Molecular Imaging - Ca2+ Imaging in Life Sciences PDF

Molecular Imaging - Ca2+ Imaging in Life Sciences - Fundamentals, Examples and Pitfalls Prof. Dr. Dr. Oliver Friedrich Institute of Medical Biotechnology (MBT) Department of Chemical and Biological Engineering Friedrich-Alexander-University Erlangen-Nürnbe...

Molecular Imaging - Ca2+ Imaging in Life Sciences - Fundamentals, Examples and Pitfalls Prof. Dr. Dr. Oliver Friedrich Institute of Medical Biotechnology (MBT) Department of Chemical and Biological Engineering Friedrich-Alexander-University Erlangen-Nürnberg www.chemiereport.at adjunct Affiliate: University of New South Wales (UNSW) Victor Chang Cardiac Research Institute, Sydney Queensland University of Technology (QUT), Brisbane creationwiki.org E-mail: [email protected] Web: http://www.mbt.tf.fau.de Environmental Dyes – Tricks and Pitfalls  Requirement for high Contrast in Life Cell Imaging It is not only about enough light….we need contrast! A ~0 B ↑ mikroskopie.de C Fluorescent environmental probes are used to detect/monitor bright field image of internal environment by increasing specific contrast an epithelial cell muscle cell stained for Ca2+ with Fluo-4 and monitored by confocal mircoscopy Fluorescent Environmental Dyes used to detect/monitor internal environment of cells and reactions to changes in, i.e. ion concentrations (Ca2+, Na+) pH membrane potential  Environmental Ca2+ Dyes ratiometric Ca2+ dyes Ratio: during fast alternating F340, F380 recordings c,d, K don‘t change non-ratiometric Ca2+ dyes Fluo-3 Problem non-ratiometric dyes: Fluorescence signal still depends on the total dye concentration which can hardly be controlled (dye may diffuse in organelles or becomes inactivated by strong light, tec.)  The Problem with Dyes as a Buffer System 1. a dye only reliably reflects changes in Ca2+ binding partner concentration (Ca2+, H+, Na+) around ±1.5 log concentration steps around Kd value 2. in living cells, many more potential binding partners competing for the ion apart from the dye  apparent dye Kd is usually greater (lower affinity) and ‚in situ‘ calibration must be attempted (at least for ratiometric dyes where dye concentration is not a concern)  In situ calibration of Dye-Ligand Relationships Jiang & Julian (1997) AJP Cell Physiol 273 Conversion of fluorescence signals towards ion concentrations  in situ calibration techniques using ion specific ionophores, followed by incubations at different ion log steps and record steady-state fluorescence Examples from Muscle Fluorescence Microscopy The Motor System – Source of Locomotion http://sitges-info.com www.lastfm.de/ Versalis  ~ cm Both et al. (2004),J Biomed Opt ~ mm < 100 µm Structure of Skeletal Muscle ~ 1 µm Friedrich (2009), In: Comparative High Pressure Biology T.Jöllenbeck, Universität Wuppertal Molecular Mechanism of Contraction Actin filament Sliding filaments troponin tropomyosin Myosin II Molecular motor HMM S1 S2 LMM (head) (neck) (shaft) lever arm regulatory light chain essential light chain www.unmc.edu/physiology/Mann/mann14.html Regulation of Muscle Contraction  Electrical stimulation leads to release of Ca2+  Ca2+ binding to troponin allows myosin-actin interaction  Ca2+-concentration: complex regulation in muscle cells Friedrich (2006) 12.12.2023 13 Who is a Player in Ca2+ Homeostasis in Muscle? Intracellular Ca2+ levels need to be tightly controlled….too much is no good and cells will die! 12.12.2023 14 Duration of Signals determine Imaging Speed  Confocal Ca2+-imaging Ca2+ dye emitted Ca2+- Fluorescence speed restricted by scan Ca2+ SR mirror actuators; conventional systems hν can do 1-2 fps @512x512 px XYT Image stacks recording @ 0.9 fps  Increase speed with XT External Field Stimulation scan (but omit one Caffeine Release coordinate) 12.12.2023 15 Measuring global Ca2+ in cardiac muscle cells Hypothesis:  Cardiac muscle cell loaded with Fluo-4 AM − Fluo-4 AM diffuses into cells, AM is cleaved by estherases, Fluo-4 trapped in cell  Electrical field stimulation by electrodes in chamber  Evaluation: peak intensity and transient decay rates XT Line-scan of global transients: XYT scan of Ca2+-wave Results: Scan line 12.12.2023 16 Friedrich et al. (2012) Prog Biophys Mol Biol  A Disease Model: Duchenne Muscular Dystrophy Duchenne Muscular Dystrophy: inherited, progressively wasting muscle disease; muscle weakness, tissue remodeling, death from respiratory failure (suffocation) or heart failure (pump failure). Molecular pthology is absence of the subsarcolemmal protein dystrophin iris.peabody.vanderbilt.edu Harper et al. (2002), Nat Med 12.12.2023 17 Hypotheses of Disease Progression in DMD Chan & Head (2011) Exp Physiol 12.12.2023 18 Ca2+ Transients in dystrophic Hearts…who‘s right? 12.12.2023 19  Examples of global Ca2+ Transients in Muscle Cells confocal Ca2+ recordings in skinned fibres: control IL-1 control IL-1 Low Mg2+-induced transients Friedrich et al. (2014), Am J Respir Cell Mol Biol caffeine-induced transients Friedrich et al. (2014), Am J Respir Cell Mol Biol 12.12.2023 20 Examples of global Ca2+ Transients in Muscle Cells confocal Ca2+ recordings in intact fibres: Teichmann et al. (2008) PLoS One Fluo-4 fluorescence in a single mdx muscle fibre loaded with Fluo-4 and challenged with hypertonic solution to put mechanical stress on membrane 12.12.2023 21  Confocal Ca2+ Microscopy of Microdomains Epifluorescence microscopy: only very limited spatial resolution. What if signals are confined to microdomains…?? transient field-stimulated spontaneous Cheng et al. (1993) Science spark Fluo-4: non ratiometric high affinity dye What is the trick? 12.12.2023 22 Imaging elementary Ca2+ Release Events („Sparks")  Requires high spatial resolution (only ~µm size)  Requires high temporal resolution (only ~ms long) xyt image series: xt line-scan: Spark is only one or two frames long only information from one line 12.12.2023 23  Line Scan recording in Muscle – Need for Speed Olympus FV 300,40xWI, 1.15 NA 60 µM fluo-4, xyt-series I=2s, N=100, axial res. 1 µm v Wegner et al. (2007), J Muscle Res Cell Motil Dt=2s Kirsch et al. (2001), J Physiol Line Scans Kirsch et al. (2001), J Physiol Advantage: much faster scanning speed compared to XYT (~2ms/line vs. ~1s/frame  ECRE dynamics & morphology can be resolved Disadvantage: One spatial dimension has to be sacrificed  two-dimensional ECRE morphology cannot be reconstructed Schnee et al. (2008), J Phys Conf Ser 12.12.2023 24 Ca2+ Sparks in dystrophic mdx Muscle Teichmann et al. (2008), PLoS One osmotic challenge transiently increases spark frequency in mdx muscle. Ca2+ elevations are not due to Ca2+ influx from exterior just count microdomain events in successive XYT stacks 12.12.2023 25 Ca2+ Sparks modulated by mechanosensitive Channels Prof. Frederick Sachs Buffalo University NY Center for Single Molecule Biophysics Physiology and Biophysical Sciences GsMTx-4: very specific cation-selective MsC blocker (Tarantula Grammostola spatulata) ‘If I had a child with MD, I would take the chance and give him GsMTx-4 rather than watch him decay’ (from an e-mail conversation 13/11/2008 ) Spiderman…. now also curing disease…? Teichmann et al. (2008), PLoS One 12.12.2023 26  Ca2+ Sparks in Sepsis and Inflammation heart muscle Zhu et al. (2005) Crit Care Med control drug Interleukin-1 blocks Ca2+ IL-1 sparks in skeletal muscle (in heart skeletal muscle muscle, IL-1 effect is opposite, increasing tetracaine spark frequency!!) Friedrich et al. (2014), Am J Respir Cell Mol Biol Multifocal Scanning  Laser beam splitted into 64 beamlets  Array of beamlets scanned  Detection on sCMOS camera TriM Scope II LaVision Biotech von Wegner et al. (2007), IEEE TMI 12.12.2023 28 Need for automated Detection and Analysis  How many sparks do you see? v Wegner et al. (2005), Biophys J 12.12.2023 29 Summary Fluorescence of environmental dyes strongly depends not only on the ligand concentration, but also on the ‚environmental condition‘ (pH, temp, pressure) Dissociation constants under in vitro conditions are always different from cellular conditions with competing natural buffers present and require a careful ‚in situ‘ calibration Dissecting the effects of environmental condition on the dissociation constant ensures correct interpretation of fluorescence signals in living cells and tissues 12.12.2023 30