Problem of size dependence in fluorescence DNA cytometry

Abstract

The relationship among fluorescence intensity, area, thickness, and DNA concentration of DNA-bearing polyacrylamide film, stained with 50 ng/ml DAPI, was studied using an epi-illumination cytofluorometer. Measurements and theoretical analysis suggest the inevitability of the size-dependent effect on human cell nuclei in which the order of the DNA concentration is approximately 10 mg/ml, and that this effect can be virtually negated if the nuclei are stretched and flattened to less than 0.33 pm in thickness on the smears. Alternative practical ways to minimize this effect are to decrease the dye concentration of a staining solution and to make smears as thin and uniform as possible by some means such as hypotonic treatment and/or using an automatic centrifuge smearing apparatus. The size-dependent effect, nevertheless, should be taiken into account particularly when determining the DNA content of different cell types and the DNA aneuploidy of tumor cells, regardless of the technique used for measurement.Key terms: DNA content, DNA concentration, area, thickness, volume, dye concentration, D:NA-polyacrylamide film Kerker et al. (24) showed that the fluorescence intensity of dye-containing microspheres varies with the size of the microsphere by flow cytometry and theoretical analysis, but they concluded that this effect is less pronounced for biological cells because of their low refractive index. However, we (and probably whoever has performed DNA cytofluorometry) have had some experiences in epiillumination DNA cytofluorometry that call into question this assumption (15). 1) The fluorescence intensity of diploid nuclear DNA changes gradually from one end of a blood smear to the other. The fluorescence intensity is higher on the thin end than on the thick end of the smear. 2) On touch smears of various tissues, the fluorescence intensity of diploid cell nuclei of the same type is almost equal in a stamp but different from stamp to stamp even on the same glass slide. 3) Fluorescence intensity of diploid nuclei differs among heterogeneous cells with different nuclear size, in particular among blood leukocytes, internal granule neurons, and Purkinje cells. These phenomena have been observed with a variety of DNA-binding dyes (26) and are enhanced at a higher concentration of the dye in staining solutions (9).These phenomena have also been noticed in absorption cytophotometry as inconsistent with the "DNA constancy theory'' (4), in particular on leukocyte DNA (3,11,13,14,29). They could be diminished, however, by stretching and flattening of the cells on the smears (1 2,22), and tlhe "compaction hypothesis" (12,13,30) was proposed ito interpret them. This hypothesis appears reasonable, but these phenomena resulted not only from the difference in chromatin coiling but also from a high concentration of the nuclear DNA itself, as is discussed below.DNA is compactly organized in mammalian cell nuclei. Since the DNA amount of the human haploid genome is 3 x 10" base pairs (11, the diploid nuclear DNA content of man is app...

Effects of fixation method on image cytometric measurement of DNA content and distribution in cells stained for fluorescence with propidium iodide.

“…We had previously observed in acetonelmethanol-fixed cells that no significant cell-size dependencies of IO1 were present over a broad range of PI concentrations (1-200 pglml). This indicated that concentration-dependent quenching or inner filtering effects were not present at the concentrations used (22).…”

Relationship between chromatin compactness and dye uptake for in situ chromatin stained with DAPI

“…In high‐resolution observation conditions (NA = 1.3), the distribution of illumination intensity shows a partial confocal effect due to the limited illumination field (29). This effect causes the nonhomogeneous illumination of the three‐dimensional sample as function of the focal position at which the optical section was acquired and the dependence on the three‐dimensional extension of the sample if it exhibits different size (30). The sample fraction that is within the depth of field of the lens, and consequently the bidimensional projected image, is dependent on the sample three‐dimensional size (30).…”

“…The fluorescence intensity, I, of light emitted perpendicularly to the incident plane by an object of height d can be expressed (30) by the following equation: where I 0 , A , η, k , and c are intensity of the excitation light, projected area of the sample, emission yield, absorption coefficient, and concentration of the substance in the sample, respectively. Therefore, when we want to estimate the average concentration (c) of a molecule (e.g., DNA) in samples of different sizes, which have been labelled with a selective probe (DAPI in our case), a simple direct evaluation of the fluorescence intensity is not sufficient if the analysis is performed by high‐resolution conventional wide‐field microscopy (generally, the conditions of the sample and the flow cytometry setup are optimized, at low resolution, to have the entire three‐dimensional sample within the depth of field of the lens in order to perform integral intensity measurements correctly).…”

Determination of DNA Content of Aquatic Bacteria by Flow Cytometry

“…The concentrations of DNA in bacteria are several times the concentration found in human diploid nuclei (1%, by volume) (25) and are sufficient to give measured attenuation of DAPI-DNA fluorescence in the larger aquatic bacteria (10) of up to 10%. Distances are too great for resonance energy transfer (13), but the complex could absorb reemitted photons.…”