Klaus W. Berndt

Fluorescence lifetime imaging

We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-to-pixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created life-time images of Yt-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.

Lifetime‐selective fluorescence imaging using an rf phase‐sensitive camera

We report the creation of two‐dimensional fluorescence lifetime images, based on a sinusoidally modulated image intensifier that is operated as a radio‐frequency phase‐sensitive camera, synchronized to a mode‐locked and cavity dumped picosecond dye laser. By combining the image intensifier with a CCD camera and applying digital image processing, lifetime‐selective signal suppression can be realized even for fluorophores with comparable lifetimes. This phase‐sensitive technique can be used to create fluorescence lifetime images, that is, images in which the contrast is based upon the fluorescence lifetimes rather than upon local probe concentration and/or intensity. Because the lifetimes of many dyes are sensitive to the chemical environments surrounding the fluorophore, fluorescence lifetime imaging (FLIM) can reveal the local chemical composition and properties of the molecular environment that surrounds the fluorophore. As an example we created images of rhodamine 6G (4 ns) and rhodamine B (1.5 ns) solu...

Electroluminescent lamp-based phase fluorometer and oxygen sensor

We have tested 454-nm violet-emitting solid state electroluminescent lamps (ELLs) as inexpensive intensity-modulated excitation light sources for phase fluorometric oxygen sensors. Compared with blue-emitting silicon carbide LEDs, planar surface ELLs can be produced in various shapes and in large sizes. Accordingly, the overall optical output power emitted by ELLs is much higher than that of blue LEDs. By arranging a large-size ELL close to a large-size fluorescent chemical sensor, we obtained a large number of fluorescence photons allowing for the use of a pin photodiode instead of a photomultiplier tube as the detector. For a sinusoidal driving voltage at a frequency f, the ELL output light is modulated at 2f and at harmonics of 2f. Because of this nonlinear modulation characteristic, we used a square wave driving signal, resulting in a pulsed light output at a repetition rate twice the square wave frequency. The shortest light pulses obtained had a FWHM close to about 1 microsecond. This means that the violet ELLs used in our tests provide modulation frequencies at twice the square wave driving frequency and at all harmonics thereof up to about 1 MHz. This would allow the use of fluorescent chemical sensors with decay times as short as 30 ns, assuming that a phase shift of 10 degrees is adequate for the application. Due to the high ELL driving voltage, effective shielding is required to avoid electromagnetic interference between the modulated light source and the photodetector. Depending on the driving frequency and voltage applied, the ELLs showed a decrease in the optical output power to 50 or even 10% during the first 100 h of operation.

Phase‐modulation fluorometry using a frequency‐doubled pulsed laser diode light source

We describe the use of a pulsed frequency‐doubled laser diode, the Hamamatsu model PLP‐01 picosecond light pulser, as a 413‐nm excitation light source for frequency‐domain fluorescence measurements. The modulated incident light, over a range of modulation frequencies, is provided by the harmonic content of the pulse train. In comparison with the more usual light source for harmonic‐content excitation, a sync‐pumped/cavity dumped/frequency doubled dye laser, the 413‐nm PLP‐01 has a longer pulse width (FWHM of 40 ps), a similar pulse repetition rate (up to 10 MHz), much less output power at a fixed wavelength (0.44‐mW peak, 220‐nW maximum average power), but is less expensive, small‐sized, and easy to handle. Using the PLP‐01, we were able to perform frequency‐domain fluorescence measurements up to an upper modulation frequency of about 2000 MHz, and to resolve mixtures of fluorophores exhibiting different lifetimes. During our tests, we observed remarkable and lasting (2 h) time drifts between the optical ...

Detection and localization of absorbers in scattering media using frequency-domain principles

We report on time-resolved reflectance imaging experiments on a scattering medium containing a spatially limited absorber. The medium is illuminated at two positions with pulses from a mode-locked and cavity dumped picosecond dye laser. Time-resolved imaging of the back-scattered light is realized by means of a RF-phase-sensitive camera, synchronized to the laser pulses. The camera consists of a sinusoidally modulated proximity-focused image intensifier, a thermo-electrically cooled CCD camera, and a digital image processor unit. In operation, at least two images are taken under different image intensifier modulation conditions, such as modulation phase or modulation degree. By processing the stored images, a final image can be created the contrast of which is based only on time differences of the back-scattered photons. We found that this image reveals the presence and, to a certain extent, the position of a spatially limited absorber within the scattering medium. These experiments have been performed to evaluate possible ways towards an eventual optical tomography in living tissue.

Fluorescence-lifetime-based sensing of blood gases and cations

Chemical sensing with fluorescent probes is usually accomplished by measuring the fluorescence intensity or using wavelength-ratiometric probes. Measurement of fluorescence decay times, rather than intensities, is advantageous because the decay times are largely independent of intensity changes due to light losses, photobleaching, or probe washout. Present technology allows measurement of nanosecond decay times by the phase-modulation method with inexpensive and robust instrumentation. This report describes the use of phase- modulation fluorometry for lifetime-based sensing of O2, pH, Ca2+ and K+.

A 4-GHz frequency-domain fluorometer with internal microchannel plate photomultiplier cross-correlation

We have developed and tested a multifrequency phase/modulation fluorometer based on the Hamamatsu Model R2024U gatable microchannel plate photomultiplier (MCP-PMT), using internal MCP-PMT cross-correlation. This internal mixing is accomplished by biasing and modulating the gating mesh which is located 0.2 mm behind the photocathode. Near the photocathode center, no high-frequency photocurrent modulation was achieved. Within a circular area near the photocathode edge, however, the R2024U allows accurate phase shift and demodulation measurements up to at least 4.5 GHz, the frequency limit of our PMT-modulation amplifier. By mixing immediately after the photocathode, there is no decrease in the time resolution due to transit time spread, and the MCP has to process only low-frequency signals. This means no low-level high-frequency signal voltages have to be handled in this fluorometer, and the problems of RF shielding become much less critical. Also, the effective output impedance of the PMT has been increased, resulting in a 43-dB increase in the PMT output signal power. In principle, more MCPs could be built into the PMT, allowing an improved fluorescence detection limit. We have used the method of reference fluorophores in order to compensate for pronounced PMT color effects, a wavelength-dependent modulation, and a wavelength-dependent time shift. No color correction is required in the case of time-dependent depolarization. The performance of the instrument was verified by measurements of the intensity decay of perylene, which showed a single-exponential decay, and by measurements of the decay of tryptophan in water, which showed a double-exponential decay, as expected.(ABSTRACT TRUNCATED AT 250 WORDS)

4‐GHz internal MCP‐photomultiplier cross correlation

We have performed sinusoidal and pulsed internal cross correlation within a Hamamatsu model R2024U gatable microchannel‐plate photomultiplier (MCP‐PMT) by biasing and modulating the gating mesh which is located 0.2 mm behind the photocathode. The light source used was a sync‐pumped and cavity‐dumped dye laser. Near the photocathode center, no effective high‐frequency photocurrent modulation was achieved. However, for a circular area near the photocathode edge, useful modulation up to 4.5 GHz, the frequency limit of our amplifier, was obtained. The modulation frequency response is characterized by resonances caused by the gating mesh which represents a strong disturbance with respect to the modulation‐cable termination. The shortest measured rise time for pulsed modulation was 178 ps at 700 nm. The measured pulse response is a ‘‘mirror image’’ of a response curve as observed normally because the gating pulse is sampled by the δ‐like photoelectron pulse. From this observation it can be reasoned that an even...