ARTICLE |
Correspondence to: Steven J. Mentzer, PBB Lobby, Brigham & Women's Hospital, 75 Francis Street, Boston MA 02115.
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Summary |
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Tracking of cell migration plays an important role in the study of morphogenesis, inflammation, and metastasis. The recent development of probes that exist as intracellular peptide-fluorescence dye adducts has offered the possibility of aldehyde fixation of these dyes for detailed anatomic studies of lymphocyte trafficking. To define the conditions for fixation of these cytoplasmic fluorescent probes, we compared fixation conditions containing formaldehyde, glutaraldehyde, paraformaldehyde, zinc formaldehyde, and glyoxylate, as well as fixation by quick-freezing in liquid nitrogen-cooled methylbutane. The efficacy of aldehyde fixation of the cell fluorescence was assessed by quantitative tissue cytometry and flow cytometry. We studied cytoplasmic fluorescent dyes with discrete emissions in the green [5-chloromethylfluorescein diacetate (CMFDA); 492 ex, 516 em] and orange [5-(and-6)-(4-chloromethyl(benzoyl)amino) tetramethylrhodamine (CMTMR); 540 ex, 566 em] spectra. The results demonstrated that aldehyde fixation preserved cell fluorescence for more than 6 months. The primary difference between the aldehyde fixatives was variability in the difference between the yield of the cell fluorescence and the relevant background fluorescence. Formaldehyde and paraformaldehyde were superior to the other fixatives in preserving cell fluorescence while limiting background fluorescence. With these fixatives, both the CMFDA and CMTMR fluorescent dyes permitted sufficient anatomic resolution for reliable localization in long-term cell tracking studies.
(J Histochem Cytochem 49:511517, 2001)
Key Words: fluorescent dyes, cell migration, aldehydes, histocytochemistry, fluorescence cytometry
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Introduction |
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Cell movement plays an important role in biological processes, from morphogenesis to lymphocyte recirculation (
Several commercially available thiol-reactive cytoplasmic probes have been recently developed for long-term cell labeling studies. As fluorescent chloromethyl derivatives, these dyes diffuse through the membranes of living cells (
To define the utility of the thiol-reactive fluorescent probes 5-chloromethylfluorescein diacetate (CMFDA) and 5-(and-6)-(4-chloromethyl(benzoyl)amino)tetramethylrhodamine (CMTMR) in histological studies of cell migration, we compared fixation conditions using glutaraldehyde, formaldehyde, zinc formaldehyde, paraformaldehyde, glyoxylate, and fixation of tissue by quick-freezing in liquid nitrogen-cooled methylbutane. Quantitative tissue cytometry and flow cytometry were used to assess cell fluorescence. Fixatives, such as formaldehyde and paraformaldehyde, that provided uniform cell fluorescence and diminished background tissue fluorescence in both the green and orange spectra produced the best anatomic resolution. Aldehyde fixation preserved readily detectable fluorescent cell tracers for more than 6 months. Both the CMFDA and CMTMR fluorescent dyes produced sufficient signal isolation for reliable localization in long-term cell tracking studies.
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Materials and Methods |
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Animals
Randomly bred male sheep weighing 2535 kg and Lewis rats weighing 200250 g were used in these studies. All animals were sacrificed before tissue procurement. The animals were excluded from the analysis if there was any gross or microscopic evidence of disease. The fluorescently labeled lymphocytes were perfused into tissues using vascular catheters. In sheep, approximately 5 x 107 lymphocytes per injection were infused through a carotid cannula at 48 hr intervals for 72 hr before sacrifice and tissue processing. In rats, the organs were flushed and perfused with labeled lymphocytes through arterial inflow vessels after sacrifice.
Tissue Processing
After sacrifice, the tissues were harvested and immediately processed by quick-freezing or aldehyde fixation. Quick-frozen tissue was sliced into 4-mm3 blocks, coated with OCT embedding medium (TissueTek; Miles, Elkhart, IL), and placed in 15-mm cryomolds. The cryomolds were placed in liquid nitrogen-cooled 2-methylbutane, followed by immersion in liquid nitrogen. The tissue was stored at -86C for less than 3 months before processing. Tissue for aldehyde fixation was placed in a glass vial and fixed with various fixatives for 24 hr to 6 days. All fixed tissues were washed overnight in 30% sucrose and quick-frozen before sectioning.
Fluorescent Dyes
The 5-chloromethylfluorescein diacetate (CMFDA; 492 ex, 516 em) and 5-(and-6)-(4-chloromethyl(benzoyl)amino)tetramethylrhodamine (CMTMR; 540 ex, 566 em) fluorescent dyes were obtained from Molecular Probes (Eugene, OR) and labeled as previously described (
Fixative Solutions
Five commercially available fixative solutions were used. Paraformaldehyde was used as a 4% potassium phosphate-buffered solution (pH 7.0) (Fisher Scientific; Fair Lawn, NJ). Formaldehyde was used as a standard 10% neutral buffered formalin solution (4% formaldehyde, 1.5% methanol, pH 7.0) (Fisher Scientific). Glutaraldehyde was used as a 5% buffered solution (1% sodium metabisulfite in 0.1 M cacodylate buffer, pH 7.5). Two additional commercial fixative solutions were used: Glyo-Fixx (1020% ethanol, <5% glyoxal, 1% 2-propanol, <1% methanol, pH 4.0) (Shandon; Pittsburgh, PA) and Z-Fix (10% zinc formalin, pH 5.5) (Anatech; Battle Creek, MI). In this report, Glyo-Fixx is referred to as glyoxalate and Z-Fix as zinc formaldehyde.
Cryostat Sectioning and Counterstains
Tissue sections were cut at a thickness of 6 µm on a Tissue Tex II cryostat (Miles) with a chamber temperature of 25C. Sections were collected on a Fisher Superfrost Plus charged glass slide without additional treatment of the slide. For fluorescence microscopy, a DAPI (1.5 µg/ml) (Vectashield mounting medium; Vector Laboratories, Burlingame, CA) counterstain was used in most experiments. Sections were mounted by placing 25 µl of mounting medium and applying a glass coverslip.
Fluorescence Microscopy
The tissue was imaged on a Nikon Optiphot-2 microscope equipped with an episcopic fluorescence attachment. The microscope was equipped with x10 binocular eyepieces tubes and x20 and x60 plan apochromat objectives. The epifluorescent filter blocks were the blue filter UV-2A (400 nm DM), green filter B-1E (510 nm DM), and orange filters G-2A (580 nm DM) (Nikon). Additonal filter sets included orange (560 nm DM) and combined (530 nm DM) filters (Omega Optical; Brattleboro, VT). The fluorescent images were recorded using a DC120 CCD camera (Kodak; Rochester, NY) with 24-bit color and 1280 x 960 picture resolution. For most fluorescent images, shutter speed (range 1/500 to 16 sec) was 1.53 sec with a x1 zoom lens. Light exposure was minimized. Repeat images were routinely obtained in reciprocal order to control for fluorescence bleaching. The images were processed by the MDS 120 system software (Kodak) and recorded as digitized TIFF files. The archived images were processed using the MetaMorph Imaging System 4.0 software (Universal Imaging; Brandywine, PA).
Flow Cytometry
The cell fluorescence was assessed by flow cytometry using a Coulter Epics XL flow cytometer with Expo 2.0 software (Miami, FL). The flow cytometric data were collected at room temperature and exported to the Microsoft Excel (Redmond, WA) spreadsheet for data analysis using WinList 3.0 (Verity; Topsham, ME). The flow cytometric experiments were calibrated daily using Sphero Rainbow Calibration Particles (SpheroTech; Libertyville, IL).
Shading Correction
Shading correction was used to remove camera- and light source-induced photometric nonlinearities (
Optical Density
Images for the assessment of optical density were obtained using a x60 plan apochromat objective which produced a 220-µm working image. After thresholding, a 150-µm x 150-µm grid overlay was used to define regions of interest. Relevant objects were classified by cross-sectional area to exclude cells out of the plane of focus. For cells included in the analysis, optical density was calculated as the inverse logarithm of the gray scale transmittance (
Texture Measurements
Regional variations in gray levels were assessed using Markov texture parameters (
Statistical Analysis
The mean ± SD of the morphometric parameters was calculated on a minimum of 400 cells. The data are expressed as mean ± one SD. The significance level for the sample distribution was defined as p<0.05.
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Results |
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Cell Fluorescence Intensity After Tissue Fixation
The effect of fixation on the fluorescence intensity of the CMFDA and CMTMR fluorophores was studied using labeled lymphocytes perfused into in a variety of tissues including skin, lung, kidney, lymph node, and spleen. Subjective assessment of the aldehyde-fixed tissue demonstrated preserved fluorescence and anatomic resolution after mounting (Fig 1A and Fig 1B). Fluorescence was readily detectable in tissues fixed more than 72 hr after the initial in vivo injection. An estimate of the fluorescence intensity of the cytoplasmic fluorescent dyes was obtained by quantitative tissue cytometry in lung and kidney. After shading correction, 8-bit digitized fluorescence photomicrographs of more than 400 cells in each of the five fixative conditions were evaluated. Optical density, calculated as the inverse logarithm of the gray scale transmittance, was consistently greater in the formaldehyde and paraformaldehyde conditions (Fig 2). Because the retained cell fluorescence was generally associated with increased background and indistinct cell margins, optical density was closely correlated with subjective anatomic resolution. Quick-frozen tissue, glutaraldehyde-, zinc formaldehyde-, and glyoxylate-fixed tissue demonstrated the most inconsistent fixation in the five tissues examined (data not shown).
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Cell Fluorescence in Suspension
To examine the effect of the fixatives in the absence of surrounding tissue, normal lymphocytes in suspension culture were labeled with the CMFDA and CMTMR fluorophores and placed in either the aldehyde fixatives or PBS for 24 hr to 8 days. Flow cytometry demonstrated that the zinc formaldehyde and paraformaldehyde fixatives preserved higher fluorescence intensity (Fig 3). Formaldehyde was less effective in preserving fluorescence intensity, but formaldehyde produced a well-defined intensity distribution. An unexpected finding was the relatively high levels of fluorescence preserved by the PBS condition. Fluorescence microscopy demonstrated that the cells in the PBS condition showed significantly less uniformity (more texture) than the aldehyde-fixed cells. Over hours to days, the cells in the PBS condition demonstrated a speckled distribution of intracellular fluorescence. Consistent with these observations, the optical density variance (ODV) in the PBS condition was significantly greater than formaldehyde (formaldehyde ODV = 0.0137 ± 0.005, TDM = 0.677 ± 0.180; PBS ODV = 0.134 ± 0.089, TDM = 0.298 ± 0.106) (p<0.05). Serial measurements over 8 days showed a statistically insignificant decrement in fluorescence intensity for all fixatives (p>0.05) (Fig 4). Despite their relatively high fluorescence intensity by microscopy, zinc formaldehyde-fixed cells could not be assessed by flow cytometry because of cell clumping.
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Background Tissue Fluorescence
Anatomic resolution requires the preservation of cell fluorescence intensity and the minimization of background fluorescence. To obtain a quantitative measure of background fluorescence, the tissue samples obtained from the same organ containing CMFDA- and CMTMR-labeled lymphocytes were processed by quick- freezing or fixation with aldehyde fixatives. Using identical illumination and image capture conditions, images of the tissue section were processed using an RGB color space model. RGB histograms obtained from these images reflected the subjective visual impression of the slides: discrete green and orange fluorescence was better preserved by formaldehyde and paraformaldehyde fixation (Fig 5). The other fixatives produced background orange fluorescence that limited anatomic resolution.
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Discussion |
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Cell migration plays an important role in organ development, immune processes, and cell metastases. The ability to localize fluorescent cells with anatomic resolution comparable to that obtained in light microscopy has been a long-standing experimental goal. The development of thiol-reactive cytoplasmic fluorescent dyes has provided a method for tracking cells in vivo and the potential for aldehyde fixation. In this work, aldehyde fixation of cell suspensions and tissue fluorescence was studied. To assess cell fluorescence, we examined fluorescence using quantitative tissue and flow cytometry. The results showed that aldehyde fixation preserved discrete cell fluorescence while retaining surrounding tissue morphology. Of the fixatives tested, formaldehyde and paraformaldehyde were the most reliable at preserving fluorescence intensity and minimizing background fluorescence. Glutaraldehyde and glyoxylate fixatives were associated with background tissue fluorescence that limited their usefulness.
Although the exact mechanism of formaldehyde fixation is unclear, the process is generally believed to depend on the presence of primary or secondary amines. In the first step of the reaction, formaldehyde reacts with amino groups to yield highly reactive methylol compounds (
The use of multicolored probes is an important potential application for fluorescent cell tracers. The ideal fluorophores for use in multicolored applications have narrow spectral bandwidth and maximal spectral separation. Particularly useful are fluorophores, such as CMFDA and CMTMR, with strong absorption at a similar excitation wavelength and distinct emission spectra. In addition, these dyes are retained by cells for days at physiological temperatures, are easily distinguishable by fluorescence microscopy, and provide effective signal isolation for histological analysis.
Observable fluorescence intensity is quantitatively dependent on several dye-dependent parameters, including the concentration and quantum yield of the fluorophore. In many situations, simply increasing the probe concentration can be counterproductive. For example, increasing the concentration of the CMFDA and CMTMR probes not only changes the dyes' optical characteristics but the increase in glutathione-dependent reactants may adversely affect cell metabolism (
A more effective approach to increasing fluorescence intensity in histological sections is to maintain measurable quantum yield by minimizing both the adverse effects of fixation and the relevant background fluorescence. To minimize tissue autofluorescence, we routinely use probes with excitation wavelengths >480 nm. Longer wavelength excitation not only reduces background fluorescence but also has the advantage of reducing light scatter in tissues. In this study, we compared the CMFDA and CMTMR dyes for the effects of various fixation techniques on quantum yield and background fluorescence. In the aldehyde conditions, the cell and background fluorescence was stable after fixation. The practical limitations of the fixatives were apparent after examining many samples of the four different tissues. The use of glutaraldehyde, for example, was consistently limited by background tissue fluorescence. In our hands, the commercially available zinc formaldehyde and glyoxylate fixatives were more variable in preserving anatomic resolution. The primary limitation of both fixatives was enhanced background. A disadvantage of formaldehyde-fixed tissue is that it is more difficult to section for histological examination than with other fixatives, such as paraformaldehyde.
The photostability of a fluorophore is especially important in histological studies because of the prolonged high-intensity illumination required for microscopic examination. Photobleaching is a complex process that can result in the irreversible destruction of the fluorophore with repeated excitation (
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Acknowledgments |
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Supported in part by NIH Grant HL47078.
Received for publication September 8, 2000; accepted December 9, 2000.
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