'Imaging' in the Tecan

ByBarrett Nehilla

Jim and I were wondering if we could “image” the membrane windows with the Tecan.

A nice thing about the Tecan is that you can specify the number of measurements per well.  For example, in this study, I acquired a 10×10 matrix of fluorescence intensities in a “filled circle” pattern (see below).  You can also do squares, lines and points.

You can think of this type of data as a picture of the bottom of the well.

I tried to detect the fluorescence of sodium fluorescein (Na-F) with this method.  To do this, I first measured the 490/525 nm fluorescence (in HBSS/10mM HEPES) with a pnc-Si transwell in the 24-well plate (manual gain = 10).  I then added 0.02% Na-F to the transwell and measured the fluorescence after 15 and 30 minutes.  I used SiO2-deposited w644 to keep the Na-F from diffusing through the membrane over this timeframe (see past results with this wafer).  To visualize, I used MATLAB to make an intensity graph.  Each square in these images is color-coded to the fluorescence intensity from the 10×10 matrix.

First note that the fluorescence intensities are very low since I set the gain so low.  It looks like there was a slight overall increase in fluorescence at the 15 minute timepoint, but it’s only ~ 1-2 RFU’s (on a scale of 10000’s).  The 30 minute timepoint matches the 0 minute timepoint which shows that no Na-F is diffusing through the membrane.  Unfortunately, there is no discrimination of fluorescence emanating from the membrane windows.  I think the membrane windows are too far from the bottom of the plate (in 3D format) for the Tecan to image fluorescence in the apical well of transwells.

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2 Comments

  1. Why is there an increase in intensity at the 15 min time point? And then intensity decreases at 30 min?
    What if the Sepcon is allowed to rest at the bottom of the well (i.e. w/o the ring support to keep it hanging) to see if height is an issue here?

  2. I think it’s just variability in the measurement (it’s only an increase of 1-2 RFUs).  This is basically nothing.  Not sure about your suggestion but I could try it out.

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