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Calcium Imaging and Caged Compounds Roger Thompson BSC 5936 Spring 2005.

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Presentation on theme: "Calcium Imaging and Caged Compounds Roger Thompson BSC 5936 Spring 2005."— Presentation transcript:

1 Calcium Imaging and Caged Compounds Roger Thompson BSC 5936 Spring 2005

2 Introduction Calcium cannot be visualized directly Specific molecules are used that have optical properties which change upon interacting with calcium Calcium concentrations can change in milli- seconds Calcium acts as a universal 2 nd messenger Calcium regulates cellular processes such as muscle contraction, fertilization, cell division, blood clotting, and synaptic transmission

3 Fluorescence Requires molecules called fluorophores or fluorescent dyes Properties include: absorbance, lifetime, intensity, & spectra Is the result of a three stage process 3 stages are: –Excitation –Excited-state lifetime –Fluorescence emission

4 Instrumentation Detection system –Fluorophore –Wavelength filters –Detector –Excitation source Types of instruments –Spectrofluorometer –Fluorescence Microscope –Flow cytometer

5 Chemical Fluorescent Indicators

6 Selection Criteria Calcium Concentration range –Near dissociation constant (K d ) (best) –Detectable at 0.1K d to 10K d Delivery method Measurement –Quantitative or qualitative –Ion concentration –Instruments –Sources of noise Indicator’s light intensity Other physiological parameters –Simultaneous patch-clamp

7 Ultraviolet Wavelength Excitation Fluorescent Indicators Fura-2, Indo-1 and derivatives Quin-2 and derivatives Intermediate Ca 2+ -binding affinity –Fura-4F, Fura-5F & Fura-6F; plus –Benzothiaza-1 & 2 Low-affinity indicators –Fura-FF, BTC, Mag-Fura-2, Mag-Fura-5, and Mag-Indo-1

8 Visible Wavelength Excitation Fluorescent Indicators Fluo-3, Rhod-2 and derivatives Low-affinity indicators –Fluo-5N, Rhod-5N, X-Rhod-5N and derivatives Calcium Green, Calcium Orange, Calcium Crimson Oregon Green 488 BAPTA indicators Fura Red indicator Calcein

9 Ratiometric vs. NonRatiometric Ratiometric –Includes indo-1 and fura-2 –Allows for correction of differences of path length and accessible volume in three dimensional specimens –Indicator has 2 excitable wavelengths Indo-1 at 405- and 485 nm Nonratiometric –Includes fluo-3, rhod-2 and the calcium green class

10 Bioluminescent Calcium Indicators

11 Bioluminescence Light produced by biological organisms Low intensity Assays are sensitive and free of background One example is Aequorin –Photoprotein isolated from luminescent jellyfish –Is not exported or secreted; not compartmentalized –Usually microinjected into cells –Has 3 Ca 2+ binding sites

12 Ca 2+ -binding Photoproteins Visible bioluminescence by an intramolecular reaction in the presence of calcium Simple instrument requirements Not affected by photobleaching Ex: Obelin and Aequorin Problems –Loading, detection and calibration

13 Green Fluorescent Protein-based Photosensitive proteins synthesized by the jellyfish Aequorea victoria Can be cloned and fused with DNA Provides marker for gene expression and protein localization in living organisms

14 Dye-loading Procedures

15 Ester Loading –Derivatized with an AM (acetoxymethyl) ester –Passively diffuses through plasma membrane –Subject to compartmentalization or incomplete hydrolysis Microinjection –Intermittent injection of indicator dissolved in cytosolic-like solution via glass micropipette –Invasive technique

16 Diffusion from Patch- Clamp pipettes –Passive microinjection; i.e. perforated patch technique using substance such as Nystatin Diffusion through Gap Junctions –Retrograde perfusion w/ a low Ca 2+ solution containing collagenase and proteases followed by mechanical dissociation of cells

17 ATP-induced permeabilization –Extracellular ATP induces cation flux and increases permeability of the plasma membrane via ATP 4- receptor Hyposmotic Shock Treatment –Several washes in Ca 2+ free solution, followed by hyposmotic solution containing indicator –Can kill cells

18 Gravity Loading –Ca 2+ free solution wash –Centrifugation –Incubation with indicator –Centrifugation Scrape Loading –Cultured cells are scraped from dish while in buffer containing indicator –Scraping rips holes in membrane

19 Lipotransfer Delivery Method –Uses membrane permeant cationic liposomes containing indicator/dye Fused Cell Hybrids –Photoproteins are loaded by fusing cells and human erythrocyte “ghosts” contain the photoprotein in a medium containing Sendai virus

20 Potential Problems of Ca 2+ Indicators and their Solutions

21 Intracellular buffering –Indicator can alter [Ca 2+ ] i when loaded in high concentration Cytotoxicity –Can damage some types of cells –Can affect redox metabolism or cell proliferation

22 Autofluorescence –Collagen fibers and calcifications can give off autofluorescence –Pyridine nucleotides may also. These include NADH, NADP, FAD, and FMN Bleaching –Too much illumination or photodamage –Can be diminished by adding O 2 or an antioxidant

23 Compartmentalization –Indicator becomes trapped within some intracellular organelles and is not homogeneous throughout the cell Binding to Other Ions & Proteins –Indicators may bind to intracellular proteins and alter their fluorescent properties including changes in spectrum, kinetics, or K d

24 Dye Leakage –Indicators may leak from the cytosol into the extracellular medium –Is regulated by anion transporter system –Can be inhibited by probenecid, sulfinpyrazone or low temperature

25 Techniques for measuring Ca 2+

26 Optical Techniques Multiparameter digitized video microscopy Confocal laser scanning microscopy –Scans specimen collecting emitted fluorescence thru a pinhole Two-photon excitation laser scanning microscopy –One fluorophore is excited by two individual photons simultaneously Pulsed-laser imaging for rapid Ca 2+ gradients –Allows submicron spatial resolution and msec temporal resolution Time-resolved fluorescence lifetime imaging microscopy (FLIM) –(  ) time in excited state to ground state Photomultiplier tube Flow cytometry –Flowing stream suspension

27 Non-Optical Techniques Electrophysiology –Currents generated by Ca 2+ -dependent ion channels Ca 2+ -selective electrodes –Ion-complexing ligand in a liquid lipophilic membrane Vibrating Ca 2+ -selective probe –Ion-selective; non-invasive; located w/in microns of the cell surface; measures ion flux

28 Caged Compounds

29 Characteristics Photosensitive chelators of ions or substances such as Ca 2+, H +, ATP, or IP 3 Exist as an inactive form due to combination with radicals such as the nitrophenyl group “chemically caged” Activated by exposure to UV illumination Release of Ca 2+ in  sec or msec

30 Ratiometric intracellular calcium imaging in the isolated beating rat heart using indo-1 fluorescence Eerbeek etal., 2004 J. Appl. Physiol. 97:2042-2050.

31 Purpose Describes an optical ratio imaging setup and an analysis method for the beat-to-beat Ca i 2+ videofluorescence images of an indo-1 loaded, isolated Tyrode-perfused beating rat heart. Possible to register different temporal Ca i 2+ transients together with left ventricle pressure changes.

32 Why? Because abnormalities in intracellular calcium (Ca i 2+ ) handling has been implicated as the underlying mechanism in a large number of pathologies in the heart; i.e., contraction, electrophysiological properties, mitochondrial function, heart failure, and cardiac hypertrophy.

33 How? Used indo-1. Requires a single-excitation wavelength of light and two emission wavelengths to be recorded Visualization of Ca i 2+ changes in time and space A multiviewer (filters/dichroic mirrors) split emission light into two wavelengths; projected the two mages on a CCD chip of a CCD camera

34 Fig. 1. A: schematic diagram of the dual- wavelength videofluorometric measurement system. The 365-nm excitation light, which is provided by a 100-W Mercury (Hg) arc lamp, is selected by a 365-nm filter (UG-1 filter), DCLP 390 is a dichroic mirror in the beam splitter, which is translucent for wavelengths higher than 385 nm. B: the dichroic mirrors DCLP 455 are translucent for wavelengths higher than 455 nm. Both light paths use filters 405_+ 10nm and 485 _+ 20 nm, respectively. Both images are projected simultaneously on the cathode (I) of a second-generation image intensifier tube by a macro lens charge-coupled device (CCD) of a cooled video camera. BP, band-pass filter; LVP, left ventricular pressure.

35 Fig. 2. A: a hear image is shown on which the 5 X 5 matrix pattern (spatial resolution 1.8 mm) is superimposed to illustrate how the spatial dependency of the cytosolic calcium (Ca i 2+ ) was analyzed. The whole matrix, including the uppermost left part, is covering the left ventricle of the heart. Symbols are the same as Fig. 6 (symbols are associated with that heart position). RP, reference point for the image processing. B: the three traces show the indo-1 ratio signals of the 3 selected areas during a whole heart cycle before filtering.

36 Fig. 3. NADH autofluorscence (NADH / uranyl) measured at 450 nm, during the first 100 ms of the heart cycle, before and after a mimicked loading procedure.

37 Fig 4. Two images are shown at 485 nm. A: NADH image (487nm). B: the same heart after loading with indo-1 at the same wavelength.

38 Fig. 5. Image at both wavelengths (405 and 485 nm) and the ratio image after a small lesion (pinching the tissue) on the epicardium of an indo-1- loaded heart.

39 Fig. 6. Time course of the indo-1 ratio (  ) and the left ventricular pressure (0) during a whole heart cycle of 200 ms (pacing frequency 300/min). The indo-1 ratio is corrected for the autofluorescence.

40 Fig. 7. Time course of the indo-1 ratio of the 3 squares o,, and  (positions shown in Fig. 2A) produced with a 5 X 5 matrix (spatial resolution 1.8 mm) during a heart cycle of 200 ms (pacing frequency 300/min). A and B with stimulation of the right atrium, and C and D, with direct stimulation of the left ventricle, show the time course of the indo-1 ratio. B and D after addition of 2,3-butanedione monoxine (BDM) (= diacetyl monoxine DAM). Inset in A and C shows the schematic heart with the position of the 3 squares in the matrix and in C also the position of the stimulation electrode (stim).

41 Fig. 8. A and B: Time course of the indo-1 ratio at different locations in 2 different hearts (positions shown in each matrix) produced with a 15 X 15 matrix (spatial resolution 0.6 mm) during the first 100 ms of the heart cycle (pacing frequency 300/min). In the horizontal direction a delay in calcium transients is present from left to right.

42 Fig. 9. A: steps taken from the acquired image from the CCD to a detailed analysis of calcium transients in a small portion of the left ventricle. The splitting of the CCD output into 2 images, representing the 405- and 485-nm wavelengths, is performed by using the 2 reference points (see MATERIALS AND METHODS ). The contrast and brightness of the ratio image (image at 405 nm divided by the image at 485 nm) is optimized to show more clearly the region of interest. It is clear that the ratio method is effective in canceling out inhomogeneities in the fluorescence at the same wavelengths. The area of interest is 4.2 by 4.2 mm and was analyzed by dividing it in 7 parts in either the horizontal or vertical direction. B: results of the analysis in both the horizontal (vertical binned) and vertical (horizontal binned) direction from 1 heart. In the horizontal direction, a delay in calcium transients is present from the left (1) to the right (7). This delay is absent in the vertical direction.

43 Results Ca i 2+ transients show that Ca i 2+ activation propagates horizontally from the left to right during sinus rhythm or from the stimulus site during direct left ventricle stimulation. The indo-1 ratiometric video technique allows the imaging of ratio changes of Ca i 2+ with a high temporal (1 ms) and spatial (0.6mm) resolution in the beating heart

44 Inhibition of inositol 1,4.5- trisphosphate-induced Ca 2+ release by cAMP-dependent protein kinase in a living cell Tertyshnikova & Fein, 1998 PNAS 95:1613-1617

45 Hypothesis Is the principal mechanism of cAMP- dependent inhibition of Ca 2+ mobilization by inhibition of IP 3 -induced Ca 2+ release or by stimulation of Ca 2+ removal from the cytoplasm?

46 Ca 2+ & cAMP Ubiquitous intracellular 2 nd messengers Believed to modulate each other cAMP can potentiate or inhibit agonist- induced Ca 2+ elevation depending on cell type

47 How tested? Used caged IP 3, caged Ca 2+, and caged cAMP to rapidly elevate their [C] i Carbacyclin = elevates cAMP ( via G s protein- dependent activation of adenylyl cyclase ) = inhibits IP 3 KT5720 and IP 20 used as inhibitors of carbacyclin SNP (sodium nitoprusside) = inhibits cGMP






53 Results Elevation of cAMP inhibits IP 3 -induced Ca 2+ release in an intact cell but does not affect the removal of Ca 2+ from the cytoplasm. Inhibition is mediated by cAMP Protein Kinase.

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