Navigation Links
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. Comerford#, Ph.D

* Department of Biochemistry and Molecular Biology,
Monash University,
Clayton campus,
Victoria 3800, Australia

# Technical Assistance,
Varian Australia,
679 Springvale Road,
Mulgrave,
Victoria 3170, Australia
E-mail: fluorescence@varianinc.com

Introduction

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 use ful 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

Yeast strains

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 35 days.

Protocol

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 l) were diluted with 1 mL 50 mM T ris/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.

Results

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 of BFP.

Discussion

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 s 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.

Conclusion

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.


'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Automated Fluorescent-Tag Cycle Sequencing
2. Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells
3. Fluorescently Labeling Long dsRNA with the Silencer siRNA Labeling Kit
4. Measuring Intracellular Enhanced Green Fluorescent Protein With the VersaFluor Fluorometer
5. Laser Capture Microdissection of Cells Labeled with Enhanced Green Fluorescent Protein
6. Cytosolic expression of Green Fluorescent Protein (GFP) and its derivatives in the yeast Saccharomyces cerevisiae: Detection in vivo using the Varian Cary Eclipse
7. Rapid, High Throughput Mutation Discovery Using Fluorescent SSCP on the ABI PRISM 3100 and 3100Avant Genetic Analyzers
8. Fluorescent pigments in living coral
9. Measurement of Green Fluorescent Protein in the SpectraMax Gemini XS Spectrofluorometer (MaxLine Application Note #44)
10. A New C-Terminal GST Vector for Protein Production in S. pombe
11. New Mammalian Two-Hybrid System Detects Protein-Protein Interactions
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/23/2016)... Calif. , June 23, 2016  Blueprint Bio, ... biological discoveries to the medical community, has closed its ... Matthew Nunez . "We have received ... with the capital we need to meet our current ... essentially provide us the runway to complete validation on ...
(Date:6/23/2016)... June 23, 2016 On Wednesday, June ... 4,833.32, down 0.22%; the Dow Jones Industrial Average edged 0.27% ... at 2,085.45, down 0.17%. Stock-Callers.com has initiated coverage on the ... Nektar Therapeutics (NASDAQ: NKTR ), Aralez Pharmaceuticals Inc. ... BIND ). Learn more about these stocks by ...
(Date:6/23/2016)... Prairie, WI (PRWEB) , ... June 23, 2016 ... ... consultancy focused on quality, regulatory and technical consulting, provides a free webinar ... is presented on July 13, 2016 at 12pm CT at no charge. , ...
(Date:6/23/2016)... June 22, 2016  Amgen (NASDAQ: AMGN ... QB3@953 life sciences incubator to accelerate the ... shared laboratory space at QB3@953 was created to help ... obstacle for many early stage organizations - access to ... sponsorship, Amgen launched two "Amgen Golden Ticket" awards, providing ...
Breaking Biology Technology:
(Date:5/9/2016)... 2016 Elevay is currently known ... freedom for high net worth professionals seeking travel for ... connected world, there is still no substitute for a ... sealing your deal with a firm handshake. This is ... advantage of citizenship via investment programs like those offered ...
(Date:4/28/2016)... and BANGALORE, India , April 28, ... Systems, a product subsidiary of Infosys (NYSE: INFY ... announced a global partnership that will provide end ... use mobile banking and payment services.      (Logo: ... key innovation area for financial services, but it also plays ...
(Date:4/26/2016)... and LONDON , April 26, ... of EdgeVerve Systems, a product subsidiary of Infosys ... announced a partnership to integrate the Onegini mobile ...      (Logo: http://photos.prnewswire.com/prnh/20151104/283829LOGO ) ... customers enhanced security to access and transact across ...
Breaking Biology News(10 mins):