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Keywords:

  • slide-based cytometry;
  • photobleaching;
  • polychromatic cytometry

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Background

Slide-based cytometry is a key technology for polychromatic cytomic investigations. Here we exploit the relocalization and merge feature of Laser Scanning Cytometry for distinguishing fluorochromes of comparable emission spectra but different photostabilities.

Methods

Blood specimens were stained with the fluorochrome pairs: FITC/ALEXA488, PE/ALEXA532, or APC/ALEXA633. Bleaching was performed by repeated laser excitation.

Results

Since ALEXA dyes are photostable as compared to the conventional fluorochromes FITC, PE, and APC, a differentiation within one fluorochrome pair is possible.

Conclusion

The sequential photobleaching method results in an increased information density on a single cell level and represents an important component to perform polychromatic cytometry. © 2006 International Society for Analytical Cytology

Multicolor cell and tissue analysis has become increasingly important in various biological settings, in particular in Systems Biology and Cytomics (1). To distinguish more cell subsets, polychromatic cytometry is of eminent usefulness. To this end photostability of the fluorochromes can be used as additional parameter for immunophenotyping (2). ALEXA Fluor dyes are substantially more photostable than their conventional color analogs (2, 3). Therefore, fluorochromes with similar emission spectra (e.g. APC and ALEXA633) can be used simultaneously for cell staining and a discrimination of both is possible in the same photomultiplier (PMT) by performing differential photobleaching.

Multicolor analysis is of special importance in two different settings: Firstly in patients with low blood volume such as neonates or critically ill infants. Secondly, since our view of the immune system gets more complex, the characterization of leukocyte subsets makes the use of more parameters necessary and even unavoidable. With the increased number of measurable characteristics of a cell and additionally its morphological evaluation, slide-based multiplex cell analysis is a powerful analytical and diagnostic tool (4, 5).

In the present study we will demonstrate for the first time the feasibility of sequential photobleaching in cytometry.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Staining

Twenty μl of EDTA anticoagulated peripheral blood from healthy adult volunteers is incubated for 20 min with each 2.5 μl of various conjugates of ALEXA dyes (antiCD4-ALEXA488 (Caltag Laboratories, Burlingame, CA), Streptavidin-ALEXA532 or ALEXA633 (Molecular Probes, Eugene, OR) bound to antiCD3-Biotin (Caltag)) and simultaneously with 2.5 μl of their conventional color analogs (antiCD19-FITC, antiCD4-PE, or antiCD8-APC, respectively (all BD Biosciences, San Jose, CA)). In the case of Streptavidin, cells were first incubated with 2.5 μl antiCD3-Biotin (Caltag) for 20 min, then washed with PBS and stained (Method details see Ref.4). After incubation erythrocytes were lysed with 1 ml FACS lysing solution (BD) for 15 min, centrifuged, washed with PBS, and centrifuged again. Supernatant was removed. Cells were pipetted onto a conventional glass slide, mounted with Fluorescent Mounting Medium (DAKO Cytomation, Carpinteria, CA) and covered with a cover slip.

Analysis and Data Display

Specimen was analyzed on an Laser Scanning Cytometer (LSC; Compucyte, Cambridge, MA) using the appropriate filter settings (6). After an initial measurement, fluorochromes are excited several times (up to 18×) with the excitation laser line (633nm for ALEXA633 and APC; all other fluorochromes 488nm, 5mW laser power) in order to perform a differential photobleaching. Following these bleaching steps, the specimen is measured a last time. The first and the last measurements are merged to one data file based on the saved x–y-positions of the measured events. In the resulting merged data file, fluorescence information of the cells before and after bleaching can be displayed as separate parameters.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The fluorescence emitted by the fluorochrome pairs could not be distinguished by different filter settings (not shown). However, if the specimen is excited several times with the laser, e.g. ALEXA633 was, as expected, more photostable than APC. This could also be observed for the other ALEXA dyes (ALEXA488, ALEXA532) as compared to their counterparts. Several excitations with the laser (as indicated in Fig. 1, central column) lead to a substantial loss of emitted fluorescence of the conventional fluorochromes (FITC, PE, APC). The lower fading rate of the ALEXA fluorescence intensity enables the discrimination of the respective fluorochrome pairs (FITC/ALEXA488, PE/ALEXA532, and APC/ALEXA633). The ability to discriminate the fluorochrome pairs is increased by each scanning (bleaching) step until a plateau is reached (Fig. 2). After bleaching of the conventional fluorochromes, the first and the last measurements are merged. In this merged data file, the fluorescence intensities of the fluorochrome pairs before and after bleaching are displayed in one dotplot. In this dotplot the discrimination of similar fluorochromes is possible (Fig. 1, right column). Since ALEXA dyes are bleached only minimally, their fluorescence is located near a diagonal line. Opposed to this, the fluorescence intensity of the conventional fluorochromes decreases much more during the bleaching; therefore their signal shifts off the diagonal towards the axis of the measurement before bleaching.

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Figure 1. Differential photobleaching for the discrimination of fluorochromes with similar emission spectra. Samples were stained with antibodies or streptavidin as indicated. Differential bleaching was performed by several excitations with the laser. In the left two columns merged files before vs. after bleaching of single stained samples, in the last column of double stained samples is shown. ALEXA dyes show higher photostability against laser excitation than their conventional analogs, leading to the alignment of the stained cells along the diagonal. Cells stained with conventional dyes clearly shift off the diagonal towards the axis of the first scan. Due to their different photostability the fluorochromes can be combined and differentiated in one sample (right column). Original data were gated on lymphocytes based on Forward scatter and cell area (details e Ref. 6); only data of lymphocytes are displayed. Dotplots show data of at least 5.000 lymphocytes.

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thumbnail image

Figure 2. Quantification of differences in photostability of ALEXA dyes and their conventional analogs. Differences of the fluorescence intensity (normalized to 100%) of each fluorochrome pair at each scanning step n is displayed. With the first scan no differentiation of the fluorochrome analogs is possible. By differentially bleaching the sample the difference of the fluorescence intensity increases as the conventional dyes loose substantially more of their fluorescence than ALEXA dyes. PE can be best bleached resulting in the best discrimination of the fluorochrome pair PE and ALEXA532 (also see figure 1) already after 5 scanning steps. Difference and therewith the capability to discriminate between ALEXA dyes and their conventional analogs was calculated as follows:

  • equation image

A0: fluorescence intensity of ALEXA dyes in the first measurement; An: fluorescence intensity of ALEXA dyes at the scanning step n; C0: fluorescence intensity of the conventional dyes in the first measurement; Cn: fluorescence intensity of conventional dyes at the scanning step n.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The advantage of slide-based systems is that cells are not lost after analysis and remain immobilized on the slide. This fact, together with the merge feature of such systems, allows further analyses of the sample, e.g. restaining of the specimen for different antigens (7), and reanalysis with different filter settings (4, 8) or, as shown here, after differential bleaching of fluorochromes. The use of photostability for highly multiplexed (Toponomic) quantitative cell and tissue analysis has already been exploited by Schubert (9). We demonstrate in the present paper, for the first time, to apply photobleaching for the discrimination of fluorochromes with comparable emission spectra. The combination of measurements after changing of filters allows the simultaneous analysis of eight different fluorochromes in blood specimens by LSC (4). Also in tissue sections, an eight color immunofluorescence protocol is feasible by spectral imaging microscopy (10). The assay presented here shows that it is possible to increase the eight color immunophenotyping by distinguishing fluorochromes with similar emission spectra but different photostabilities as additional parameter. In SBC systems, reanalysis of specimens allows the combination of fluorochromes which cannot be discriminated by flow cytometry (e.g. FITC and ALEXA488, PE and ALEXA532, APC and ALEXA633). With this method, six different fluorochromes can be detected and displayed as separate parameters in only three channels. Differential bleaching of several fluorochromes, in combination with the merge feature of SBC, opens the way to polychromatic or even hyperchromatic cytometry and could be a key cytomic technology.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED