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Optimization of DCAB (Diffusion Contrast After Bleaching) Fluorescence Microscopy in Living Cells

Tyler Hull, Jacob Ritz, and Dr. Elias M. Puchner

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Outline

    • Background
    • Problem
    • Methods
    • Results
    • Conclusion

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Background

  • What is cell signaling?
  • What is a signaling pathway?
  • Why does this matter?

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Background

Mechanical

  • Pressure
  • Voltage
  • Temperature
  • Light

Chemical

  • Molecules
  • Atoms
  • Peptides
  • Gas

What is cell signaling?

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Background

Lots of different signaling pathways.

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Lots of places for things to go wrong.

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  • Diabetes (Insulin signaling pathway shown)
  • Alzheimer's
  • Cancer

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Background

  • We can tag proteins we want to study with photo-fluorescent molecules.
  • Recording the signal from these proteins allows us to study these interactions.

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Problem

Signal from the non - interacting protein drowns out signal from the interacting protein.

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PALM imaging

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Methods

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dcPALM

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DCAB

Bleach the freely diffusing molecules to reduce the signal enough that we can distinguish between bound/unbound molecules.

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Methods

Develop a stain of yeast cells with

  • SEC63 – HaloTag
    • Stain the ER around the nucleus and cell wall, showing the region to avoid during the DMA screening process
  • NIC96 – mEos2
    • Simulate bound molecules
  • PGAL – mE0s2
    • Simulate unbound molecules

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Results

PALM imaging without the freely diffusing PGAL shows a clearly defined nucleus,

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Results

PALM imaging without bleaching, signal from the bound NIC96 is masked by the signal from the freely diffusing PGAL.

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Results

Almost all of the freely diffusing PGAL has been bleached, leaving only signal from the bound NIC96

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Results

NIC96 + PGAL

PALM

NIC96 + PGAL

DCAB, PALM

NIC96

PALM

σnuc - σcyto

(localizations/pixel

σnuc

σcyto

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Conclusion

    • DCAB is an effective technique in reducing the signal from non interacting localizations
    • More work can be done with the dyeing process, time the freely diffusing protein is bleached, size of the bleaching area
    • Eventually move on to non-simulated proteins

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  • Questions?