FLIM/Hyperspectral Confocal Microscope for Ultrafast Live Cell Imaging

Client : Sunnybrook Research Institute
Industries: Life sciences, Medical
Technologies: Fluorescence Microscope, Hyperspectral Imaging

Context

INO provides integrated, high-end services for the design and build of unique, personalized multi-modal microscopes. Here is an example of our capabilities: a custom ultra-fast automated FLIM-FRET hyperspectral system to study complex live cell protein-protein interactions and biochemical phenomena in drug development with diffraction limited resolution. 

Ever more rapid imaging of live cells and biomolecular interactions is required for high performance life sciences applications, such as drug development. Image-based High Content Screening (HCS) is a technique frequently used in this area of research. The combination of Fluorescence Lifetime Imaging Microscopy (FLIM) and hyperspectral imaging modes in drug screening can provide information on the molecular specificity as well as the mechanism of action of candidate molecules. 

Solution

INO has developed an ultra-fast FLIM-FRET hyperspectral system to study complex live cell protein-protein interactions and biochemical phenomena in drug development. The objective is to drastically reduce the timeline of the drug discovery pipeline using a custom-designed, automated multimodal microscope that combines FLIM and hyperspectral images with diffraction limited resolution.

Flim Hs Ino

The system features a greater speed of detection than conventional systems, making it possible to carry out a FLIM-based drug screen. The robustness of its optomechanical design enables long-term imaging without any drift or misalignments due to thermal or vibrational perturbations. The software enables automatic binning over regions of interest using a plug-in-like feature that allows the user to modify the binning parameters and the style of region of interest detected. A selection of fitting algorithm enables the user to analyze the data with the particular needs of each experiment. 

Key Features

  • Enables acquisition of a 384 wells plate with 20 images @ 400x400 pixels per well in less than 24 hours with full TCSPC and Hyperspectral channels
  • 16Mcts/sec could be recorded allowing images of 400x400 in less than 10 seconds for mCerulean expressing cells
  • FLIM and hyperspectral images can be simultaneously acquired (interleave excitation pulses)
  • Automatic and customizable binning scheme and fit algorithm (compatible with .dll) in FLIM-FRET and hyperspectral analysis

The following preliminary results demonstrate the use of the system to detect the disruption of a known interaction upon addition of a drug.

Acquisition / Analysis Software

  • Auto-selection of ROI (custom .dll can be used) to bin only meaningful photons for FLIM or hyperspectral analysis
  • Powerful MLE fitting algorithm (custom .dll can be used)
  • HCS capabilities readily interfaced to Nikon Imaging Software
  • Batch analysis

FLIM FRET Software Screenshot 1.1

Acquisition/configuration window: the software allows the user to select the acquisition parameters according to a particular experiment need (resolution, FOV, frame rate, wavelength, etc.). An intuitive interface allows the visualization of the filter selection, as well as the spectrum of its favorite fluorophore.  

Soft 2 Flimfret

Visualization/analysis window: Here BMK-d3 cells expressing mCerulean-BCL-XL and Venus Bad recorded by the system. The software automatically selects ROI, shown by color region in the TCSPC image, and grey in the hyperspectral image.

Soft 3 Flimfret

ROI window: For each ROI selected, the user can visualize the lifetime fit and the associated value, as well as the associated spectrum.  

Preliminary results: BCL-XL unbinding BAD by ABT263 drug

Results obtained by Dr. David Andrews and his team at Sunnybrook Research Institute.

FLIM FRET Results Screenshot 1

Baby Mouse Kidney (BMK)-d3 cells stably expressing mCerulean3, the donor fluorophore, fused to BCLXL: “mCerulean3-BCL-XL”, were transiently transfected with Acceptor fluorophore (Venus) fused to BAD: “Venus-BAD”; or a mutant of BAD: “Venus-BAD2A”. Venus-BAD binds to mCerulean3-BCL-XL, causing proximity FRET between the Donor and Acceptor fluorophores (positive control for binding). Venus-BAD2A does not bind and no FRET is observed (negative control for binding).

Results 2 Flimfret

With ABT-263 added 5 hours post transfection, preliminary results indicate ABT-263 can disrupt the interaction between BCL-XL and BAD, as seen by a decrease in FRET efficiency similar to what was observed by Liu et al., 2012.

 

We are grateful for financial support of this work from CQDM, CFI and the BL-NCE program. This project has been made possible thanks to a collaboration with the research groups of Dr. David Andrews, Sunnybrook Research Institute, and Prof. Qiyin Fang, McMaster University.

 

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