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.
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.
Brian Research, 921(1-2): 1-11, 2001. Courtesy of Dr. G. Brewer
Click on image to enlarge.
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
PNAS 101 (35), 13062-13067. 2004 Courtesy of Dr. S. Chen
Click on image to enlarge.
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)
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).
J Neurophysiol 90: 3283-3294, 2003. Courtesy of Dr. S. Kolesnikov
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
Plant Cell, 1:1731-1742, 1999. Courtesy of Dr. Z. Yang
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.
The J. of Neuroscience, 20(8):2800-2808, 2000. Courtesy of Dr. M. Salter
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
J.l of Neuroscience, 2003, 23(17):6728-6739. Courtesy of Dr. Michael W. Salter
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
The Journal of Cell Biology, 150 (6), 1349-1360. 2000. Courtesy of Dr. R. Boucher
Nature cell biology, 2 (7): 392-398, 2000. Courtesy of Dr. Hofer.
Click on image to enlarge.
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).
(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.
British Journal of Pharmacology 132:1084-1094 (2001). Courtesy of James, I
Biophys J, 79 (5): 2509-2525, 2000.
Courtesy of Dr. I, Pessah
Click on image to enlarge.
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
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
J Cell Sci 2003;116:2857-2865.
Courtesy of Dr. Dewitt, S. et al.
Correlation of Oxidative Activation with Ca2+ and Phagocytosis
J Cell Sci 2003;116:2857-2865. Courtesy of Dr. Dewitt, S. et al.
Click on image to enlarge.
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.
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.
J Cell Sci 2003;116:2857-2865.
Courtesy of Dr. Dewitt, S. et al.
Influx of Presynaptically Released Zn2+ into Postsynaptic Neuron
J Neurophysiol 86: 2597-2604, 2001;
Courtesy of Dr. Frederickson C.
Click on image to enlarge.
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.
Harootunian A.T. Kao J.P.Y. Eckert B.K. and Tsien R.Y., J. Biol. Chem. 1989, 264, 19458-19467
Click on image to enlarge.
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).
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.
Am. J. Respir. Cell Mol. Biol., 23 (1),2000 27-36.
Courtesy of Dr. Plopper, C.
Dose-dependence of Trehalose Response in S2-Gr5a Cells
PNAS 100 (suppl. 2):14526-14530, 2003.
Courtesy of Dr. Carlson, J
Click on image to enlarge.
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.
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).
IP3-induced Intracellular [Ca2+]c and Mitochondrial [Ca2+]m Responses
EMBO, 18(1): 96-108, Courtesy of Dr. Hajnoczky
Click on image to enlarge.
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).
