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EasyRatioPro
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Fluorescence Imaging
 

Applications

EasyRatioPro Is Up For The Task For Many Applications

As a scientist, you don't want to make the wrong decision when buying an imaging system. Many systems on the market claim to be able to do all fluorescence imaging applications. However this is simply not true. Other systems are complex and offer so many options that will never even be used for Ion applications. This clutters and confuses researchers. EasyRatioPro has been designed for Ion Imaging applications. It is finely tuned for kinetic Imaging. Some applications are listed below. Simply do a scholar Google search for Photon Technology, and you will find thousands of applications where our fluorescence products have been used.

Calcium response from Fura2  loaded cells stimulated with glutamate. Data collected using an EasyRatioPro from PTI during McMaster University Biophotonics course.

 

 

Caffeine-induced Ca2+ release in HEK293 cells

Caffeine-induced Ca2+
Caffeine-induced Ca2+

Caffeine-induced Ca2+ release in HEK293 cells transfected with wild-type ryanodine receptor cDNA. Ca2+ release was measured by intracellular Fluo-4 fluorescence before (A) and after the addition (B) of 8 mM caffeine. Fluorescence change of one cell in B (arrow) was analyzed with PTI EasyRatioPro program (C).

The skeletal muscle ryanodine receptor ion channel (RyR1) is essential for muscle function by releasing from an intracellular membrane compartment, the sarcoplasmic reticulum, Ca2+ required for muscle contraction. To uncover the molecular determinants of gating, ion conductance and selectivity of RyR1 and understand the etiology of core myopathies, RyR1 single and multiple mutants are expressed in HEK293 cells. Their expression levels and function are investigated by immunoblot analysis and single channel measurements using the planar lipid bilayer method.  Retention of function in HEK293 cells is probed  by cellular Ca2+ release in response to the Ca2+ releasing drug caffeine using PTI EasyRatioPro instrumentation and software (Figure).

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Fura-2 Fluorescence

Fluorescence Imaging
Fluorescence Imaging
  • Fura-2/AM loaded DIV 7 neocortical culture
  • Illuminator: PTI DeltaScan
  • Camera: Hamamatsu c2400 SIT video camera
  • Software: PTI ImageMaster™

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In-Situ Calibration of Intracellular [Ca2+]i

Ratioing fluorescence intensities can be performed on fluorescence images as well. Cells loaded with fluorescent dye are illuminated by alternating 340 and 380 nm light and the resultant fluorescent images are captured by a sensitive video camera and ratioed pixel to pixel. The ratio image is displayed subsequently in pseudo-color with the ratio scale mapped to calcium concentration.

Fluorescence Imaging
Fluorescence Imaging

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Intracellular Calcium

Fluorescence Imaging
Fluorescence Imaging
  • Fura-2/AM loaded neurons
  • Illuminator: PTI DeltaRAM
  • Camera: Sensys CCD
  • Software: PTI ImageMaster™
  • Imaging: Typical Fura-2-fluorescence ratio imaging for intracellular Ca2+ in hippocampal neurons from old rats before NMDA (A) and after NMDA (B), scale values in nM

 

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Intracellular Ca2+ Measurements

  • Fura-2 loaded mice cells
  • Illuminator: PTI DeltaScan
  • PTI ImageMaster™ software
  • Cells were stimulated
  • The vertical colored scale shows the fluorescence ratio (bound Fura-2/unbound Fura-2)
Fluorescence Imaging
Fluorescence Imaging
Fluorescence Imaging
Fluorescence Imaging

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Single HEK293 Cell Ca2+ Imaging

Fluorescence Imaging
Fluorescence Imaging
  • HEK293 cell loaded with Fura-2.
  • Illuminator: PTI DeltaRAM
  • Software: PTI ImageMaster™
  • In the picture:(A) Single-cell fluorescent Ca2+ images in the presence (Upper) or absence (Lower) of 0.3 mM caffeine at various [Ca2+]o (0-1.0 mM).
    (B) Fura-2 ratios of representative RyR2(wt) cells in the absence (green trace) and presence (blue trace) of 0.3 mM caffeine and a HEK293 parental cell expressing no RyR2 (pink trace)

 

 

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Dopamine-induced Ca2+ Transients in Rat MSN

  • EGFP transfected rat MSN cultures loaded with Fura2/AM
  • Illuminator: PTI DeltaRAM
  • Camera: PTI IC-300
  • Software: PTI ImageMaster™ Pro
  • Fura-2 340/380 nm ratios in rat MSN before (-1 min) and after (0–28 min) application of 400 µM dopamine
Fluorescence Imaging
Fluorescence Imaging

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Intracellular Calcium in Human Neuronal Cell Cultures

Fluorescence Imaging
Fluorescence Imaging
  • Fura-2AM loaded human neurons
  • Illuminator: PTI DeltaRAM
  • Camera: Photometrics CCCD
  • Software: PTI ImageMaster™
  • Imaging: Fura-2 imaging in SDF-1 treated (B) and control (A) neuronal cells. SDF-1 activate intracellular calcium

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Mitochondria [Ca2+]m in Single Cells

  • Fura2FF-loaded single permeabilized RBL cells
  • PTI DeltaRAM illuminator
  • Photometrics PXL CCCD camera
Fluorescence Imaging
Fluorescence Imaging

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Measurement of Extracellular Near-membrane [Ca2+]

Fluorescence Imaging
Fluorescence Imaging
  • Fura-C18-loaded HEK CaR cells
  • Illuminator: PTI DeltaRAM
  • Camera: PTI IC-100
  • Images a–c: ratio images taken at different time points
  • Image d shows fluorescence at 340 nm excitation (510 nm emission) of the same cells

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Fura-2 Imaging of Taste Bud

  • Fura-2 AM loaded, isolated taste buds and individual taste cells
  • Illuminator: PTI DeltaScan
  • Camera: PTI ICC200
  • Images: (A) 2 fluorescent sequential images of a taste bud in control (a) and after the application of 10 µM ATP (b). Color palette in a shows the pixel intensity mapping.
    (C) 2 sequential fluorescent images of an isolated taste cell in control (a) and after the application of 10 µM ATP (b).
Fluorescence Imaging
Fluorescence Imaging

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Tip-Focused Ca2+ Gradient in Pea Pollen Tubes

  • Fura-2 dextran injected, in vitro germinated pea pollen tubes
  • PTI imaging system
  • Images: The arbitrary color scale at right indicates relative levels of Ca2+, with purple and blue representing the high and low ends of Ca2+ levels, respectively
Fluorescence Imaging
Fluorescence Imaging

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Calcium Wave

Fluorescence Imaging
Fluorescence Imaging
  • Fura-2 labeled 1321N1 astrocytoma cells expressing P2Y2 (labeled with yellow circles)
  • Illuminator: PTI monochromator
  • Camera: PTI ICCD
  • A Ca2+ wave was initiated by touching the cell indicated by the arrow at t = 0 sec
  • The five subsequent panels show 340/380 ratiometric images of the cells before stimulation (t = 0) and at 5, 10, 15, and 20 sec after stimulation
  • Ca2+ waves propagate in these cells

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Ca2+ Waves in 1321N1-P2Y1 Cells

  • Fura-2 loaded 1321N1-P2Y1 cells
  • Illuminator: PTI monochromator
  • Camera: PTI ICCD
  • Software: PTI ImageMaster™
  • Images: Cell 1 was stimulated in the absence of antagonist (Panel a), 10 min later in the presence of bath-applied A3P5PS (10 µM) (Panel b), and10 min after washout of A3P5PS (Panel c)
  • Black arrows: Cell 1 was mechanically stimulated at the times indicated
  • White arrow indicates the cell directly simulated
  • The color bar on the right indicates the scale of ratio intensity.
Fluorescence Imaging
Fluorescence Imaging

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Quantitation of Mechanically Induced Ca2+ Waves

Fluorescence Imaging
Fluorescence Imaging
  • Fura-2 loaded immortalized nasal epithelial cells
  • PTI ImageMaster™ System
  • Images:a, Ca2+ waves: mechanical stimulation resulted in a radially propagating Ca2+ wave
  • Subsequent exposure to carbachol (Carb) induced a second Ca2+ response, indicating that the cells remain viable and responsive
  • b, All cell (Except 1 from the EthD-1 staining) are alive and were lysed with digitonin
  • c, Quantitation of Ca2+ waves in 3 AOIs

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Mechanical Stimulation Increases Ca2+ Waves

  • Fura-2 AM loaded rat alveolar epithelial cells (AECs)
  • Illuminator: PTI DeltaRAM Camera: PTI ICCD camera
  • Software: PTI ImageMaster™
  • Images: A-D: Mechanical stimulation resulted in a Ca2+ wave that averaged slightly over 4 cells
  • E–H: in the presence of the gap junction-inhibiting peptide Gap 27, [Ca2+]i increase restricted to the stimulated cell
  • I–L: Apyrase did not significantly reduce Ca2+ wave propagation
  • Arrow: Cell that was briefly stimulated with a glass micropipette
  • White lines, cell borders
  • Color bar, approximate [Ca2+]i
Fluorescence Imaging
Fluorescence Imaging

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Mechanical Wounding Increases Ca2+ Waves

Fluorescence Imaging
Fluorescence Imaging
  • Fura-2 AM loaded rat alveolar epithelial cells (AECs)
  • Illuminator: PTI DeltaRAM
  • Camera: PTI ICCD camera
  • Software: PTI ImageMaster™
  • Images: A–D: mechanical wound-induced Ca2+ waves
  • E–H: gap junction inhibitor does not affect this Ca2+ waves
  • I–L: apyrase restricts this Ca2+ waves
  • Arrow: Cell that was mechanically wounded
  • White lines, cell borders
  • Color bar, approximate [Ca2+]i

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Increase or Block Ca2+ Wave Propagation

  • Mixture of 1321N1 cells stably expressing P2Y2 (labeled with yellow circles) or P2Y
  • a: The spread of the Ca2+ wave in the same field of cells at 5, 10, 15, and 20 sec after stimulation before (Control) and after incubation with apyrase (30 U/ml) for 15 min (Apyrase)
  • b, c, Representative traces of the 340/380 Fura-2 emission ratios from individual control cells (b) or apyrase treated cells (c) during a Ca2+ wave
  • Conclusion: Apyrase blocks Ca2+ wave propagation in P2Y2-1321N1 cells but increases the Ca2+ wave propagation in P2Y1-1321N1 cells
Fluorescence Imaging
Fluorescence Imaging

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Ca2+ Wave Propagation in Not-contacting Cells

Fluorescence Imaging
Fluorescence Imaging
  • Fura-2 loaded immortalized nasal epithelial cells
  • PTI ImageMaster™ System
  • Images: Top: Mechanical stimulation resulted in a Ca2+ wave propagation in cells that were not in physical contact with the stimulated cell
  • Bottom: Apyrase abolished the spread of Ca2+ wave
  • Bar graph on the right depicts summary data for multiple experiments

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Ca2+ Wave Propagates Over a Physical Gap

  • Fura-2 loaded immortalized nasal epithelial cells
  • PTI ImageMaster™ System
  • Images: The gap in the imaging was generated by scrapping a confluent culture with a micropipette. The red arrow indicates a single cell close to the scrape to be mechanically stimulated
  • Top: in the absence of apyrase ,the Ca2+ wave propagated over the gap (top) indicating that the signal transmission mechanism involves extracellular substance(s)
  • Bottom: Apyrase inhibit this wave propagation, indicated the mediator was sensitive to apyrase
Fluorescence Imaging
Fluorescence Imaging

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Ca2+ Mobilization from BHK-21 Cells

Fluorescence Imaging
Fluorescence Imaging
  • System: PTI DeltaScan-based Imaging system
  • Fura-2 AM loaded BHK-21 fibroblasts cells co-culture with HEK-CaR cells
  • Images: Both cell types show increases in intracellular [Ca2+] following histamine treatment, shown at three different time points (1, 2, 3), each separated by 12 s
  • The final panel (Mesh) shows the configuration of cells (red, HEK-CaR; blue, BHK-21) and region covered by polypropylene (gray shading).

 

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Ca2+ Homeostasis After Metabolic Inhibition

  • Fura-2 loaded myocytes treated with metabolic inhibition (MI Tyrode)
  • System: PTI monochromator based Imaging system
  • Images: Recordings (Left) and images (Right) of cells exposed to MI and reperfusion without (D) and with (E) diazoxide pretreatment
  • MI led to [Ca2+]i increase in both cells
  • Image shows a field of cells 10 min after the removal of MI
  • Most control cells developed high [Ca2+]i,
  • Diazoxide provides complete protection of [Ca2+]i in 7/10 cells ( appear green)
Fluorescence Imaging
Fluorescence Imaging

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Ca2+ Spike Induced by cADPr Microinjection

Fluorescence Imaging
Fluorescence Imaging
  • Fura-2/AM Jurkat T-lymphocytes
  • Illuminator: PTI power filter Hamamatsu SIT CCD camera
  • ImageMaster™ software
  • Images shows Ca2+ spikes after microinjection of cADPr (10 µM) in the presence of 1 mM extracellular Ca2+

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Hg2+ Causes Fura-2 Efflux

  • Illuminator: PTI monochromator
  • Camera (Hamamatsu C-2400)
  • Images: Hot red colors indicate high ratios and a high intracellular Ca2+ concentration
  • Images: Pseudo-colored images captured at different time points after Hg2+ addition
  • Top panel: After approximately 5 min, Ca2+ begins to leak into the cells, followed by a rapid efflux of the loaded fluorophore
  • Middle Panel: The course of events is independent of extracellular Ca2+
  • Bottom: Addition of EGTA abolishes the inhibitory effects of subsequent addition of Hg2+
  • Bright field images: Hg2+ does not cause cell lysis.
Fluorescence Imaging
Fluorescence Imaging

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External K+ Induced Calcium Oscillations

Fluorescence Imaging
Fluorescence Imaging
  • Fura-2 labeled AtT-20 cells
  • Illuminator: PTI DeltaScan
  • Camera: Hamamatsu C2400-SIT
  • Images: (1): Bright Field
  • (2)–(4): Calcium oscillations before (2), during (3) elevated external K+ and after washing in control solution.

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Effect of Heat on [Ca2+]i

  • Fura-2 labeled VR1-expressing CHO cells cells
  • Illuminator: PTI Illuminator
  • Camera: CCCD (Photometrics)
  • (a) human and (b) rat VR1 CHO cells show a rapid increase in [Ca2+]i when the temperature rises above around 40°C. Each trace on graphs (a) and (b) represent a single cell in the same representative experiment.
Fluorescence Imaging
Fluorescence Imaging

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Intracellular Ca2+ Concentration of ROG Cells in Response to FSH and ATP

Fluorescence Imaging
Fluorescence Imaging
  • Fura-2 AM loaded ROG cells
  • Illuminator: PTI DeltaRAM
  • Camera: ICCD camera
  • Software: PTI ImageMaster™

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[Ca2+]i Response to ATP

  • Fura-2/AM loaded ROG cells
  • Illuminator: PTI DeltaRAM
  • Camera: ICCD camera
  • Software: PTI ImageMaster™

 

Fluorescence Imaging
Fluorescence Imaging

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Regenerative Calcium Oscillations

Fluorescence Imaging
Fluorescence Imaging
  • Fura-2/AM loaded differentiated 1B5 myotubes
  • Illuminator: PTI DeltaRAM
  • Camera: ICCD 300 camera
  • Software: PTI ImageMaster™
  • Images: (A) Cells stimulated with 3 mM caffeine. After 2 s, a calcium wave begins from a discrete region and spreads across the cell. After ~2 s more, the calcium wave occurs again. (C)The corresponding change in the Fura-2 340/380 ratio
  • (B) Ratio images from the same cell in A stimulated with 40 mM caffeine. Calcium increases globally throughout the cell, and no calcium waves or oscillations are observed. (D) The corresponding change in the Fura-2 340/380 ratio

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Simultaneous Measurement of Phagocytosis and [Ca2+]i

  • Illuminator: PTI DeltaRAM
  • Camera: PTI ICCD100
  • Fura-2 labeled human neutrophils were presented with a DCDHF-labelled C3bi-opsonised particle for phagocytosis
  • Images: Phase contract (top) and corresponding fura2 signal (middle). 90 s: micropipette presenting the particle to the cell; 102 s:adhesion of the particle to the cell without Ca2+ signaling; 123 s: formation of the phagocytic cup; 141s: closure of the phagosome 180 s: completion of the event and the return of cytosolic free Ca2+ to baseline
Fluorescence Imaging
Fluorescence Imaging

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Correlation of Oxidative Activation with Ca2+ and Phagocytosis

Fluorescence Imaging
Fluorescence Imaging
  • Illuminator: PTI DeltaRAM
  • Camera: PTI ICCD100
  • Images: Top: Phase contract to show the phagocytic event
  • Middle row: corresponding fura2 signal to show cytosolic free Ca2+ changes.
  • Bottom row: DCDHF fluorescent intensity of the internalized zymosan particle to assess oxidative activity The graph at the bottom shows the complete time course for cytosolic free Ca2+ change (black) and DCDHF intensity (SI) with the point of phagosomal closure marked by the arrow
  • Conclusion: the onset of oxidative activity correlates with the second phase of the Ca2+ signal.

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Local Oxidase Activation and Ca2+ Signal Reported by Fura2-dextran Conjugate

  • Illuminator: PTI DeltaRAM
  • Camera: PTI ICCD100
  • The Fura-2 dextran conjugate micro-injected neutrophils was challenged with an opsonised particle
  • Images: Phase contract (top) and corresponding Fura-2 dextran signal (bottom) show the phagocytic cup (270 seconds), phagosome closure (340 seconds) and completion of the Ca2+ signal (380 seconds)
  • The graph on the right shows the complete Ca2+ data, with the point of phagosome closure marked by the downward arrow.
Fluorescence Imaging
Fluorescence Imaging

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Influx of Presynaptically Released Zn2+ into Postsynaptic Neuron

Fluorescence Imaging
Fluorescence Imaging
  • Hippocampal slice loaded with Newport Green (NG) in culture.
  • Illuminator: PTI monochromator
  • Camera: PTI ICCD 100
  • Software: PTI ImageMaster™
  • Images: A typical image of the dentate hilus region (1). The square region in the hilus is enlarged (2) and shows an increase in NG fluorescence (3) after electrical stimulation.

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Stimulation-induced Release of Zn2+ from Nerve Terminal

Fluorescence Imaging
Fluorescence Imaging
  • Hippocampal slice loaded with Newport Green (NG) in culture
  • Illuminator: PTI monochromator
  • Camera: PTI ICCD 100
  • Software: PTI ImageMaster™
  • Images of the hilus of hippocampal dentate gyrus before (1) and 0.5 s after (2) electrical stimulation
  • Arrow: placement of the stimulation electrodes

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Store-Operated Sr2+ Entry

  • Fura-2/AM loaded CHO cells transfected with CD20 or vector
  • System: PTI DeltaRAM based-ImageMaster™ system
  • Images: (A) Treatment of the cells
  • (B) Images at a, b, and c time points:
  • Before store depletion, no difference in base-line fluorescence, and no Sr2+ entry
  • ATP depleted Ca2+ stores and sharply increased [Ca2+]c in both cell lines
  • Subsequent perfusion of Sr2+ induced a large increase in the CD20-transfected but not the control cells
Fluorescence Imaging
Fluorescence Imaging

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In-Situ Calibration of Intracellular [Na+]i

Fluorescence Imaging
Fluorescence Imaging

For this composite image, rat aortic vascular smooth muscle cells were clamped at calibrating Na+ concentrations using gramicidin, monensin and nigericin. The ratio scale was calibrated using a LUT constructed on the basis of Region of Interest photometry from the nuclear region (left scale) as well as based on the concentration equation (right scale).

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Fluorescence-based determination of LIP

  • CA-loaded hepatocytes Hepatocytes
  • Illuminator: PTI Monochromator
  • Camera: Hamamatsu ICCD camera
  • Software: PTI ImageMaster™ 3.0
Fluorescence Imaging
Fluorescence Imaging

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Heterogeneity of Glutathione (GSH) in Clara Cell

  • Clara Cell and epithelial cells lining intrapulmonary conducting airways
  • Illuminator: PTI DeltaScan
  • Camera: Hamamatsu ICCD
  • Images: Second-generation (A) and Terminal (B) bronchus
  • Clara cell after 4 days (C) and 7 (D) days in culture
  • (*): cells with high signal
  • (**) cells with moderate to low signal
  • Conclusion: Intracellular GSH of Clara cells is highly heterogeneous within the population. This heterogeneity corresponds closely to the response of Clara cells to injury.

 

Fluorescence Imaging
Fluorescence Imaging

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Interaction of Liposomes With Cells

Fluorescence Imaging
Fluorescence Imaging
  • Differentiated THP-1 cells incubated with calcein-loaded liposomes labeled with Rh-PE
  • PTI Ratio/Imaging system
  • Software: PTI ImageMaster™
  • Images taken before (left) and after (right) the cells are treated inhibitors of endocytosis
  • Top: Inhibitors of endocytosis drastically reduced the levels of calcein fluorescence intensity in the cytoplasm and at the cell periphery
  • Middle: Inhibitors cause liposome aggregation

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A TM Cell Changing Area Upon Exposure to Hypotonicity

  • Illuminator: PTI illuminator
  • Camera: PTI IC-200
  • Software: PTI ImageMaster™
  • Freshly harvested trabecular meshwork (TM) cell loaded with calcein-AM
  • Images: (A) In isotonic solution without BDM
  • (B) After application of hypotonic solution, cell area was maximum
  • (C) after ~20 min in hypotonic solution. The cell area was smaller, reflecting a regulatory volume decrease (RVD)
  • (D–F) Effect of the thresholding on the same imagings (A–C)

 

Fluorescence Imaging
Fluorescence Imaging

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PI Staining to Determine Hippocampal Cell Injury

Fluorescence Imaging
Fluorescence Imaging
  • Hippocampal slice loaded with PI in culture
  • Illuminator: PTI monochromator
  • Camera: Hamamatsu low-light CCD camera
  • Software: PTI ImageMaster™
  • Images: Top: PI signal after 120 min of hypoxia with normoxic reoxygenation
  • Bottom: PI signal after 120 min of hypoxia with hyperoxic reoxygenation

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Use of DCDHF as an Oxidative Indicator During Phagocytosis

Fluorescence Imaging
Fluorescence Imaging
  • Illuminator: PTI DeltaRAM
  • Camera: PTI ICCD100
  • Images: (a) DCDHF-conjugated zymosan particles before (left) and after (right) addition of H2O2
  • The traces below show the time courses for the increase in fluorescence with the arrow indicating the addition of H2O2
  • (b) Fluorescence intensity of internalized (arrowed) and adherent (asterisk) DCDHF-conjugated zymosan particles
  • The DCDHF intensity image and the phase contrast image have been superimposed for clarity.

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Ratio Imaging of Rat Medium Spiny Neurons

  • GFP expression S2 cells loaded with Fura-2
  • Illuminator: PTI DeltaRAM
  • Camera: PTI IC-300
  • Software: PTI ImageMaster™ Pro
Fluorescence Imaging
Fluorescence Imaging

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Dose-dependence of Trehalose Response in S2-Gr5a Cells

Fluorescence Imaging
Fluorescence Imaging
  • GFP expression S2 cells loaded with Fura-2
  • Illuminator: PTI DeltaRAM
  • Camera: PTI IC-200
  • Software: PTI ImageMaster™
  • Images: Upper: Divided panels of S2-Gr5a cells (Left and Center) or negative controls, transfected with GFP vector alone (Right), before and after application of either trehalose (Left and Right) or maltose (Center)
  • Lower: Images of fields of S2-Gr5a cells taken on application of different concentrations of trehalose.

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Time Course of Trehalose Response in S2-Gr5a Cells

  • GFP expression S2 cells loaded with Fura-2
  • Illuminator: PTI DeltaRAM
  • Camera: PTI IC-200
  • Software: PTI ImageMaster™
  • Images: Upper: A series of images of a single fura 2-loaded S2-Gr5a cell, taken at 5 s intervals
  • Lower: A quantitative representation of the response of the same cell. Bar indicates stimulus period.
Fluorescence Imaging
Fluorescence Imaging

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Cells Expression Redo-GFP

Fluorescence Imaging
Fluorescence Imaging
  • Redo-GFP expressing cells
  • PTI DeltaRAM illuminator
  • Roper Sensy camera
  • PTI ImageMaster™ 3.0 software

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Visualization of Mitochondria by mitoGFP

  • mitoGFP transfected intact (A) and permeabilized (B-D) mast cell
  • Cells were also loaded with MitoTracker Red (C) or rhod2/AM (D)
  • PTI DeltaRAM illuminator.
  • Photometrics PXL CCCD camera
  • The green images (left panels) show the distribution of mitoGFP, the red images (middle panels) show the distribution of MitoTracker Red (C) or compartmentalized rhod2 (D). These images are overlaid in the right panels to show the coincidence of the labeled organelles (overlay).

 

Fluorescence Imaging
Fluorescence Imaging

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IP3-induced Intracellular [Ca2+]c and Mitochondrial [Ca2+]m Responses

Fluorescence Imaging
Fluorescence Imaging
  • Fura2FF-loaded permeabilized cell
  • PTI DeltaRAM illuminator
  • Photometrics PXL CCCD camera
  • Left: the overlaid images show the distribution of the membrane-bound CaGreen-C18 (image i, purple) and the mitochondrially compartmentalized Fura2FF (image i, green), and the changes in the Fura2FF fluorescence (images ii-v, 380 nm green/340 nm red) upon addition of 100 nM IP3 (ii versus iii), 12.5 M IP3 (iii versus iv) and ionomycin (iv versus v)
  • Right: time courses of the global [Ca2+]pm response (vi) and the average [Ca2+]m response (vii, thick line), and the [Ca2+]m responses of the marked (1–6 on image i) individual mitochondria (vii, thin lines).

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