, 10 axotomized and 8 intact one day after injury, and 7 axotomized and 6 intact two days after injury. The IB cell type was excluded from subsequent analyses.
  • Changes in intrinsic properties of regular-spiking neurons suggest modifications of potassium channels Action potential amplitude was measured with respect to the -60 mV resting membrane potential (RMP), where the cells were held with small constant depolarizing or hyperpolarizing currents when necessary. The action potential amplitude was increased in axotomized neurons one and two days after injury and in intact neurons one day after injury (A, B). Lower dashed line in A indicates -60 mV resting membrane potential, while upper dashed line identifies the peak for the control cell shown. There was no change in either action potential rise time (C) or action potential threshold (D), measures sensitive to changes in numbers or density of sodium channels. The after-hyperpolarization (note only non-doublet RS cells were used for this measure) was decreased in the same groups showing an increase in action potential amplitude (E, F), suggesting a possible reduction in potassium channels. Data shown are from the following number of neurons: 15 control, 9 axotomized and 6 intact one day after injury, and 6 axotomized and 6 intact two days after injury.
  • Spinning disc confocal microscope Molecular Expressions - http://www.microscopy.fsu.edu/
  • Cyclosporin A attenuates both mitochondrial injury…
  •  
  • Fluorescence-based Detection
    • Advantages
    • sensitive
    • low background
    • rapid results
    • polychromatic, multiple spectral identities
    • wide dynamic range
    • quantifiable
    • non-hazardous
    • good spatial resolution
    • Disadvantages
    • background from autofluorescence
    • non-permanent preparation
    • reporter altered by imaging
  • Fluorescent molecules DAPI DiI FITC Fura-2 Hoechst Indo-1 Propidium Iodide Texas Red Alexa 488
  • Fluorescent Proteins GFP fluorescent protein emission GFP http://tsienlab.ucsd.edu/
  • Immunofluorescence FITC Alexa 488 Cy2 rhodamine Alexa 568 Cy3 Texas Red
  • Fluorescent molecule properties
    • Quantum yield (fluorescence efficiency) (F) - ratio of photon emission / photon absorption
    • Extinction co-efficient (e) - absorption of reference wavelength of light by 1 M solution of molecule over 1 cm (measure of ability of fluorophor to absorb light)
      • (high extinction co-efficients have a high probability of fluorescence)
    • Lifetime (t) - time molecule remains in excited state before returning to ground state
    • Environmental sensitivity - pH, O 2 , solvent polarity
  • Digital Imaging
    • Types of detectors
      • CCD
      • PMT
    • Image resolution
    • Temporal resolution (speed)
    • Quantum efficiency (sensitivity)
    • Spectral properties of detector
    • Nyquist sampling
    • Binning (CCD)
    • Noise reduction / averaging
    • Bit depth (gray level discrimination)
  • Confocal Microscopy
  • Spinning disc confocal microscope Molecular Expressions - http://www.microscopy.fsu.edu/
  • Confocal - Spinning disc vs. Laser scanning
    • Spinning disc
      • better speed (acquisition speed limited by detector rate)
        • ( i.e. CCD off-loading)
      • compatible with variety of illumination sources
      • poor light throughput
      • fixed pinhole size
    • Laser scanning
      • better lateral resolution
      • better light throughput
      • pinhole optimization
      • PMT detector
        • large detection area
        • amplify signal
        • low dark current
        • high dynamic range
        • rapid refresh rate
      • slower acquisition time
      • laser dependant
  • Optimization of confocal images
    • Choice of mounting & immersion media
    • Choice of coverslip thickness
    • Selection of high N.A. objective lens
    • Pinhole adjustment
    • Correction for cross-talk
    • Selection of scan resolution
    • Selection of acquisition bit depth
    • Selection of scan speed
    • Averaging of acquired image
    • Elimination of background autofluorescence
  • Cyclosporin A attenuates both mitochondrial injury…
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  • Mitochondria: Death Switch of the Cell? MPTP Smac/Diablo AIF P. Sullivan Death Triggers Ca 2+ Glutamate Bax Oxidants Caspases Cyto C Cyto C Caspase 9 Apaf-1 dATP Downstream Caspases APOPTOSIS NECROSIS ATP  ROS