Interfacing of An Analog Spectrofluorometer With a Personal Computer

ABSTRACT

By acquiring data in the form of an excitation - emission matrix (EEM), fluorescence spectroscopy is capable of analyzing multicomponent samples. However due to electronic limitations, earlier designed fluorometers are incapable of acquiring EEM data and new instruments which can acquire EEM data are relatively expensive. To circumvent this problem, an economical hardware interface was constructed, between an older analog fluorometer and a personal computer. The excitation and emission monochromators can now be electronically controlled, monochromator position continuously tracked, and detector output digitized so as to acquire EEM data in a reasonable period of time. Multicomponent EEM spectra of polynuclear aromatic hydrocarbons were acquired as a system test.     

Multiphoton microscopy in life sciences

Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three‐dimensional fluorescence imaging based on non‐resonant two‐photon or three‐photon fluorophor excitation requires light intensities in the range of MW cm^?2 to GW cm^?2, which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi‐gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non‐invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth‐resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non‐invasive fluorophore loading into single living plant cells. Non‐destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis‐like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two‐photon excitation process rather than a one‐photon or three‐photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two‐photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid‐induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm^?2 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non‐invasive NIR laser surgery within a living cell or within an organelle is therefore possible.     

Fibre‐optical microendoscopy

Microendoscopy has been an essential tool in exploring micro/nano mechanisms in vivo due to high‐quality imaging performance, compact size and flexible movement. The investigations into optical fibres, micro‐scanners and miniature lens have boosted efficiencies of remote light delivery to sample site and signal collection. Given the light interaction with materials in the fluorescence imaging regime, this paper reviews two classes of compact microendoscopy based on a single fibre: linear optical microendoscopy and nonlinear optical microendoscopy. Due to the fact that fluorescence occurs only in the focal volume, nonlinear optical microendoscopy can provide stronger optical sectioning ability than linear optical microendoscopy, and is a good candidate for deep tissue imaging. Moreover, one‐photon excited fluorescence microendoscopy as the linear optical microendoscopy suffers from severe photobleaching owing to the linear dependence of photobleaching rate on excitation laser power. On the contrary, nonlinear optical microendoscopy, including two‐photon excited fluorescence microendoscopy and second harmonic generation microendoscopy, has the capability to minimize or avoid the photobleaching effect at a high excitation power and generate high image contrast. The combination of various nonlinear signals gained by the nonlinear optical microendoscopy provides a comprehensive insight into biophenomena in internal organs. Fibre‐optical microendoscopy overcomes physical limitations of traditional microscopy and opens up a new path to achieve early cancer diagnosis and microsurgery in a minimally invasive and localized manner.     

Ultrafast optical pulse delivery with fibers for nonlinear microscopy

Nonlinear microscopies including multiphoton excitation fluorescence microscopy and multiple-harmonic generation microscopy have recently gained popularity for cellular and tissue imaging. The optimization of these imaging methods for minimally invasive use requires optical fibers to conduct light into tight space, where free-space delivery is difficult. The delivery of high-peak power laser pulses with optical fibers is limited by dispersion resulting from nonlinear refractive index responses. In this article, we characterize a variety of commonly used optical fibers in terms of how they affect pulse profile and imaging performance of nonlinear microscopy; the following parameters are quantified: spectral bandwidth and temporal pulse width, two-photon excitation efficiency, and optical resolution. A theoretical explanation for the measured performance of these fibers is also provided.     

Photon Counter System With a Rotating Interference Filter for Measuring Time-Resolved Electroluminescence Spectra.

ABSTRACT

An automatic system for measuring electroluminescence spectra is described. The instrument works in photon counting mode to record low-intensity light. Wavelength separation is done by a rotating interference filter. The instrument is controlled by a microprocessor, and is capable of generating arbitrary pulse wave-forms for the excitation of electroluminescence. The temporal variation of the spectra can be recorded at millisecond timescale. Application to electrogenerated chemiluminescence of luminol and fluorescein is shown.     

A Multifrequency Phase Fluorometer Using the Harmonic Content of a Mode-Locked Laser

ABSTRACT

We describe the construction and operation of a cross-correlation phase and modulation fluorometer which uses the harmonic content of a high repetition rate mode-locked laser as the excitation source.

A mode-locked argon ion laser is used to synchronously pump a dye laser. The pulse train output from the dye laser is amplitude modulated by an acousto-optic modulator and then frequency doubled with an angle tuned frequency doubler. With the particular dye utilized in these studies, the ultraviolet light obtained was continuously tunable over the range 280-310 nm. In the frequency domain the high repetition rate pulsed source gives a large series of equally spaced harmonic frequencies. The frequency spacing of the harmonics is determined by the repetition frequency of the laser. Amplitude modulation of the pulse train permits variation of the frequency quasi-continuously from a few hertz to gigahertz. Use of cross-correlation techniques permits precise isolation of individual frequencies. The cross-correlation frequency required for the analysis of the phase delay and modulation ratio is obtained using coupled frequency synthesizers. In the present instrument three synthesizers are used. One drives the pump mode-locker head, a second drives the acousto-optic modulator and the third is used to modulate the response of the photomultiplier tubes which detect the signal. The accuracy, reproducibility and sensitivity of the instrumentation have been determined. Experimental data are provided to show use of this high frequency cross-correlation phase-modulation fluorometer for the determination of fluorescence lifetimes and rotational motions of tryptophan in solution and in proteins.     

Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation

A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes. Importantly, the penetration depth of the two-photon exciting (infra)red light is substantially greater than for the corresponding single-photon wavelength while photobleaching is significantly reduced. The time structure of the Ti:Sa laser can be employed in a straightforward way for the realization of fluorescence lifetime imaging. The fluorescence lifetime is sensitive to the local environment of the fluorescent molecule. This behaviour can be used for example to quantify concentrations of ions, such as pH and Ca²⁺, or pO₂ and pCO₂. In the set-up presented here the fluorescence lifetime imaging is accomplished by time-gated single photon counting. The performance and optical properties of the microscope are investigated by a number of test measurements on fluorescent test beads. Point-spread functions calculated from measurements on 230-nm beads using an iterative restoration procedure compare well with theoretical expectations. Lifetime imaging experiments on a test target containing two different types of test bead in a fluorescent buffer all with different lifetimes (2.15 ns, 2.56 ns and 3.34 ns) show excellent quantitative agreement with reference values obtained from time correlated single photon counting measurements. Moreover, the standard deviation in the results can be wholly ascribed to the photon statistics. Measurements of acridine orange stained biofilms are presented as an example of the potential of two-photon excitation combined with fluorescence lifetime contrast. Fluorescence lifetime and intensity images were recorded over the whole sample depth of 100 μm. Fluorescence intensity imaging is seriously hampered by the rapid decrease of the fluorescence signal as a function of the depth into the sample. Fluorescence lifetime imaging on the other hand is not affected by the decrease of the fluorescence intensity.     

Development of a standing-wave fluorescence microscope with high nodal plane flatness

This article reports about the development and application of a standing-wave fluorescence microscope (SWFM) with high nodal plane flatness. As opposed to the uniform excitation field in conventional fluorescence microscopes an SWFM uses a standing-wave pattern of laser light. This pattern consists of alternating planar nodes and antinodes. By shifting it along the axis of the microscope a set of different fluorescent structures can be distinguished. Their axial separation may just be a fraction of a wavelength so that an SWFM allows distinction of structures which would appear axially unresolved in a conventional or confocal fluorescence microscope. An SWFM is most powerful when the axial extension of the specimen is comparable to the wavelength of light. Otherwise several planes are illuminated simultaneously and their separation is hardly feasible. The objective of this work was to develop a new SWFM instrument which allows standing-wave fluorescence microscopy with controlled high nodal plane flatness. Earlier SWFMs did not allow such a controlled flatness, which impeded image interpretation and processing. Another design goal was to build a compact, easy-to-use instrument to foster a more widespread use of this new technique. The instrument developed uses a green-emitting helium–neon laser as the light source, a piezoelectric movable beamsplitter to generate two mutually coherent laser beams of variable relative phase and two single-mode fibres to transmit these beams to the microscope. Each beam is passed on to the specimen by a planoconvex lens and an objective lens. The only reflective surface whose residual curvature could cause wavefront deformations is a dichroic beamsplitter. Nodal plane flatness is controlled via interference fringes by a procedure which is similar to the interferometric test of optical surfaces. The performance of the instrument was tested using dried and fluorescently labelled cardiac muscle cells of rats. The SWFM enabled the distinction of layers of stress fibres whose axial separation was just a fraction of a wavelength. Layers at such a small distance would lie completely within the depth-of-field of a conventional or confocal fluorescence microscope and could therefore not be distinguished by these two methods. To obtain futher information from the SWFM images it would be advantageous to use the images as input-data to image processing algorithms such as conceived by Krishnamurthi et al. (Proc. SPIE, 2655, 1996, 18–25). To minimize specimen-caused nodal plane distortion, the specimen should be embedded in a medium of closely matched refractive index. The proper match of the refractive indices could be checked via the method presented here for the measurement of nodal plane flatness. For this purpose the fluorescent layer of latex beads would simply be replaced by the specimen. A combination of the developed SWFM with a specimen embedded in a medium of matched refractive index and further image processing would exploit the full potential of standing-wave fluorescence microscopy.     

A Phosphorimeter for Studying Delayed Fluorescence from Samples Immersed in a Magnetic Field

SUMMARY

The design and construction of a phosphorimeter especially suited for investigating the effect of a magnetic field on delayed luminescence processes is described. Although it is designed for the light levels and time domains usually pertinent to fluid-solution delayed fluorescence studies, it can be easily adapted to phosphorescence work. In order to avoid the influence of the applied field on the excitation and detection elements, these components are removed from the field and are heavily shielded. A medium-pressure mercury arc is used as an excitation source, and either frontal or right-angular illumination of the sample can be employed. Filters in both the excitation and emission beams provide the means of wavelength selection. The use of a single, rotating, slotted disk to time both beams gives the instrument a compact design and enhances its physical stability. Tests of instrument performance show that the phosphorimeter is useful for measurements at fields up to 8000 G.     

Single Photon Counting With Synchronously Pumped Dye Laser Excitation

ABSTRACT

This article reviews the advances that have been made in the technique of pulse fluorometry with time-correlated single photon counting detection brought about by the introduction of mode-locked synchronously pumped dye laser excitation. High repetition rates and small pulse width permit high data collection rates and excellent time-resolution. A modern pulse fluorometer which allows efficient measurement of fluorescence decay curves as well as automated measurement of time-resolved fluorescence spectra and of fluorescence anisotropy decays is described in detail.     

Construction of a Microcomputer Controlled Photoacoustic Spectrometer

ABSTRACT

We have developed a UV-Visible photoacoustic spectrophotometer using relatively inexpensive individual optical components. The instrument was initially developed due to the absence of a commercial photoacoustic instrument in the UV-Visible range. We have found that this custom built instrument allows numerous software generated options that are unavailable on any commercial spectrometric instruments. In addition to recording routine intensity versus wavelength spectra, we can record the following: 1) resolution of the spectra in layered samples; 2) quantitative depth profiles in samples; 3) spectra and spectral fragments can be smoothed and joined into continuous spectra; and 4) signal averaging of spectra can be carried out. The construction of this custom built instrument allowed the maximum flexibility in experimental application with minimum cost to the investigator.     

In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy

In this study, we use combined two‐photon excitation fluorescence (TPEF), second‐harmonic generation (SHG) and third‐harmonic generation (THG) measurements to image cellular structures of the nematode Caenorhabditis elegans, in vivo. To our knowledge, this is the first time that a THG modality is employed to image live C. elegans specimens. Femtosecond laser pulses (1028 nm) were utilized for excitation. Detailed and specific structural and anatomical features can be visualized, by recording THG signals. Thus, the combination of three image‐contrast modes (TPEF‐SHG‐THG) in a single instrument has the potential to provide unique and complementary information about the structure and function of tissues and individual cells of live biological specimens.     

A Phosphorimeter for Measurements of Triplet Diffusion

ABSTRACT

A phosphorimeter is described which is intended to be used for delayed fluorescence measurements in which the excitation light is introduced in a spatially nonhomogeneous manner. The excitation optics produce a reduced image of the space intermittency pattern at a 1 mm layer of the test solution. The delayed emission is isolated from prompt fluorescence by an adjustable phasing between excitation and emission choppers. Solutions of anthracene and 9, 10-diphenyl anthracene in ethanol both show changes in delayed fluorescence intensity with changing dimensions of the space intermittency pattern. The effects observed are in agreement with theoretical expectations.     

Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging

Two‐photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580–1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two‐photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two‐photon and the one‐photon emission spectra. However, a blue‐shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non‐linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.     

A Strategy to Probe Air-Liquid Interfaces by Confocal Fluorescence Microscopy

Abstract

Confocal fluorescence microscopy has been seldom applied to air-liquid interfaces due to technical difficulties. Satellite lines of an excitation laser beam can be used as an inherent reference for a confocal microscope to align and calibrate the setup. This strategy is especially useful and important to a liquid surface, and makes it possible to observe very weak fluorescence without time-consuming alignment procedures.     

Independent Component Analysis Applied to Raman Spectra for Classification of In Vitro Human Coronary Arteries

Abstract

Optical diagnostic methods, such as near‐infrared Raman spectroscopy allow quantification and evaluation of human affecting diseases, which could be useful in identifying and diagnosing atherosclerosis in coronary arteries. The goal of the present work is to apply Independent Component Analysis (ICA) for data reduction and feature extraction of Raman spectra and to perform the Mahalanobis distance for group classification according to histopathology, obtaining feasible diagnostic information to detect atheromatous plaque. An 830 nm Ti:sapphire laser pumped by an argon laser provides near‐infrared excitation. A spectrograph disperses light scattered from arterial tissues over a liquid‐nitrogen cooled CCD to detect the Raman spectra. A total of 111 spectra from arterial fragments were utilized.     

The light microscope on its way from an analytical to a preparative tool

Light intensities of up to 10¹³ W/cm² can be generated by focusing light, particularly laser pulses, into a microscope. Such power densities can be used to cut, perforate, or fuse microscopic objects with submicrometre accuracy. Suitable light sources for such a ‘microbeam’ are nitrogen lasers with a working wavelength of 337 nm, frequency-multiplied Neodym YAG lasers (266 or 355 nm) or excimer lasers (308 nm). In combination with dye lasers, tunable microbeams covering the wavelength range from the ultraviolet to the infra-red can be constructed. Such laser microbeams can be used to modify microchip substrates. Micro-injection of materials into biological cells or fusion of selected cell pairs under total microscopic control is also possible. Using the same equipment, elongated biological objects can be microdissected with submicrometre precision, for example in attempts to isolate DNA from a specific region of the human genome. In addition to the use of high-power pulsed lasers, the light of a continuous-wave infra-red laser can be used for the transport of microscopic objects. There, light pressure and the inhomogeneity of the electric field in a light pulse are used to trap microscopic objects in the focused laser beam, using the beam as ultrafine non-mechanical tweezers. Unlike mechanical microtools the optical trap is gentle and absolutely sterile. A combined laser microbeam and optical trap (a microbeam-trap) converts the light microscope, which is usually regarded as an analytical instrument, into a universal preparative instrument that allows micromanipulation of microscopic objects without mechanical contact. In contrast to any other micromanipulator, the microbeam-trap can work in the depth of an object without opening it.     

Remote Detection of Organochlorides With a Fiber Optic Based Sensor II. a Dedicated Portable Fluorimeter

ABSTRACT

A dedicated portable fluorimeter, for use with fiber optic chemical sensors (FOCS) has been designed, constructed, tested, and calibrated. This represents a major advance in the development of a FOCS system suitable for in-field use. The portable fluorimeter uses an incandescent lamp, instead of a laser, for FOCS excitation and a photodiode, in place of a photomultiplier tube for detecting the fluorescence signal. It uses an optical system which is internally connected to a unitized optical block by 600 μm core optical fibers, to minimize alignment problems and increase overall system ruggedness. The system noise is less than .1.5 mV and the long-term drift is less than ±2 mV/hour. Measurements of organochloride were made at concentrations as low as 80 parts-per-billion with a signal to noise ratio of 40:1.     

Second harmonic generation imaging of the deep shade plant Selaginella erythropus using multifunctional two‐photon laser scanning microscopy

Background : Multifunctional two‐photon laser scanning microscopy provides attractive advantages over conventional two‐photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were achieved from the deep shade plant Selaginella erythropus. Results : These measurements show that the S. erythropus leaves produce high SHG signals in both directions and the SHG signals strongly depend on the laser's status of polarization and the orientation of the dipole moment in the molecules that interact with the laser light. The novelty of this work is (1) uncovering the unusual structure of S. erythropus leaves, including diverse chloroplasts, various cell types and micromophology, which are consistent with observations from general electron microscopy; and (2) using the multifunctional two‐photon laser scanning microscopy by combining three platforms of laser scanning microscopy, fluorescence microscopy, harmonic generation microscopy and polarizing microscopy for detecting the SHG signals in the forward and backward directions, as well as two photon excitation fluorescence. Conclusions : With the multifunctional two‐photon laser scanning microscopy, one can use noninvasive SHG imaging to reveal the true architecture of the sample, without photodamage or photobleaching, by utilizing the fact that the SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation characteristic.