Even though fluorescence detection is a broadly applied technology in biological and clinical research, the illumination and detection techniques have remained fairly limited; that is, fluorophores absorb light over a narrow spectral range, and the excitation light reflected from a sample is usually filtered out of the fluorescence signal in the frequency domain using spectral filters. For multicolor fluorescence detection, which allows simultaneous monitoring of different physical and biological cellular functions, the number of excitation sources and narrowband detectors is usually increased, causing complexity and higher cost.
However, by using an ultrafast broadband supercontinuum source, researchers at the Center for Ultrafast Optical Science and the Michigan Nanotechnology Institute for Medicine and the Biological Sciences at the University of Michigan (both in Ann Arbor, MI) are able to excite multiple fluorophores simultaneously and remove scattered pump light in the time domain, while simplifying the optical design by eliminating the need for filters and dichroic mirrors.1
The supercontinuum white light generated from a Coherent (Santa Clara, CA) Mira 900 ultrafast pulsed laser is used as the excitation source. The 800 nm, 70 mW, 50 fs pulses at a 76 MHz repetition rate are pumped into a 1-m-long nonlinear photonic-crystal fiber with a zero-dispersion wavelength of 750 nm. Nonlinear interactions inside the fiber (including self-phase modulation, four-wave mixing, and stimulated Raman scattering) produce a broad subpicosecond supercontinuum with a spectral density that is sufficient to excite fluorophores between 460 and 850 nm.
To demonstrate the technique, the supercontinuum is focused into a sample cuvette containing a mixture of different fluorophore dyes. The emission from the sample-containing both scattered excitation light and the fluorescence signal-is focused into a spectrometer and then analyzed using a time-resolved detector. The scattered excitation light (with a time duration of less than 40 ps) is separated from the fluorescence signals (with durations on the order of hundreds of picoseconds to tens of nanoseconds) in the time domain using a Hamamatsu (Bridgewater, NJ) streak camera in synchroscan mode. In this mode, the electron beam in the camera is swept over the detector screen synchronously with the 76 MHz excitation pulse train.
Because the streak trace is synchronized with the laser pulse, the scattered source light is gated out by adjusting the delay (phase) of the streak-camera sweep voltage relative to the laser such that the scattered excitation light falls out of the detection time window, while the fluorescence signal falls within the time window.
The output of the streak camera is detected either with a cooled CCD detector to record the time-resolved fluorescence spectrum (see figure) or with a photon-counting multiplier tube coupled to a multichannel scaler detector to detect rapidly changing signals. The successful, simultaneous analysis of multiple fluorophores in a sample opens up a range of new applications that are difficult to address even with multiple laser excitation systems and multiple detection channels.
“Because this invention establishes a general fluorescence-detection approach based on a new mechanism that is fundamentally different from conventional methods, the successful development of this technology will lead to significant improvements in many different kinds of fluorescence-based instruments such as flow cytometers, fluorescence microscopes, microplate readers, and endoscopes,” says Jing Yong Ye, a researcher at the University of Michigan. He notes that the technology is now patented and his group wants to commercialize the technology by working with interested companies.
1. J.Y. Ye et al., Optics Express 15, 10439 (Aug. 6, 2007).