We present a fluorescence/luminescence imaging system for use in high-throughput screening of samples in microplates. High efficiency imaging of microplates is an optical challenge usually involving performance compromises. Conventional microplate imagers use large refractive systems. These systems typically have many large lenses and suffer from their disadvantages such as low light transmission efficiency, field shading (low uniformity), and weight. Our optical design enables simultaneous detection of light from all of the samples in a microplate with high collection efficiency, high transmission efficiency, low chromatic aberration, high uniformity, and sub-well (submillimeter) resolution all in a relatively small package. Our optical system includes a primary mirror and an on-axis secondary mirror with a central aperture (a reverse-conjugate Schwarzchild arrangement) along with a small set of refractive field correctors. The field correctors compensate for the aberrations induced by the wide field (108×72 mm) without resorting to aspheric mirrors. The prototype of this design met our goals of high transmission, high uniformity and low crosstalk.
Compounds that interact with the cytoskeleton affect mobility and division, making them useful for treatment of certain types of cancer. Actin binding drugs such as the phallotoxins (small, bicyclic peptides) bind to and stabilize actin polymers (F-actin) without binding to actin monomers (G-actin). It has been shown that the intensity of fluorescently labeled phallotoxins such as fluorescein- phalloidin and rhodamine-phalloidin increases upon bind F- actin. We used LJL BioSystems' new FLARe<SUP>TM</SUP> technology to measure excited state lifetime changes of fluorescein- phalloidin and rhodamine-phalloidin upon binding to F- actin.
We report the implementation of intensity modulated diode lasers in frequency-domain pump-probe studies, diode lasers are compact, stable, and economical units that require little maintenance. In our study, a 365 nm diode laser is used as the excitation source and the output of a 680 nm unit induces stimulated emission from excited state fluorophores. By modulating the intensities of the two diode lasers at slightly different frequencies, and detecting the fluorescence signal at the cross-correlation frequency, both time-resolved and high spatial resolution imaging can be achieved. The laser diodes are modulated in the 100 MHz cross-correlation signal has been used for time-resolved imaging of fluorescent microspheres and mouse fibroblasts labeled with nucleic acid stains TOTO-3. These results demonstrate and feasibility of using intensity modulated diode lasers for frequency-domain, pump-probe studies.
Fluorescence instrumentation for high-throughput screening (HTS) must be sensitive, accurate and reliable. An instrument must be capable of robust measurement as well. HTS assays are often complicated by interfering signals from background fluorescence, scattering, absorption and quenching. Traditional fluorescence intensity methods do not remove the effect of interferences well. Fluorescence lifetime methods, however, have the ability to retrieve the true assay signal from a signal plagued with interferences. We have developed fluorescence-lifetime methods specifically for high-throughput screening with a high-frequency time- resolved fluorometer created from the optics of a high- throughput screening instrument, a high-speed LED or laser diode light source and the phase and modulation method of fluorescence lifetime measurement. The prototype instrument is capable of measuring the lifetime of samples from 1-1000 ns, able to measure a single frequency in less than one second and able to distinguish nanomolar concentrations of fluorophores in small volumes. Furthermore, the often complex and troublesome fluorescence lifetime measurement is made simple and reliable with this prototype. To demonstrate robustness and reliability, we used lifetime-based methods to measure a model system with background fluorescence form biological membranes. Specifically, methods based upon long- lifetime fluorophores can significantly improve immunity to assay interferences.
Fluorescence lifetime, the mean interval between absorption and emission, is as fundamental a characteristic of fluorescence as excitation and emission wavelengths and quantum yield. Yet, with the exception of time-resolved fluorescence assays utilizing lanthanide chelates, the analytical possibilities of methods based on fluorescence lifetime are virtually unexploited outside the academic research laboratory. We discuss the potential use of fluorescence-lifetime technologies in high-throughput screening from the standpoint of assay reagents and instrumentation. Among these applications are fluorescence- polarization assays based on long-lifetime probes and fluorescence-intensity assays using lifetime-resolved detection to reject background. We find that fluorescence- lifetime technologies offer significant practical advantages over existing methods.
Microfabricated channels are widely thought to be the key to realizing chemical analysis on a microscopic scale. Chemical and biological information in the microchannels is often probed with optical techniques such as fluorescence, Raman and absorption spectroscopy. However, the optical effects of a microchannel are not well characterized. For example, it is important to understand the optics of the channel in order to optimize optical coupling efficiency. We consider various designs for enhancing the sensitivity of fluorescence detection in a microchannel.
Time-resolved fluorescence imaging can enhance the contrast of microscope images and it can also provide important information about the micro-environment in cellular systems. We have developed a fluorescence microscope which can measure fluorescence lifetimes over an entire image. Fluorescence lifetimes are measured by using heterodyne frequency domain techniques. Heterodyning is accomplished by using an intensity modulated laser light source and a fast scan CCD camera coupled with a gain modulated microchannel plate as the detector. The high duty cycle of this method allows us to generate a phase resolved image with about five seconds integration time. Operating in the fast scan mode, the systematic uncertainties in lifetime determination caused by photobleaching are less severe than those of slow-scan cameras. The microchannel plate can be modulated at frequencies up to 300 MHz, which allows us to measure lifetimes as short as 500 ps with resolution of 50 ps. The modulation of the microchannel plate only slightly degrades the spatial resolution of the image from the diffraction limit; 0.8 micron resolution is maintained with 500 nm laser excitation.
Our laboratory has developed a modular laser tomography system, with pulsed or amplitude modulated (MHz to GHz), near infrared lasers that deliver a probing beam to the tissue of interest through a fiber optic. After the incoming light is scattered and attenuated by the tissue, a detector or imaging fiber optic bundle delivers it to a point detector (photomultiplier tube) which is heterodyned with the modulation frequency to yield the phase delay and demodulation resulting from the light-tissue interaction. The CCD electronics are phase-locked with those of the digitizer to minimize pixel jitter and, in addition, an external clock synchronizes the detector units with the modulated laser source. The digitized time slices are integrated into four bins corresponding to four quadrants of the cross correlation period. The final processing step, a fast Fourier transform, generates the phase shift, demodulation, and average intensity data suitable for image reconstruction.
Imaging of thick tissue has been an area of active research during the past several years. Among the methods proposed to deal with the high scattering of biological tissues, the time resolution of a short light probe traversing a tissue seems to be the most promising. Time resolution can be achieved in the time domain using correlated single photon counting techniques or in the frequency domain using phase resolved methods. We have developed a CCD camera system which provides ultra high time resolution on the entire field of view. The phase of the photon diffusion wave traveling in the highly turbid medium can be measured with an accuracy of about one degree at each pixel. The camera has been successfully modulated at frequencies on the order of 100 MHz. At this frequency, one degree of phase shift corresponds to about 30 ps maximum time resolution. Powerful image processing software displays in real time the phase resolved image on the computer screen.