Journal of Histochemistry and Cytochemistry, Vol. 49, 511-518, April 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Aldehyde Fixation of Thiol-reactive Fluorescent Cytoplasmic Probes for Tracking Cell Migration

Charles A. Westa, Chufa Hea, Mei Sua, Scott J. Swansona, and Steven J. Mentzera
a Laboratory of Immunophysiology, the Dana-Farber Cancer Institute, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts

Correspondence to: Steven J. Mentzer, PBB Lobby, Brigham & Women's Hospital, 75 Francis Street, Boston MA 02115.


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 49:511–517, 2001)

Key Words: fluorescent dyes, cell migration, aldehydes, histocytochemistry, fluorescence cytometry


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Cell movement plays an important role in biological processes, from morphogenesis to lymphocyte recirculation (Butcher et al. 1980 ; Edelman 1987 ; Behrens et al. 1992 ; Springer 1994 ). Most experimental approaches to tracking lymphocytes in vivo have relied on radionuclide markers (Gowans 1959 ). A major disadvantage of radionuclide markers is that anatomic resolution relies on cumbersome autoradiography. In addition, commonly used radionuclide markers significantly overlap in their emission spectra, preventing the use of multiple simultaneous probes. In contrast, the discrete emission spectra of most fluorescent dyes permit the use of multiple simultaneous probes (Li et al. 1996 ) while providing remarkable structural specificity. For cytoplasmic fluorescent dyes, the primary disadvantage has been the spontaneous release of the dye within a few hours at physiological temperature (Weston and Parish 1992 ). Membrane-bound fluorescent labels can persist for days in vivo (Melnicoff et al. 1988a , Melnicoff et al. 1988b ; Horan and Slezak 1989 ; Melnicoff et al. 1989 ; Slezak and Horan 1989 ; Teare et al. 1991 ; Van de Langerijt et al. 1994 ), but fixation is technically demanding and time-consuming (Basse et al. 1991 ). The fluorescence of membrane-bound fluorescent probes is quenched by aldehyde fixation.

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 (Merrilees et al. 1995 ). The cytoplasmic dye is believed to undergo several thiol-dependent reactions, including a glutathione S-transferase-mediated reaction (Haugland 1996 ). The reaction products are membrane-impermeant and persist for several cell divisions (Baker et al. 1997 ). An additional advantage of these dyes is that, unlike free dyes, these probes exist in the cell as peptide-fluorescent dye adducts. Although the successful use of aldehyde fixatives has not been reported, the expression of primary amines by these adducts offers the potential for stable aldehyde fixation.

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.


  Materials and Methods
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Materials and Methods
Results
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Literature Cited

Animals
Randomly bred male sheep weighing 25–35 kg and Lewis rats weighing 200–250 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 4–8 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 (West et al. in press ). The CMFDA and CMTMR dyes were sterilely dissolved in anhydrous DMSO, aliquotted, and stored as a 10 mM stock solution at -20C. The cells were labeled with fluorescent dyes (final concentration 1–10 µM) in the dark at a concentration of 5 x 107 cells/ml in pre-warmed protein-free Dulbecco's minimal essential medium (MEM). The cells and fluorescent dyes were incubated for 30 min at 37C with frequent agitation. The labeled cells were washed twice before intravascular infusion. Before injection, aliquots of the labeled cells were taken for flow cytometric analysis to confirm adequate fluorescence labeling of the CMFDA- and CMTMR-labeled cells.

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 (10–20% 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 1.5–3 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 (Benson et al. 1987 ; Jericevic et al. 1989 ). To correct for shading in the fluorescent images, appropriate fluorescent calibration microscopic slides (Applied Precision; Seattle WA) were imaged with excitation light that almost saturated the brightest pixel of the CCD camera. A background reference was acquired from a plain glass slide. The shading reference was the difference between these two images (Harris 1985 ). MetaMorph 4.0 was used to correct the test image pixel by pixel: scaling factor * (specimen image - background reference)/shading reference (McNamara 1994 ). The brightest gray scale value of the shading reference image was used as a scaling factor.

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 (Doudkine et al. 1995 ). Specific pixel transmittance was defined as the gray scale value divided by the maximal possible number of gray scale levels. Because each monochrome image was 8-bit, pixel transmittance was calculated as the gray scale value divided by 256.

Texture Measurements
Regional variations in gray levels were assessed using Markov texture parameters (Pressman 1976 ). Texture difference moment (TDM) was used as a measure of the uniformity of gray scale levels in an object. Objects with a uniform gray level have a TDM approaching zero. Objects with a greater variation of gray level have a larger TDM. Using the MetaMorph system, the texture parameters are calculated as the sum of the elements of a weighted conditional gray level transition probability matrix. Optical density variance (ODV) is a measure of the optical density distribution of an object. For objects of uniform density, the variance approaches 0. The variance has a maximal value of 1.0.

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.


  Results
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Materials and Methods
Results
Discussion
Literature Cited

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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Figure 1. Fluorescence microscopy of CMFDA- and CMTMR-labeled lymphocytes injected into a sheep carotid artery for 72 hr before fixation in formaldehyde and harvested in the skin and lymph node. The digital photomicrograph was obtained 6 months after sectioning and mounting. (A) The formaldehyde-fixed tissue was stained with DAPI and sequential digital images were obtained using blue (400-nm DM), green (510-nm DM), and red (580-nm DM) fluorescence filters. After application of selective filter masks, the green and red images were overlaid on the DAPI background. The combined image is shown. Bar = 100 µm. (B) Similarly fixed lymph node was sectioned and mounted without DAPI. The image was obtained using a 510-nm longpass fluorescence filter and is presented without modification. Bar = 20 µm. The yellow cells are believed to reflect dual labeling as a result of recirculation (West et al. in press ).



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Figure 2. Comparison of the optical density of cellular fluorescence of several different fixation conditions: QF, quick frozen; Form, formaldehyde; ZF, zinc formaldehyde; Glut, glutaraldehyde; Glyox, glyoxylate; Para, paraformaldehyde. The tissue was prepared and processed as described (see Materials and Methods). The optical densities of the (A) CMFDA and (B) CMTMR dyes are shown. The mean optical density is shown ± one SD. Quantitative cytometry of a minimum of 400 cells was assessed for each data point.

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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Figure 3. Serial flow cytometry histograms assessing relative fluorescence intensity of fluorescent cells in suspension: unlabeled lymphocytes (black line), PBS (dashed line), formaldehyde (Form; gray line), zinc formaldehyde (ZF; black dots), paraformaldehyde (Para, gray dots). Quantitative flow cytometry was performed using Rainbow Calibration Particles. The flow cytometry histograms are shown for both the CMFDA (A,B) and CMTMR (C,D) dyes. The histograms show intensity profiles after 24 hr (A,C) and 8 days (B,D) of culture in the fixative concentrations used for tissue fixation (see Materials and Methods).



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Figure 4. The stability of the mean relative fluorescence intensity of the fluorescent dyes over eight days: PBS (diamond), formaldehyde (square), zinc formaldehyde (circle), paraformaldehyde (triangle). Serial measurements of the zinc formaldehyde condition by flow cytometry were limited because of aggregation of the cells. Concentrations of the fixatives are described in Materials and Methods.

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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Figure 5. RGB color space analysis of lymph node sections containing CMFDA- and CMTMR-labeled lymphocytes. Six conditions were analyzed: (A) quick-frozen, (B) formaldehyde, (C) zinc formaldehyde, (D) glyoxylate, (E) glutaraldehyde, and (F) paraformaldehyde. A combined fluorescence filter showing both green and red fluorescence (DM = 510 nm) was used under identical illumination and optical conditions. Histograms are shown for green (black) and red (gray) color channels.


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Friedman 1992 ). In the presence of a favorable steric environment, methylol groups condense with amides or other groups to form methylene bridges that crosslink polypeptide chains (Fox et al. 1985 ). The distinct advantage of the thiol-reactive dyes is that they appear to exist in the cytoplasm as fluorescent-peptide adducts (Haugland 1996 ). The fluorescent-peptide biomolecules play an important role not only in retaining dye in the cytoplasm but also in exposing amino groups for potential aldehyde fixation. In our studies, the difference between protein-linked tracers and freely diffusible fluorochromes was particularly apparent days after cell death. In the PBS condition, the CMFDA and CMTMR dyes persisted in the remnants of the cytoplasm. The condensed dye resulted in preserved fluorescence intensity by flow cytometry, but the less uniform fluorescence distribution limited the recognition of single-cell fluorescence in the tissues.

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 (Haugland 1996 ). Another consideration is the fluorescence-quenching effects of increasing dye concentration (Wehry 1973 ). Finally, recorded fluorescence intensity is not proportional to tissue fluorophore concentration because of the so-called inner-filter effect (Kubista et al. 1994 ). The tissue structure absorbs excitation light before it reaches the fluorophore (primary inner-filter effect) and reabsorbs some of the emitted light (secondary inner-filter effect) (Parker and Rees 1962 ). These limitations suggest that increasing dye concentration does not reliably increase detection sensitivity or recordable fluorescence intensity.

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 (Song et al. 1995 , Song et al. 1996 ). This process is considerably more complex with labeled biological specimens that have undergone histological fixation. One approach to improving photostability is the use of mounting resins after fixation and counterstain. Mounting resins can exclude oxygen and minimize the dye-to-oxygen mechanism of photobleaching (Usui et al. 1965 ).


  Acknowledgments

Supported in part by NIH Grant HL47078.

Received for publication September 8, 2000; accepted December 9, 2000.


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Materials and Methods
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