Use of a new violet-excitable AmCyan variant as a label in cell analysis

Here we report a new variant of AmCyan fluorescent protein that has been specifically designed for multicolor cell analysis. AmCyan is one of the existing violet fluorochromes for use in flow cytometers equipped with a violet (405 nm) laser. It is also widely used as a label in fluorescent spectroscopy. Limitations on its use are due to the significant AmCyan fluorescence spillover into the FITC detector, due to excitation of AmCyan by the blue (488 nm) laser. In order to resolve this problem, we modified the excitation profile of AmCyan. The new fluorescent protein that we developed, AmCyan100, has an emission profile similar to AmCyan with an emission maximum at 500 nm, but its excitation maximum is shifted to 395 nm, which coincides more closely with the violet laser line and decreases the excitation with the blue laser, thus reducing the spillover observed with the original AmCyan. Moreover, this new protein has a Stokes shift of more than 100 nm compared to the Stokes shift of 31 nm in its precursor. Our data also suggests that AmCyan100-mAb conjugates have brightness similar to AmCyan-mAb conjugates. In summary, AmCyan100 conjugates have minimum spillover into the FITC detector, and can potentially replace existing AmCyan conjugates in multicolor flow cytometry without any changes in instrumental setup and existing reagent panel design.     

Human lymphocyte subpopulations identified by using three-color immunofluorescence and flow cytometry analysis: correlation of Leu-2, Leu-3, Leu-7, Leu-8, and Leu-11 cell surface antigen expression

Three-color immunofluorescence has been used to determine the co-expression of cell surface antigens on human peripheral blood lymphocytes. Monoclonal antibodies or avidin were coupled to either FITC (green), phycoerythrin (orange), or Texas Red (red) fluorochromes. These three fluorochromes could be independently measured by using a dual laser FACS IV system equipped with an argon ion laser (488 nm) and a dye laser (600 nm). Human peripheral blood lymphocytes were stained with the following combinations of reagents: (1) FITC anti-Leu-11a + PE anti-Leu-2a + TR avidin/biotin anti-Leu-7; (2) FITC anti-Leu-11a + PE anti-Leu-3a + TR avidin/biotin anti-Leu-7; (3) FITC anti-Leu-8 + PE anti-Leu-2a + TR avidin/biotin anti-Leu-7; and (4) FITC anti-Leu-11a + PE anti-Leu-2 + TR avidin/biotin anti-Leu-8. The light scatter, green fluorescence, orange fluorescence, and red fluorescence signals for each sample were stored by a Consort 40 PDP/11 computer in list mode files. Sequential reanalysis of the data directly demonstrated the existence of several unrecognized subpopulations of lymphocytes. Previously, we reported that the anti-Leu-7 and anti-Leu-11 antibodies can be used to identify discrete subsets of human NK cells with distinct functional capacities. In this report, we show that these subsets can be further subdivided on the basis of Leu-8 and Leu-2 expression. Thus, these studies illustrate how multicolor and multiparameter flow cytometry can further our understanding of cellular heterogeneity within this group of lymphocytes.     

Triple immunofluorescence confocal laser scanning microscopy: spatial correlation of novel cellular differentiation markers in human muscle biopsies.

Two different methods for triple immunofluorescence imaging with a confocal laser scanning microscope (CLSM) are described. The methods enable spatial "mapping" of 3 different epitope distribution patterns simultaneously in one tissue section. The key to triple imaging includes: (a) specific immunolabeling with 3 different mouse monoclonal antibodies (mAbs), (b) localization of the antibody binding sites by 3 different dyes, (c) spectral isolation of each dye by using selective band pass or long pass emission filters, (d) computerized imaging of the fluorescences as colored overlays or as selective signals in each optical section through the tissue in the z-direction. Method 1 consists of the combination of fluorescein isothiocyanate (FITC), phycoerythrin R (PE), and Texas Red (TR) as fluorescent markers. These dyes can be imaged by using 488 nm and 543 nm excitation laser lines. In method 2 aminomethyl coumarin acetic acid (AMCA) was combined with FITC and PE. For this application the CLSM was adapted to ultraviolet microscopy that enabled the use of 3 laser lines (364 nm, 488 nm, 543 nm) for excitations. Cryostat sections of diagnostic human muscle biopsies (n = 9) were studied which were normal by ordinary light microscopic examination. Sections were incubated with mAbs specific for: (1) the fast myosin heavy chain (My32); (2) the major histocompatibility class II antigen HLA-DR; (3) the lymph node homing receptor Leu8, and (4) the cell adhesion receptor OKM5 (CD36). By combining these mAbs in triple staining procedures, 3 capillary types and 4 different phenotypes expressed by muscle fibers were identified simultaneously. The mAbs Leu8 and OKM5, widely used as leukocyte typing antibodies in the blood, exhibit hitherto unrecognized specificities for antigens displayed by muscle fibers. At the level of these markers, specific spatial correlations between OKM5 reactive capillaries and both OKM5 reactive and nonreactive muscle fiber types become visible. The presented results provide direct evidence for cellular complexity and novel insight into the immunoanatomical architecture of skeletal muscle. The methods may be of general significance for the construction and quantification of three-dimensional multiparameter "maps" of cells and tissues.     

Increased immunofluorescence sensitivity using 532 nm laser excitation.

OBJECTIVE: We evaluated the use of a high power, diode pulsed solid-state laser emitting 532 nm light for immunofluorescence applications. We compared the sensitivity and utility of this laser with the standard 488 nm excitation. METHODS: A flow cytometer was equipped with both a 488 nm and a 532 nm laser; fluorescence emissions from each laser were collected using the same filters and the same detector system. Cells or compensation beads (e.g. latex beads coated with anti-kappa antibodies) were stained with monoclonal antibodies conjugated to phycoerythrin (PE) as well as the PE tandem dyes TRPE, Cy5PE, Cy5.5PE, and Cy7PE. The sensitivity of detection of these reagents as well as those in heavily compensated channels was quantified by measuring the spreading error for a primary detector into a secondary detector. RESULTS: Measurement of the fluorescence emission of PE and PE-tandem dyes was considerably more sensitive when using 532 nm excitation (150 mW) as compared with 488 nm excitation (20 mW). In addition, as the absolute number of photoelectrons collected was greater, there was less measurement-error-induced spread into the compensated channels. As an example, when comparing the spreading error of PE labeled cells into the TRPE detector, the green laser was found to be 15-fold more sensitive as compared with the blue laser. In addition, the blue laser produced more autofluoresent signal from cells as compared with the green laser. Together, these advantages of the 532 nm excitation line provides for a significantly improved detection of immunofluorescence staining.     

Multiplex FISH and three-dimensional DNA imaging with near infrared femtosecond laser pulses

We report on a novel technology for multicolor gene and chromosome detection as well as for three-dimensional (3D) DNA imaging by multiphoton excitation of multiple FISH fluorophores and DNA stains. Near infrared femtosecond laser pulses at 770 nm were used to simultaneously excite the visible fluorescence of a wide range of FISH fluorophores, such as FITC, DAC, Cy3, Cy5, Cy5.5, rhodamine, spectrum aqua, spectrum green, spectrum orange, Jenfluor, and Texas red as well as of DNA/chromosome stains, for example Hoechst 33342, DAPI, SYBR green, propidium iodide, ethidium homodimer, and Giemsa. In addition to the advantage of using only one excitation wavelength for a variety of fluorophores, multiphoton excitation provided the intrinsic possibility of 3D fluorescence imaging. The technology has been used in human genetics for the diagnosis of numerical chromosome aberrations and microdeletions. In particular, multicolor 3D images of the intranuclear localization of FISH-labeled chromosome territories in interphase nuclei of amniotic fluid cells have been obtained. Using the high light penetration depth at 770 nm, optical sectioning of Hoechst 33342-labeled DNA within living culture cells and within tissue of living tumor-bearing mice was performed.     

Analysis of CD36 expression on human monocytic cells and atherosclerotic tissue sections with quantum dots: investigation by flow cytometry and spectral imaging microscopy

OBJECTIVE: To demonstrate CD36 expression with quantum dots (QDs) 525 and/or 605 on human monocytic U937 cells and atherosclerotic tissue sections by means of flow cytometry (FCM) and/or confocal laser scanning microscopy (CLSM). STUDY DESIGN: U937 cells and tissue sections were analyzed by means of FCM and/or CLSM. FCM was performed, using different ultraviolet (UV) and visible (488/532 nm) excitation modes. In the visible mode, fluorescence intensities of QDs, phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were compared. Three-dimensional (3-D) sequences of images were obtained by spectral analysis in a CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, providing factor curves and images. Factor images are the result of the FAMIS image processing method, which differentiates emission spectra from 3D sequences of images. In CLSM analysis, preparations are screened in a UV excitation mode to optimize the possibilities of QDs and have the benefit of 4',6-diamino-2-phenylindole or Hoechst 33342 counterstaining of nuclei. RESULTS: FCM and CLSM revealed CD36 expression by means of QDs 525 and/or 605. Fluorescence intensity of PE and of FITC was higher than that of QDs 525 and of 605. As factor curves and images show the red emission of QDs 605 only, subsequent reliable identification and localization of CD36 was obtained. CONCLUSION: QDs 525 and 605 are useful to analyze antigenic expression. Following FCM, which is well adapted to detect fluorescence emission of QDs in the UV or visible excitation mode, CLSM and subsequent spectral analysis assess more specific characterization of QD fluorescent emissions.     

Fluoro-Gold: An alternative viability stain for multicolor flow cytometric analysis.

BACKGROUND: The viability stains propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) are excited at 488 nm, as are the commonly used antibody conjugates fluorescein isothiocyanate (FITC), phycoerythrin (PE), and cyanine 5 dye covalently coupled to R-phycoerythrin (RPE-Cy5). When excited by a single laser, spectral overlap in the emission of PI and 7-AAD with RPE-Cy5 precludes the use of these viability stains for three-color immunophenotyping, particularly when evaluating low levels of marker expression in viable target cells. The ultraviolet excitable dye hydroxystilbamidine methanesulfonate (Fluoro-Gold, or FG) binds to DNA at the A-T-rich regions of the minor groove in permeabilized or dead cells. We assessed the suitability of this dye as a viability stain. METHODS: The ability of FG to detect nonviable cells in fresh and cryopreserved human apheresed peripheral blood cells was compared with that of PI and 7-AAD. The stability of FG staining and the effects of dye and cell concentration on the discrimination of nonviable cells was determined by measuring changes in the median fluorescence of viable and nonviable cells. RESULTS: FG labeling at dye concentrations of 2-8 microM is stable for at least 3 h over a wide range of cell concentrations (4 x 10(5) to 4 x 10(7) cells/ml). Costaining studies and linear regression analysis show that cell viability as determined by FG is strongly correlated with estimates using PI (r = 0.9636) and 7-AAD (r = 0.9879). CONCLUSIONS: FG is a reliable, alternative viability stain that can be used in conjunction with fluorochromes including FITC, PE, and RPE-Cy5 for multicolor analysis using dual-laser instruments.     

Single-nucleotide polymorphism analysis using fluorescence resonance energy transfer between DNA-labeling fluorophore,fluorescein isothiocyanate,and DNA intercalator,POPO-3, on bacterial magnetic particles

To develop an analytical system for single-nucleotide polymorphisms (SNPs), the fluorescence resonance energy transfer (FRET) technique was employed on a bacterial magnetic particle (BMP) surface. A combination of fluorescein isothiocyanate (FITC; excitation 490 nm/emission 520 nm) labeled at the 5' end of DNA and an intercalating compound (POPO-3, excitation 534 nm/emission 570 nm) was used to avoid the interference from light scattering caused by nanoparticles. After hybridization between target DNA immobilized onto BMPs and FITC-labeled probes, fluorescence from POPO-3, which was excited by the energy from the FITC, was detected. The major homozygous (ALDH2*1), heterozygous (ALDH2*1/*2), and minor homozygous (ALDH2*2) genotypes in the blood samples were discriminated by this method. The assay described herein allows for a simple and rapid SNP analysis using a fully automated system.     

Improved BrdUrd-Hoechst bivariate cell kinetic analysis by helium-cadmium single laser excitation.

In laser based flow cytometers, UV excitation of Hoechst 33258 and propidium iodide (PI) or ethidium bromide (EB) is performed with 351/364 nm high power lines of UV-capable argon ion lasers, which are expensive and short-lived. In this paper we note for the first time that helium-cadmium lasers emitting 10 to 30 mW at 325 nm are even more superior for cell kinetic bivariate bromodeoxyuridine (BrdUrd)/Hoechst PI or EB cell cycle analysis. HeCd single laser UV excitation gives comparable CVs for cell cycle distributions, and almost normal G2M/G1 ratios of 1.9 to 2.0 for all cell cycles. This is shown for synchronous and asynchronous cell populations on a FACStar+ and an Ortho Cytofluorograf. Therefore we recommend helium-cadmium lasers as low-power, cheap, and long-lived UV excitation sources for the cytochemically simple but high resolution multiparameter BrdUrd-Hoechst cell kinetic analysis.     

Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins

Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp. in conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP. In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signals that could be electronically compensated in real time using dual-laser excitation at 458 and 568 nm. Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-laser excitation at 488 nm. Since many flow cytometers are equipped with an argon-ion laser that can be tuned to 488 nm, the DsRed/EYFP/EGFP combination is expected to have broad utility for facile monitoring of gene transfer and expression in mammalian cells. The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four fluorescent gene products within living cells.     

Multicolor immunofluorescence and flow cytometry utilizing cascade blue to purify murine hematopoietic stem cells from fetal liver and bone marrow.

BACKGROUND: Here we demonstrate the utility of cascade blue (CB), to purify hematopoietic stem cells by flow cytometry. Multicolor immunofluorescence and the sensitivity (signal-to-noise) of the fluorochromes are essential for the identification and isolation of rare stem cell populations. METHODS: We isolated hematopoietic stem cells utilizing a 407 nm laser line to excite CB and propidium iodide (PI) in combination with FITC, PE, and Red670 which were excited at 488 nm. RESULTS: CB is maximally excited using a 407 nm laser line, when compared to UV or 413 nm excitation. The increase in sensitivity of CB at 407 nm can be contributed to higher absorption of CB and a reduction of autofluorescence at this excitation wavelength (Ropp et al.: Cytometry 21: 309-317, 1995). CONCLUSIONS: Despite the fact that the CB antibody conjugate has a tendency to adhere specifically to a B cell subpopulation in bone marrow, we nevertheless could purify stem cells by using CB for the detection and elimination of lineage positive cells. Isolated stem cells from mouse fetal liver (Lin-CD34(+)Sca-1(+)c-Kit(high)) and adult bone marrow (Lin-CD34(-/low)Sca-1(+)c-Kit(+)) were transplanted into lethally irradiated mice, and the sorted stem cells had the ability to efficiently repopulate all mature hematopoietic lineages in recipient mice.     

A method for immunofluorescent demonstration of three coexisting neurotransmitters in rat brain and spinal cord, using the fluorophores fluorescein, lissamine rhodamine, and 7-amino-4-methylcoumarin-3-acetic acid.

Coexistence of neurotransmitters within single nerve fibers or terminals can be convincingly demonstrated by the use of multicolor immunofluorescence. The present study examined whether three-color immunocytochemical localization of coexisting neurotransmitters can be performed using the blue fluorophore AMCA. Spectrofluorometric examination of secondary antibodies conjugated with AMCA, fluorescein, and lissamine rhodamine showed that the peaks of excitation and emission were well separated and that dots of AMCA-conjugated IgG dried on slides were not visible when viewed using microscope filters for rhodamine and fluorescein. These findings suggest that AMCA might be suitable for three-color immunofluorescence. The usefulness of AMCA for triple labeling was tested directly by staining sections of rat brainstem and spinal cord for serotonin (5HT), substance P (SP), and either enkephalin (ENK) or prepro-thyrotropin-releasing hormone 160-169 (ppT), a marker peptide for thyrotropin-releasing hormone. Triple labeling for 5HT, SP, and ppT was observed in both brainstem and spinal cord but was only very rarely observed for 5HT,SP, and ENK. No evidence was found for artifactual triple labeling, although false negatives appeared to be possible in some circumstances. We conclude that AMCA can be combined with fluorescein and lissamine rhodamine for three-color immunofluorescent studies of coexisting neurotransmitters. In addition, the coexistence of 5HT with ENK appears to be much less common than the coexistence of 5HT with either SP or ppT.     

Determination of gabapentin in human plasma by capillary electrophoresis with laser-induced fluorescence detection and acetonitrile stacking technique

A sensitive analytical method for gabapentin [1-(aminomethyl) cyclohexaneacetic acid] (GBP) in human plasma based on capillary electrophoretic separation and laser-induced fluorescence (LIF) detection has been developed. 6-Carboxyfluorescein succinimidyl ester (CFSE) was used for precolumn derivatization of the non-fluorescent drug in plasma. Optimal separation and detection were obtained with an electrophoretic buffer of 50mM sodium borate (pH 9.5) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). A calibration curve ranging from 0.3 to 150 microM was shown to be linear. The concentration limit of detection (LOD) in plasma was 60 nM. We also demonstrate how the detection limit can be enhanced by using acetonitrile stacking technique. With stacking, the limit of detection for gabapentin in plasma was 4.8 nM. A calibration curve ranging from 0.03 to 15 microM was shown to be linear. Both the within-day and day-to-day reproducibility and accuracy were 相似文献   

Heterokaryon identification through simultaneous fluorescence of tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate labelled protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emission spectrum, but not both, was possible for any single filter set.     

Heterokaryon Identification Through Simultaneous Fluorescence of Tetramethylrhodamine Isothiocyanate and Fluorescein Isothiocyanate Labelled Protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein iso-thiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emisson spectrum, but not both, was possible for any single filter set.     

Fluorescence properties of free and protein bound fluorescein dyes. I. Macrospectrofluorometric measurements.

Excitation and emission properties of fluorescein derivatives were studied macrofluorometrically. Measurements were performed with solutions of various concentrations (0.07-100 microgram/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit gamma-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FITC. (2) The fluorescence properties of FITC upon excitation with blue light (lambda = 496 nm) are only slightly altered by the conjugation to rabbit gamma-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (lambda = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 microgram/ml. Beginning at 5 microgram/ml (10-(5) M/1) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.     

Simultaneous identification and banding of human chromosome material in somatic cell hybrids

We have developed a method that identifies human chromosomes in human x hamster somatic cell hybrids and simultaneously bands these same metaphases. Other methods generally require separate slides for banding and detection of human chromosome material, making the precise characterization of human material difficult. Our procedure involves denaturing metaphase chromosomes, followed by in situ hybridization of biotinylated whole human DNA. Fluoresceinated avidin is then bound to the biotinylated DNA, staining the human chromosomes yellow-green when excited with UV light. Chromosome banding is achieved by staining the slides with DAPI and actinomycin D. The fluorescein and DAPI excite maximally at 488 and 355 nm and emit at 520 and 450 nm, respectively. This permits identification of the human material at one excitation wavelength and visualization of the banding patterns at another wavelength. With this procedure, we have successfully identified both intact and broken human chromosomes, as well as human material involved in human x hamster translocations. The results indicate that this procedure is more accurate and considerably more rapid than previous methods and can be routinely employed for the cytogenetic analysis of human x rodent hybrids.     

A comparison of low-power helium-cadmium and argon ultraviolet lasers in commercial flow cytometers

The feasibility of installing a low power ultraviolet (UV) laser in a commercial flow cytometer was evaluated by testing an Ortho Cytofluorograf 50HH and a Coulter Epics V. Both instruments were equipped with two argon ion lasers, one emitting at 488 nm and the other in the UV region and were tested by measuring the DNA content of cells stained with Hoechst 33342 or DAPI. The coefficient of variation (CV) of the G0/G1 peak of the DNA histograms produced by each instrument did not deteriorate markedly when results obtained at 100-125 mW were compared to those obtained at 10 mW. These tests indicated that a helium-cadmium laser (He-Cd) which can produce 10 mW at 325 nm should work well as a UV laser in these instruments. An Ortho Cytofluorograf IIs was purchased with a 10 mW He-Cd laser installed in the forward position. Studies of DNA content have confirmed that this low power UV laser can produce CVs of 2.2% with DAPI stained fixed cells and 3.6% with Hoechst 33342 stained viable lymphocytes. Thus, the He-Cd laser should provide a reasonable alternative as a UV source for flow cytometers.     

Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5.

Monoclonal antibodies specific for phycoerythrin (PE) were covalently labeled with the fluorescent dye cyanine 5 (Cy5). Excitation at 488 nm of immune complexes obtained by mixing Cy5-anti-PE with PE resulted in a 4-fold reduction of PE fluorescence measured at 565 nm and an increase of fluorescence measured at 655 nm. The observed energy transfer between PE and Cy5-anti-PE was used to develop three color immunofluorescence staining procedures for flow cytometers equipped with an Argon laser tuned at 488 nm. Mouse IgG1 monoclonal antibodies specific for cell surface antigens were cross-linked with either unlabeled or Cy5 labeled mouse IgG1 anti-PE using F(ab')2 fragments of monoclonal rat anti-mouse IgG1. PE was added to these immune complexes in sufficient amounts to saturate all PE binding sites. Cells were incubated with PE-labeled and PE/Cy5-labeled tetrameric antibody complexes together with FITC labeled antibodies and analyzed by flow cytometry. The emission from FITC, PE and PE/Cy5 could be readily separated and bright three color immunofluorescence staining of mononuclear cells from human peripheral blood and bone marrow was observed. The results of these experiments demonstrate that useful probes for single laser three color staining of cell surface antigens can be readily obtained by mixing of selected reagents. Compared to standard procedures for the covalent labeling of PE (tandem) molecules to antibodies, the non-covalent procedures described in this report provide significant advantages in terms of the amount of reagents, time and equipment required to obtain suitable reagents for three color immunofluorescence staining.     

Aminomethyl coumarin acetic acid: A new fluorescent labelling agent for proteins

Summary A new fluorescent protein labelling agent, 7-amino-4-methyl coumarin-3-acetic acid (AMCA), emits in the blue region (440–460 nm) on activation with UV light (350 nm). The active reagent is theN-hydroxysuccinimide ester which reacts with lysine residues under mild conditions to form photostable amide links.The Stokes shift of 100 nm compared to 30 nm for Fluorescein isothiocyanate (FITC) allows easy filter discrimination of exciting and emitting radiation. The agent has been demonstrated in use for fluorohistochemical examination of human kidney glomeruli, using the sandwich technique and compared with the same procedure using FITC-labelled antibodies. The good quantum yield coupled with convenient emission lines in the mercury spectrum allows photographic exposure time of fluorescent labelled sections to be reduced to a quarter of that required for a corresponding FITC conjugate.AMCA—immunoglobulin conjugates were not susceptible to photobleaching and have a storage life at – 20° C of more than two years.To whom correspondence and reprint requests should be addressed.