Minimizing Photobleaching of Blue Fluorescent Protein (BFP) using
the Varian Cary Eclipse Fluorescence Spectrophotometer
Paul Gavin*, Mark Prescott*, Ph.D, Daren J. Fyfe#, Ph.D and Jeffrey J.
* Department of Biochemistry and Molecular Biology,
Victoria 3800, Australia
# Technical Assistance,
679 Springvale Road,
Victoria 3170, Australia
The term photobleaching refers to loss of fluorescence emission from
samples due to prolonged exposure to exciting radiation. While many fluorophores
retain stable fluorescence after extended periods of illumination, some
photobleach after short periods of time.
In order to maintain optimal fluorescence emission, it is important to
minimize photobleaching. Strategies to achieve this usually involve limiting
one or both of the following: (1) the time of exposure to, or (2) the
intensity of, the exciting light. However, either of these strategies
may compromise the quality of the results or limit the types of analyses
that can be performed because the signal to noise ratio (S/N) is unavoidably
decreased. Furthermore, kinetics-based assays performed over an extended
period of time may not be possible due to increased exposure of the fluorophore
to the excitation light which results in photobleaching.
Blue Fluorescent Protein (BFP) provides an example of a fluorophore that
is susceptible to photobleaching: “…even with folding improvements, BFP
still suffers from a relatively low fluorescence quantum yield [compared
to green fluorescent protein] and relatively easy bleaching.” (Tsien,
1998)1. Despite the useful spectral properties of BFP for use
in multi-labelling studies and techniques such as FRET (fluorescence resonance
energy transfer),2 it is likely that its low quantum yield
and susceptibility to photobleaching could limit ongoing detection in
instruments that irradiate the sample continuously, or too intensely,
or both. Accordingly, the present study aimed to determine whether using
the Varian Cary Eclipse fluorescence spectrophotometer, which utilizes
a special xenon flash lamp, photobleaching of BFP could be eliminated,
or at least reduced, compared to other commercially available instruments
having continuous or pulsed light sources.
Materials and Methods
Equipment (For part numbers, see reference 3.)
• Varian Cary Eclipse fluorescence spectrophotometer
• Peltier-thermostatted multicell holder (with electromagnetic stirring)
• Temperature controller
• Temperature probes
• Magnetic stirrer bars
YRD15 (MATa, his3, ura3, leu2, rho+) of the yeast S. cerevisiae was the
parental strain used in this study. The open reading frame encoding BFP
was cloned into the yeast expression plasmid pAS1N and transformed into
the yeast strain YRD15 as previously described4. Transformants were plated
out on yeast minimal medium (0.75% yeast minimal medium w/o amino acids,
2% glucose, 1.5% agar) with selective markers as required and grown at
28 °C for 3–5 days.
Yeast cells were washed twice in 1 mL MilliQ water before being lysed
using Y-PER (Progen) as per manufacturers instructions. Y-PER lysates
(5 µl) were diluted with 1 mL 50 mM Tris/HCl pH 8 and placed in disposable
fluorescence cuvettes (Sarstedt) in the multicell holder positioned within
the sample compartment of the Cary Eclipse fluorescence spectrophotometer.
Using the ‘Scan’ application, cell suspensions were repeatedly excited
using ‘cycle mode’ with UV light of 370 nm, specific for BFP excitation,
and the emission monitored from 400 to 550 nm. Slow scan speeds were used
(120 nm/min) for best S/N, however, this maximized the exposure of BFP
to the excitation light source. Operating parameters for the ‘cycle mode’
of BFP excitation scans are given in Figure 1.
Raw data showing superimposed emission spectra of BFP for 10 successive
scans using the Varian Cary Eclipse are shown in Figure 2a. Spectra were
also recorded in an identical manner on a commercially available fluorescence
spectrophotometer with a continuous xenon arc lamp (Figure 2b). Continuous
excitation from the Varian Cary Eclipse xenon flashlamp resulted in a
2.4% decrease in BFP emission over a total scan time of 21.5 minutes.
In comparison, spectra recorded on the other instrument showed a much
greater loss of signal (19.1%), indicating a high degree of photobleaching
Photobleaching can be a major limiting factor in the time-based fluorescence
measurements of living systems. The Varian Cary Eclipse fluorescence spectrophotometer
allows users to measure photosensitive samples without compromising the
quality of data, as demonstrated by Figures 2 (a-b). The exposure time
of the sample to the excitation light is minimized because the lamp is
only on when the instrument is taking a reading. This feature not only
reduces photobleaching, but also significantly increases the lamp life.
Using a narrow pulse-width flash lamp (2 µs pulse width at half peak height),
high intensity excitation can be delivered to the sample resulting in
excellent S/N over varying periods of time. The lamp flashes at 80 Hz,
providing the flexibility to measure extremely fast reactions (in the
order of milliseconds) and much slower responses (in the order of hours).
This combination of electronics and optics makes the Varian Cary Eclipse
an ideal platform for fluorescence studies in vivo of systems
involving BFP and other fluorescent probes that are susceptible to photobleaching.
In addition, having the lamp on when only taking a reading significantly
increases lamp life.
The characteristics of the xenon flashlamp in the Varian Cary Eclipse
minimizes photobleaching in biological samples by irradiation the sample
only when readings are being taken. This is a considerable benefit compared
to other instruments fitted with xenon flash lamps or continuous arc lamps
that continuously irradiate the sample.