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Correlative Confocal - QPI Imaging

This protocol describes correlative fluorescence (confocal) and label-free (quantitative phase) imaging of cells tagged with one or more fluorescent reporters. The following protocol was developed for imaging cell lines from the Organelle Box resource.

Sample preparation

  • 150,000 of A549 cells were plated in 24 well glass bottom plate.
  • Next day, nucleus was stain with Hoechst in 1:10,000 dilution in PBS for 10 mins.
  • Cells were fixed with either 4% of PFA or cold MtOH for 20 mins.
  • Then, PBS was used during imaging.

Our microscope

We use a confocal with a transmitted-light path for correlative fluorescence and label-free imaging.

Microscope body

Leica DMi8 inverted microscope with an Okolab cage incubator.

Objective

  • Magnification - 63X
  • Numerical aperture - 1.47
  • Immersion media - Oil

Imaging channels

  • DAPI: (Hoechst), 358 nm excitation, 461 nm emission.
  • FITC: (nuclear translocation sensor), 495 nm excitation, 519 nm emission.
  • Label-free channels: BF (brightfield), 450 nm illumination.

Imaging software

We used Micro-Manager to automate the imaging. We acquired Multi-channel 3D (C, Z, Y, X ) images using Multi-D acquisition dialog.

Conditions imaged

  • Live,
  • PFA fixed,
  • Methanol fixed

Spatial resolution

  • z-slices at 0.2 um spacing
  • x-y pixel at 0.103 um resolution

Imaging conditions

Live cells are imaged under incubated conditions at 37 degrees Celsius and 5% CO2, 90% humidity. Fixed cells were imaged at room temperature.

Laser intensity for various markers were optimized for best SNR. The intensity of the markers in live condition is ranked as follows , increasing through the list:

ATP1B3 < RPL36 < NCLN < SEC61B < LAMP1 < PNPLA2 < EDC4 < ACTB < EEA1 < TOMM20 < PEX3 < CLTA < SLC3A2 < ATG101 < VSP35 < G3BP1 < MAP1LC3B < RAB11A < H2BC21 < NPM1

The ranking of intensity of the markers can differ with fixation conditions.