Emission Fingerprinting and FRET

An application example for FRET detection and analysis with LSM 510 META by taking the advantage of the Emission Fingerprinting method is Yellow-Cameleon-2 (YC2, A. Miyawaki, RIKEN, Wako, Japan).

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YC2 is a tool that applies FRET for the visualization of Ca²⁺-concentrations within living cells. YC2 is a fusion protein of CFP (Cyan Fluorescence Protein) and YFP (Yellow Fluorescence Protein) with Calmodulin (CaM) and its binding-site M13. Cells can be transfected with YC2 and will then express an internal Ca²⁺-sensor.

Yellow Cameleon2 (YC2) as internal Ca²⁺ -sensor:
	low Ca²⁺: no FRET signal; high Ca²⁺: FRET signal
A. Miyawaki, RIKEN, Wako, Japan

Step 1:

Complete Emission Detection via Lambda Stack Acquisition

Lambda Stack acquisition with LSM 510 META can be combined with time series to increase the information density. The output is a 4 dimensional (xy-lambda-t) image stack with a spectral readout at any pixel of the image as a function of time.

Cytoplasmic expression of YC2 in a cultured cell:

xy-lambda-t image stack

Spectral readout from a Region Of Interest (ROI) over time.

Step 2:

Linear Unmixing Using Reference Spectra

A high-dynamic 2 channel (CFP, YFP) time series is generated by applying Linear Unmixing to the Lambda Stacks over time.

Input for Linear Unmixing: Lambda Stacks and reference spectra (e.g. taken from the database)
Output of Linear Unmixing: Clearly separated fluorescence signals of different dyes

Cytoplasmic expression of YC2 in a cultured cell:

2 channel (CFP, YFP) time series generated by Linear Unmixing.

Intensity readout from a region of interest (ROI) over time

Step 3:

YFP (acceptor) / CFP (donor) Ratio

Finally, the ratio function of the LSM 510 META is applied to the CFP, YFP time series to directly visualize the spectral shift between CFP and YFP emission. This shift is due to FRET of YC2 in response to an increased intracellular Ca2+ -concentration.

Cytoplasmic expression of YC2 in a cultured cell:

Pseudo-coloured YFP/CFP ratio time series; YFP/CFP ratio readout from a region of interest (ROI) over time

Lambda Stack acquisition as first step of the Emission Fingerprinting method is particularly advantageous for FRET detection.
Conventionally, small detection bands are applied to separate CFP (donor) from YFP (acceptor) emission. Some emitted fluorescence signal is cut-off and not detected. In contrast, Lambda Stack acquisition of LSM 510 META applies complete emission detection without discarding signal.


Detection of two narrow emission bands Some signal is discarded	Complete emission detection No signal is discarded

A direct comparison of conventional emission band detection of CFP and YFP signal with the Emission Fingerprinting method demonstrates the advantages of the LSM 510 META concept for FRET.

Emission Fingerprinting with Lambda Stack acquisition for complete emission detection and Linear Unmxing for clear separation of the fluorescent signals, leads to a greater dynamic range and a better signal-to-noise ratio resulting in a higher sensitive of FRET detection than emission band based methods.

A completly new approach to FRET - Emission Fingerprinting with LSM 510 META:

Complete emission detection (including possible crosstalk) via Lambda Stacks
Crosstalk (emission and/or excitation) is no problem
Direct spectral readout from any pixel of the image
Less (no) bleaching artefacts in the spectral pattern due to fast parallel Lambda stack acquisition
Better dynamic range, better signal-to-noise ratio than any emission band- or filter-based systems.