A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents

Optogenetics is an excellent tool for noninvasive activation and silencing of neurons and muscles. Although they have been widely adopted, illumination techniques for optogenetic tools remain limited and relatively nonstandardized. We present a protocol for constructing an illumination system capable of dynamic multispectral optical targeting of micrometer-sized structures in both stationary and moving objects. The initial steps of the protocol describe how to modify an off-the-shelf video projector by insertion of optical filters and modification of projector optics. Subsequent steps involve altering the microscope's epifluorescence optical train as well as alignment and characterization of the system. When fully assembled, the illumination system is capable of dynamically projecting multispectral patterns with a resolution better than 10 μm at medium magnifications. Compared with other custom-assembled systems and commercially available products, this protocol allows a researcher to assemble the illumination system for a fraction of the cost and can be completed within a few days.     

Comparison of three types of reagents for detection of Bacteroides fragilis by direct immunofluorescence

Three different methods were used to prepare conjugates for the detection of rods of the Bacteroides fragilis group by direct immunofluorescence. Lyophilized conjugates were prepared. Three sets (five in each) of monovalent conjugates against serotype strains of B. fragilis (including conjugate E/E1 + E2) and polyvalent conjugate (A + B + C + D + E1 + E2) were obtained. Each conjugate was prepared in two variants: 1. unabsorbed, 2. absorbed with tissue powder prior to lyophilization. Conjugates obtained by precipitation of sera with 50% ethanol and direct coupling of gammaglobulins with stain were found to meet the requirement for good fluorescence reagents and are well suited for the detection of B. fragilis by direct immunofluorescence. Absorption of the conjugates with tissue powder before lyophilization did not affect their quality.     

Improved double immunofluorescence for confocal laser scanning microscopy.

Reliable double immunofluorescence labeling for confocal laser scanning microscopy requires good separation of the signals generated by the fluorochromes. We have successfully overcome the limitation of a single argon ion laser in achieving effective excitation of dyes with well-separated emission spectra by employing the novel sulfonated rhodamine fluorochromes designated Alexa 488 and Alexa 568. The more abundant antigen was visualized using the red-emitting Alexa 568, with amplification of the signal by a biotinylated bridging antibody and labeled streptavidin. This was combined with the green-emitting Alexa 488, which yielded brighter images than fluorescein but exhibited comparable photodegradation. With appropriate controls to ensure the absence of crosstalk between fluorescence channels, these dyes permitted unequivocal demonstration of co-localization. This combination of fluorochromes may also offer advantages for users of instruments equipped with alternative laser systems.     

Improvement of immunofluorescence for diagnosis of AIDS using laser microscopy

One of the most commonly used methods for demonstration of HIV antibodies is indirect immunofluorescence employing HIV-infected, CD4-positive lymphoid cell lines as antigenic substrate. Immunofluorescence with conventional optic equipment is reported to be slightly less sensitive than enzyme-linked immunoassay (ELISA). We have developed an immunofluorescence microscope which is equipped with an argon laser that has the advantages of much brighter fluorescence than conventional techniques, the prevention of fluorescence bleaching, and the possibility of distinguishing specific from nonspecific staining by comparative analysis of the kinetics of the bleaching curves. This microscope has now been used for demonstration of HIV antibodies in indirect immunofluorescence tests on the H9 lymphoid cell line, which is highly efficient in expressing HIV after infection. Titers of ELISA and Western blot-verified HIV-positive patients and appropriate normal controls were compared using four types of microscopic equipment, including the laser immunofluorescence microscope. The latter afforded significantly higher titers than those obtained with conventional immunofluorescence microscopes, and also made possible the distinction between specific and nonspecific staining.     

A comparison of mercury arc lamp and laser illumination for flow cytometers.

Optical differences between a mercury arc lamp and a laser-illuminated flow cytometer are compared. The distributions of spectral intensities of the two light sources are shown in relation to the excitation characteristics of the fluorescent dyes acriflavine, chromomycin A3, mithramycin, ethidium bromide, Hoechst 33258, and 4,6-diamidino-2-phenylindole (DAPI). Fluorescence intensities of microspheres and Hoechst 33258-stained mouse sperm are compared in the two cytometers. The optical efficiencies are similar and depend on the match of the excitation characteristics of the stain with the emission spectra of the light source.     

Flow injection analysis with immobilized reagents.

Immobilized reagent phase flow injection analysis can be configured as discrete reagent cells upstream of the sensor element or as an integral reagent/transduction system (flow injection analysis-biosensor). The former approach has attracted greater attention because several assays can be assembled with greater versatility in reagent column units employing a single sensor, than can be co-immobilized on the surface of a transducer.     

Violet laser diodes as light sources for cytometry

BACKGROUND: Violet laser diodes have recently become commercially available. These devices emit 5-25 mW in the range of 395-415 nm, and are available in systems that incorporate the diodes with collimating optics and regulated power supplies in housing incorporating thermoelectric coolers, which are necessary to maintain stable output. Such systems now cost several thousand dollars, but are expected to drop substantially in price. Materials and Methods A 4-mW, 397-nm violet diode system was used in a laboratory-built flow cytometer to excite fluorescence of DAPI and Hoechst dyes in permeabilized and intact cells. Forward and orthogonal light scattering were also measured. RESULTS: DNA content histograms with good precision (G(0)/G(1) coefficient of variation 1.7%) were obtained with DAPI staining; precision was lower using Hoechst 33342. Hoechst 34580, with an excitation maximum nearer 400 nm, yielded the highest fluorescence intensity, but appeared to decompose after a short time in solution. Scatter signals exhibited relatively broad distributions. CONCLUSIONS: Violet laser diodes are relatively inexpensive, compact, efficient, and quiet light sources for DNA fluorescence measurement using DAPI and Hoechst dyes; they can also excite several other fluorescent probes.     

A method of laser dark-field illumination for equal-probability excitation and photobleaching of randomly oriented fluorescent molecules

Calculations have been done that allow developing a new type of illuminators optimized for dark-field microscopy and fluorescent nanoscopy, a new type of ultrahigh-resolution microscopy for which we have patent priority, and its later analogs. The concept proposed here substantiates the feasibility of uniformly illuminating different parts of an object to ensure equal probability of excitation and, in due time, simultaneous photobleaching of fluorescent molecules despite the random orientation of their light-absorbing oscillators. The latter is important because non-bleached fluorophores that accumulate in the specimen produce background noise and make it too difficult to locate the centers of spots produced by new fluorescent molecules.     

Evaluation of novel derivatisation reagents for the analysis of oxysterols

Oxysterols are oxidised forms of cholesterol that are intermediates in the synthesis of bile acids and steroid hormones. They are also ligands to nuclear and G protein-coupled receptors. Analysis of oxysterols in biological systems is challenging due to their low abundance coupled with their lack of a strong chromophore and poor ionisation characteristics in mass spectrometry (MS). We have previously used enzyme-assisted derivatisation for sterol analysis (EADSA) to identify and quantitate oxysterols in biological samples. This technique relies on tagging sterols with the Girard P reagent to introduce a charged quaternary ammonium group. Here, we have compared several modified Girard-like reagents and show that the permanent charge is vital for efficient MSⁿ fragmentation. However, we find that the reagent can be extended to include sites for potential stable isotope labels without a loss of performance.     

Two-photon excitation and photolysis by pulsed laser illumination modelled by spatially non-uniform reactions with simultaneous diffusion

Two-photon absorption in the focus of a pulsed laser has the potential for localized photolysis of caged compounds, generating high concentrations of neurotransmitters, hormones and messengers. The concentrations of cage, intermediates and products in the femtolitre focal volume depend on reaction rates and diffusional exchange with the external volume. This problem of reaction with diffusion was analysed with analytical and numerical methods to determine simple relations between parameters useful in the design and interpretation of experiments. The diffraction-limited laser spot is approximated well by a sphere, radius A, in diffusional exchange with either an infinite uniform medium, representing extracellular photolysis, or within a non-permeable sphere, a "cell" of radius B, representing intracellular photolysis. Photolysis is modelled as sequential irreversible reactions, with either the excitation step alone, rate constant k(e), or with a subsequent "dark" reaction, rate constant k(p). For extracellular photolysis, steady-state depletion of a cage averaged in a spherical spot increases hyperbolically with k(e) with half-maximum depletion at k(e) = K0.5 = 2.5 D/A2, where D is the diffusion coefficient. With measured parameters for spot size A = 0.3 microm and diffusion D = 800 microm2/s, K0.5 = 22,200 s(-1). The optimal exposure for localized photolysis is the characteristic diffusion time tau = A2/D, 113 micros in this example, and is the time taken to reach 57% of steady state in the diffusion-limited case. In the two-step model, with excitation and "dark" reaction steps, rate constants both exceeding K0.5 are necessary to generate 50% of maximal product concentration in the illuminated volume. High concentrations of photolysis products depend particularly on a high excitation rate constant (k(e) > K0.5), and localization of the products requires fast dark reactions (k(p) > K0.5). If products diffuse faster than the cage, their steady-state concentrations are decreased, and concentration transients may occur. For localized intracellular photolysis, the duration of exposure that generates product concentration at the cell boundary, B, less than 10% of the spot concentration should be shorter than 0.043(B/A)3tau, and is determined by diffusion.     

Dual-parameter scatter-flow immunofluorescence analysis of Bacillus spores

Using a commercial flow cytometer (Cyto-fluorograf), narrow-forward-angle (NFA) light-scatter signals were detected for spore preparations of Bacillus anthracis Vollum, B. anthracis Sterne, B. cereus NCTC 8035, and B. subtilis var niger. In the flow immunofluorescence (FIF) analysis of spores stained with fluorescein-conjugated hyperimmune antibody to B. anthracis Vollum spores, fluorescence histograms could be acquired by selecting on NFA scatter. Fluorescence data selected on ninety degree scatter were rather noisier. Fluorescence analysis by dual parameter NFA scatter-FIF techniques was shown to have several advantages over the subtraction FIF method reported earlier. The implication from FIF analysis of spore suspensions and corresponding cell-free supernatants that the peak in the fluorescence histogram was caused by signals from fluorescing spores, was confirmed by use of the cell sorter and subsequent microscopy of the sorted samples. Although a proportion of spore aggregates was present in samples sorted from the right-hand tail of the fluorescence histogram, it was demonstrated that the majority of the observed distribution of fluorescence was not due to the formation of aggregates but was rather an expression of variation in the degree of staining of individual spores.     

Simultaneous enumeration of T-cell subsets and macrophages in bronchoalveolar lavage fluids by immunoenzyme double staining. Comparison with conventional immunofluorescence

Bronchoalveolar lavage is a common research and clinical tool for the retrieval of cells from the lower respiratory tract. In addition to conventional morphologic study of these cells, the subtyping of T lymphocytes is often important for reaching a diagnosis of a disease or assessing its activity; subtyping is usually done by a standard immunofluorescence assay on cell suspensions requiring about 5 X 10(5) cells. Since the number of leukocytes in the lavage fluid from many patients is too small to obtain reliable information by this assay, a double immunoenzyme staining of T-lymphocyte subtypes on Cytospin preparations was utilized. This method, which requires a small number of cells, was compared with the standard immunofluorescence assay for the identification and quantitation of lymphocyte subtypes in the lavage fluids of patients with different disorders. Although the immunoenzyme double staining assay is somewhat more laborious, it provides important advantages: (1) simultaneous observation of two lymphocyte subsets and macrophages on the same slide; (2) a considerably smaller number of cells (2 X 10(4) instead of 5 X 10(5] is necessary; (3) the availability of permanent preparations; (4) the possibility of storing the Cytospin slides before staining; and (5) conventional light microscopy can be used. Since the reliability of both techniques appeared to be the same, the double staining assay for routine usage with bronchoalveolar lavage fluids appears to be preferable.     

Immunoglobulin-producing cells in lymphatic tissues of germfree and conventional rabbits as detected by an immunofluorescence method

Lymphatic tissues of GF and CV rabbits were observed. No cells producing IgA and IgM antibodies were detected in appendix, sacculus rotundus, ileum terminale and thymu s of GF rabbits. IgA cells were found in lymph nodes of GF rabbits.     

MEK inhibitor U0126 interferes with immunofluorescence analysis of apoptotic cell death

BACKGROUND: Binding of extracellular growth factors to cell surface receptors often results in activation of the mitogen-activated protein kinase (MAPK). MAPK is regulated by MAPK kinase, also called MEK. Deprivation of growth factors during cell culture or intracellular MEK inhibition leads to inhibition of proliferation and apoptotic cell death. Besides other techniques, apoptotic cells can be identified by phosphatidylserine (PS) exposure and exclusion of membrane-impermeant propidium iodide (PI). We investigated the limitations of detection of apoptotic cell death and cytofluorometry in cells cultured in the presence of the MEK inhibitor U0126. METHODS: Apoptotic cell death was induced in the plasmacytoma cell line INA-6, in peripheral blood mononuclear cells (PBMC), and in cultured T lymphoblasts by deprivation of interleukin-6 (IL-6) or by incubation with the MEK inhibitor U0126. Apoptotic cell death was quantified by flow cytometry using annexin V/propidium iodide (AxV/PI) double staining. RESULTS: U0126-treated cells dramatically changed their fluorescence pattern during cell culture. If AxV/PI staining is employed to detect apoptotic cell death, the background fluorescence mimicks PS exposure on viable cells. The compound itself has no intrinsic fluorescence in vitro but develops an intensive fluorescence during cell culture which can be observed in all fluorescence channels with a predominance in the FL1 channel (525 nm). We further demonstrate that at least some of the U0126-induced background fluorescence is dependent on cellular uptake and intracellular modifications or cellular responses. CONCLUSIONS: These results demonstrate that appropriate controls for every single time point are necessary if fluorescence analyses are performed in the presence of chemical enzyme inhibitors. In the case of MEK inhibitors, either the use of PD098059 or PD184352 as an alternative for U0126 or nonfluorometric methods for detection of apoptosis should be considered.     

Use of the indirect immunofluorescence method for the comparative analysis of histones

With the help of indirect immunofluorescence on the model systems--a ploid line of wheat, haploid and diploid cells of Chlamydomonas reinhardii, glass beads with adsorbed histones--a study was made of the dependence of ultimate dilutions (UD) of antihistone sera on the quantity, density and immunochemical properties of histones--antigens. The UD value in the artificial model system (glass beads) increased with the rise in the quantity and density of histones on bead to some definite limits, and then the UD remains constant to be determined only by the titre of antiserum. In natural model system (wheat, Chlamydomonas reinhardii), with the rise in quantities of DNA and histones in the nucleus their densities remain constant, with no changes of UD values being observed. The results obtained are discussed in terms of establishing the dependence of UD on immunochemical properties of histones-antigens. Thus, the method of indirect immunofluorescence may be used for comparative analysis of immunochemical properties of histones in various objects.     

Enhancement of biodegradation of phenol and a nongrowth substrate 4-chlorophenol by medium augmentation with conventional carbon sources

The enhancement of biodegradation of phenol and 4-chlorophenol (4-cp) as a cometabolised compound by Pseudomonas putida ATCC 49451 was accomplished by augmenting the medium with conventional carbon sources such as sodium glutamate and glucose. Compared with phenol as the sole carbon source, the addition of 1 gl(-1) sodium glutamate increased the toxicity tolerance of cells toward 4-cp and significantly improved the biodegradation rates of both phenol and 4-cp even when the initial concentration of 4-cp was as high as 200 mgl(-1). On the other hand, supplementation of glucose caused a significant drop in the medium pH from 7.2 to 4.3 resulting in a reduction of degradation rate, leaving a considerable amount of 4-cp undegraded when the initial concentration of 4-cp was higher than 100 mgl(-1). By regulating the pH of the medium, however, enhancement of degradation rates of phenol and 4-cp in the presence of glucose was achieved with a concomitant complete degradation of phenol and 4-cp.     

Enhancement of biodegradation of phenol and a nongrowth substrate 4-chlorophenol by medium augmentation with conventional carbon sources

The enhancement of biodegradation of phenol and4-chlorophenol (4-cp) as a cometabolised compound byPseudomonas putida ATCC 49451 was accomplishedby augmenting the medium with conventional carbonsources such as sodium glutamate and glucose. Comparedwith phenol as the sole carbon source, the addition of1 gl^-1 sodium glutamate increased the toxicitytolerance of cells toward 4-cp and significantlyimproved the biodegradation rates of both phenol and4-cp even when the initial concentration of 4-cp wasas high as 200 mgl^-1. On the other hand,supplementation of glucose caused a significant dropin the medium pH from 7.2 to 4.3 resulting in areduction of degradation rate, leaving a considerableamount of 4-cp undegraded when the initialconcentration of 4-cp was higher than 100 mgl^-1.By regulating the pH of the medium, however,enhancement of degradation rates of phenol and 4-cp inthe presence of glucose was achieved with aconcomitant complete degradation of phenol and 4-cp.