FLIM with PURETIME ® fluorescent dyes
Key features of FLIM with PURETIME ® dyes
 |
No photobleaching |
|
Longer fluorescence lifetimes |
|
Homogeneous cell labelling |
|
Targeted labelling |
FLIM (fluorescence lifetime imaging microscopy) is now firmly established as a valuable research tool in cell biology. It has particularly been used to measure the effects of FRET between two fluorophores, independently of fluorescence intensity. Fluorescent proteins such as CFP and YFP can undergo FRET when the chromophores are in close proximity. FRET therefore indicates co-localisation of expressed fluorescent proteins on a molecular level, supplementing the spatial information gained by microscopy.
Fluorescent proteins and conventional fluorescent dyes such as fluorescein and rhodamine (also used in FLIM) are widely used, but suffer from photobleaching. All PURETIME ® fluorescent dyes are highly photostable, have high purity (>95%) and have well-defined fluorescence lifetimes. As the figure below shows, PURETIME22, PURETIME20 and PURETIME14 all show stable absorption after intense white light illumination from a quartz tungsten halogen source. Fluorescein on the other hand is rapidly bleached.
Even higher intensities are used in FLIM, where conventional dyes can show photobleaching in just a few seconds.
PURETIME ® dyes have relatively long fluorescence lifetimes compared to other dyes (fluorescein, GFP, YFP, CFP etc typically have lifetimes of 3-4ns), offering the possibility of FLIM images with increased dynamic range. The longer fluorescence lifetimes also assist in reducing the effects of unwanted background fluorescence.
Homogeneous labelling of fixed cells may be carried out quickly and easily. PURETIME ® reactive NHS esters offer an attractive way to chemically bond the dyes to primary amines and N-terminal position of peptides and proteins in and on cells. The fluorescence image below shows good contrast against background and demonstrates that permeablised MG63 osteoblasts can be homogeneously labelled with PURETIME22
Fluorescence image of MG63 osteoblasts labelled with PURETIME22 NHS ester after fixing and permeablising with ethanol/water (false colour)
Fluorescence intensity images can however be misleading when a uniform stain or dye label is used. It is unclear from the intensity image below whether the upper right hand feature is a differentiated cell or some adventitious material.
False colour PURETIME22 fluorescence intensity image of MG63 cell field
When the colour is mapped to fluorescence lifetime rather than intensity, the adventitious nature of the feature is clear (red indicates a long lifetime, whereas green represents a short lifetime quenched by the cell cytosol).
PURETIME22 FLIM image of MG63 cells and adventitious feature with distinctive long lifetime
Selective labelling of cells and sub-cellular structures can be accomplished with PURETIME ® labelled antibodies. The FLIM image of MG63 cells below was obtained with PURETIME®22 labelled anti-actin. Fluorescence emission was detected at 530nm and the false colour image represents the fluorescence lifetime values of the detected emission from 2ns (blue) through 6ns (green) to 11ns (red).
FLIM image of MG63 cells showing both localisation and general distribution of PURETIME22 labelled anti-actin
It can immediately be seen that there are two different chemical environments for the actin antibody. In the cytosol, the antibody experiences uniform surroundings that greatly reduce the fluorescence lifetime of the PURETIME ® dye probe. At the cell boundary however, there are different forms of actin (shown by the red punctate flecks) that exhibit much lower fluorescence quenching of the PURETIME ®22 emission.
The long fluorescence lifetime of PURETIME ®22 therefore permits easy, single label determination of actin in different chemical environments and readily distinguishes dye probe fluorescence from endogenous species.
Products
| Product code | Dye | Derivative | Abs. (nm) | Emission (nm) | Fluorescence lifetime (ns) | Amount |
| PT22-N | PURETIME ®22 | NHS ester | 506 | 556 | 22 | 1mg |
| PT14-N | PURETIME ®14 | NHS ester | 394 | 426 | 14 | 1mg |
Ordering
We ship direct from the UK all over the world. Please e-mail your request for a quotation or more information to sales@assaymetrics.com. Before placing an order with us, please ensure you have read our Terms and Conditions.
Acknowledgements
MG63 cells were kindly provided by the Welsh School of Pharmacy, Cardiff University. AssayMetrics also gratefully acknowledges the FLIM measurements, performed at the Department of Chemical Engineering, Cambridge University. FLIM images were obtained using a Lambert Instruments frequency domain camera system (for further information contact Photon Lines Ltd.)