Measurements of fluorescence lifetimes, rather than intensity or intensity ratios, offer many advantages in clinical chemistry and imaging. However, measurements of time-resolved fluorescence are normally associated with complex laser light sources and instrumentation. In this lecture, we show how emerging technology is enabling the design and use of simple instrumentation for time-resolved fluorescence. In particular, it is now possible to imagine lifetime-based measurements of blood gases and blood glucose, and lifetime imaging of calcium and other ions in microscopic samples.
As a result of a resurgence of interest in analytical ultracentrifugation a new instrument, the Beckman XLA, was released recently. This instrument automates spectrophotometric measurements of the concentration distribution of molecules in a gravitational field. In order for the intrinsic merits of analytical ultracentrifugation to be realized fully, this instrument must also include a Rayleigh interference optical system. A prototype design and preliminary performance test of a Rayleigh interferometer for the XLA ultracentrifuge is described here.
This chapter describes the principles and practice of the optical recording of cellular metabolism. Redox imaging of cells, tissues, and organs based on intrinsic fluorescent probes of cellular metabolism. Cellular metabolism may be noninvasively interrogated through the `optical method' based on the fluorescence intensity of intrinsic probe molecules (Change, 1991). The intrinsic fluorescent probes, which report on cellular metabolism, are the reduced pyridine nucleotides, NAD(P)H, and the oxidized flavoproteins.
Chemiluminescence (CL) detection offers potential for high sensitivity immunoassays (CLIAs). Several approaches were attempted to automate CL measurements. Those include the use of photographic film, clear microtitration plates, and magnetic separation. We describe a photon counting detection apparatus that performs (CLIA) measurements. The CL detector moves toward a disposable reaction vessel to create a light-tight seal and then triggers and integrates a CL signal. The capture uses antibody coated polystyrene microparticles. A porous matrix, which is a part of a disposable reaction tray, entraps the microparticle-captured reaction product. The CL signal emanated off the immune complex immobilized by the porous matrix is detected. The detection system is a part of a fully automated immunoassay analyzer. Methods of achieving high sensitivities are discussed.
A confocal image processing system is developed for automatic identification (recognition) and characterization of confocal fluorescent images (serial optical sections). The system is capable of identifying a large percentage of structures (e.g., DNA replication sites) in the presence of background noise and non specific staining of cellular structures. A combination of image processing techniques are applied to successively refine the input image and is so organized as to find the surfaces of highly visible structures first, using simple image processing techniques, and then to adjust and fill in the missing areas of these object surfaces using a number of more complex image processing techniques. As a result, the image analysis system is capable of obtaining morphometric parameters such as surface area, volume, and position of structures of interest automatically. The system provides a powerful tool for biomedical research such as micro-structure characterization, morphogenesis, cell differentiation, tissue organization, and embryo development. We illustrate the performance of the confocal image analysis system by using an image of DNA replication sites in a mammalian 3T3 cell.
Individual, stained DNA fragments were sized using a modified flow cytometer with high sensitivity fluorescence detection. The fluorescent intercalating dye ethidium homodimer was used to stain stoichiometrically lambda phage DNA and a Kpn I digest of lambda DNA. Stained, individual fragments of DNA were passed through a low average power, focused, mode-locked laser beam, and the fluorescence from each fragment was collected and quantified. Time-gated detection was used to discriminate against Raman scattering from the water solvent. The fluorescence burst from each fragment was related directly to its length, thus providing a means to size small quantities of kilobase lengths of DNA quickly. Improvements of several orders of magnitude in analysis time and sample size over current gel electrophoresis techniques were realized. Fragments of 17.1, 29.9, and 48.5 thousand base pairs were well resolved, and were sized in 164 seconds. Less than one pg of DNA was required for analysis.
We report here on the detection and fluorescence lifetime measurement of single Rhodamine- 110 molecules in a flowing, aqueous sample stream. Time-correlated photon counting (TCPC) used in combination with pulsed excitation allows for the detection, in the presence of significant prompt Raman and Rayleigh background, of photon bursts due to single fluorescent molecules passing through a small detection volume (approximately 1 pL). The fluorescence lifetime of a detected molecule is estimated from the decay curve complied from photon arrival times in the burst.
Optical correlation systems can be used to measure blood flow and tissue motion in any direction in the plane of an ultrasound image. A detailed description of a joint transform correlator (JTC) used to track the movement of speckle is presented. The JTC system required placing a filter at the Fourier plane to enable accurate tracking. Results obtained from this system demonstrate that optical correlation can measure the speckle movement with an accuracy comparable to that of digital methods currently being used. Moreover, optical methods have potential advantages in speed, size, and power consumption. In addition, results from measuring the velocity of blood flow and tissue motion are presented.
The continued development of the vacuum tube results in image intensifiers with ever increasing capabilities in spatial resolution, and time resolution. Detectors with resolution exceeding 3000 X 2000 pixels have been developed for ground base astronomy, and space telescopes, where the extreme sensitivity in the visible, UV, X-, and Gamma parts of the spectrum are of primary consideration. Such detectors are now finding applications in biomedicine. In particular beta-labelled autoradiography and fluorescent imaging of sensitive samples at very high resolution with very low stimulation are possible. The time resolution of the detectors can be exploited in new areas such as fluorescent lifetime imaging and transillumination of tissues. It is confidently expected that the new detectors will find many more applications and this paper is an attempt to stimulate discussion.
The ability to possess detection sensitivity at the single molecule level is a technically challenging task and will have important applications for the analysis of minute quantities of DNA in applications such as Sanger dideoxy sequencing, restriction maps and scanning confocal microscopy. The ability to detect single visible fluorescent dye molecules in solution has recently been demonstrated. We wish to discuss the first report concerning the detection of single near infrared (NIR) dye molecules in solution using photon burst detection and its application for the analysis of minute quantities of DNA. Near infrared excitation and detection was used to reduce the fluorescent impurity contribution to the background, which temporal and spectral filtering cannot overcome in most cases, allowing sensitive detection in complex biological matrices.
New techniques and approaches to cellular analysis are being developed at the Los Alamos National Flow Cytometry Resource. These developments can be divided into those that improve sensitivity through the implementation of new measurement techniques and those that move the technology into new areas by refining existing approaches. An example of the first category is a flow cytometric system being assembled that is capable of measuring the phase shift of fluorescence emitted by fluorophors bound to cells. This phase sensitive cytometer is capable of quantifying fluorescence life time on a cell-by-cell basis as well as using the phase sensitive detection to separate fluorescence emissions that overlap spectrally but have different lifetimes. A Fourier transform flow cytometer capable of measuring the fluorescence emission spectrum of individual labeled cells at rates approaching several hundred per second is also in the new technology category. The current implementation is capable of resolving the visible region of the spectrum into 8 bands. With this instrument, it is possible to resolve the contributions of fluorophors with overlapping emission spectra and to determine the emission spectra of dyes such as calcium concentration indicators that are sensitive to the physiological environment.
Use of the short-wave near infrared (NIR) region for fluorescence spectroscopy is shown to afford enhanced freedom, relative to the visible spectrum, from background signals and noise resulting from scattering and endogenous fluorescence from the sample matrix itself. The detection of 0.1 attomole (10-13 mole liter-1) of two cyanine dyes in methanol solution is demonstrated using diode laser excitation at 780 nm and CCD spectrometric detection. The fluorophores exhibit excellent photostability in this solvent even under prolonged laser illumination. Limitation of sensitivity in this system is imposed by shot noise associated with the solvent Raman signals. When the same dyes are placed in aqueous solutions containing bovine serum albumin (BSA), endogenous fluorescence from the BSA and photobleaching or photolysis of the cyanine raise the practical detection limits to 10-9 moles liter-1. Despite these difficulties, the NIR region affords signal to background ratios which are superior to those observed in similar experiments performed in the visible region.
Optical raytrace analysis of instrument performance offers a powerful tool for evaluating the optical design of instruments utilizing a spectrograph and multichannel array detectors. Methods for collecting light from a source, projecting the light to the sample, collecting the light from the sample, and projecting it to a spectrograph for analysis can be evaluated. The consequences of various design options on spectrograph performance can be used to optimize overall system performance by choosing the best solution for the application. Five optical systems using different methods for light collection from a sample and projection to a spectrograph are compared. The benefits and drawbacks with respect to spectrograph optical performance are compared for both one dimensional and two-dimensional array detectors. The five optical systems being compared utilize elliptical collection optics, a single lens, a lens pair, no collection optics, and an improved lens pair option.
The application of a rapid time correlated single photon counting (TCSPC) apparatus on the investigation of fluorescent labelled DNA is presented. It could be shown that the investigated dye coumarin 120 can be used as a marker for a one dye-one lane electrophoresis-concept in DNA sequencing. This is possible because of the interaction between the dye and the four different DNA bases, which is based on a photoinduced electron transfer. A distinguishing of the bases is possible because of the different decay times. A fluorescein derivative also showed excellent fluorescence after labelling. It can be used as a marker for common electrophoresis- concepts, but the interaction was too weak for a secure base distinguishing a one dye-one lane. Now further investigations are under way to find new labeling positions resulting in stronger interactions. Additionally, we are looking for dyes in the red spectral range to improve the sensitivity of detection.