This document describes how to minimize photobleaching of Blue Fluorescent Protein (BFP) using the Agilent Cary Eclipse fluorescence spectrophotometer. Photobleaching refers to the loss of fluorescence emission from samples due to prolonged exposure to exciting radiation. To maintain optimal fluorescence emission, it is important to minimize photobleaching. Strategies to achieve this usually involve limiting the time of exposure to, or the intensity of, the exciting light. However, these strategies may compromise the signal to noise ratio (S/N), affecting the quality of results or limiting the types of analyses that can be performed. Additionally, extended exposure to excitation light can result in photobleaching, making it challenging to perform kinetics-based assays over a long period of time.
This document is an application note titled "Minimizing photobleaching of Blue Fluorescent Protein (BFP) using the Agilent Cary Eclipse fluorescence spectrophotometer." It details a study comparing the performance of the Cary Eclipse against other commercially available instruments regarding the stability of BFP fluorescence.
The study highlights that the Cary Eclipse utilizes a special xenon flash lamp that only illuminates the sample when taking a reading, unlike continuous or pulsed light sources that irradiate the sample constantly. This feature significantly reduces photobleaching and extends lamp life. The note includes experimental data showing a negligible drop in BFP emission (2.4%) over 21.5 minutes with the Cary Eclipse, compared to a 19.1% loss with a continuous xenon arc lamp. It also lists part numbers for the spectrophotometer and associated accessories.
| Excitation Lamp Type | Xenon flash lamp |
|---|---|
| Lamp Flash Frequency | 80 Hz |
| Pulse Width | 2 us |
| Part Number | 00 100752 00 |
It minimizes photobleaching by using a xenon flash lamp that only illuminates the sample when readings are being taken, rather than continuously irradiating it.
The Cary Eclipse showed a 2.4% decrease in BFP emission over 21.5 minutes, whereas a continuous xenon arc lamp showed a 19.1% loss.