Cell Friendly Excitation
In many cases, the act of fluorescent excitation of a living cell under a microscope can effect its activity and influence results. One of the greatest contributing factors is the photobleaching and phototoxic effects of this excitation light. The researcher will want to minimise the time which the sample is exposed to excitation light as well as minimise the intensity to the lowest level acceptable for imaging. CoolLED LED excitation systems offers the researcher a more appropriate light source and also a higher level of control to minimise these adverse effects.
LED's have a Narrowband source of light;
Broadband “white” light sources such as Xenon, mercury and metal halide provide a wide spectrum of excitation light that must be filtered to excite only the probe of interest. It is common for an excitation filter to have a passband of around 30nm and attempt to block the rest of the wide spectrum from the lamp which may be well over 400nm. Any unwanted light that leaks through these blocking filters will add to phototoxicity and bleaching problems, leading to unhappy live cells.
Blocking filters are not prefect;
All unwanted out-of-band light may also add to background through increased stray light and auto-fluorescence from the sample. For the narrower more appropriate LED spectrum this is not an issue allowing the researcher to gain clearer images.
Simply better control with LEDs;
CoolLED systems can respond in microseconds without vibration, far quicker than the best mechanical shutters. With precise 1% intensity control, the crude level of intensity adjustment offered by the use of ND filters is also superceded .
Accumulation of delays;
Over-exposing cells to the excitation light is a common problem in live cell microscopy. Often for an image to be taken, the sequence shown below may apply:
Open shutter -> Delay -> Image taken by camera -> Delay -> Close shutter
These delays can be very significant. An image capture measured in single milliseconds can require sample excitation in tens or hundreds of milliseconds. Since the CoolLED system requires no shutters and instantly provides an even illumination over the FOV it can be triggered by TTL pulse to (1) Switch on, (2) Excite, (3) Switch off - all within microseconds if required. Significantly reduced sample damage can be achieved.
With a CoolLED LED system for Cell Viability, you have....
- Excitation only during image capture
- Stability & Repeatability
- Independent wavelength intensity control
- No need for an excitation filter wheel or shutters
- Less heat reduces focus drift
Click here to watch a Time Lapse movie using the CoolLED system as the excitation source.
Movie provided by courtesy of Mike Davidson,
National High Magnetic Field Laboratory, Florida State University.
Example: Cell Viability with LED fluorescence excitation for 4 wavelengths
CoolLED pE-2 excitation system with excitation at 365, 470, 550 and 635nm