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Keywords:

  • deep-UV;
  • mass mapping;
  • mammalian cells;
  • CHO cells;
  • Jurkat cells;
  • hepatocellular carcinoma;
  • Swiss 3T3;
  • mouse embryos;
  • melanoma

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Literature Cited

We present measurements by deep-ultraviolet mass mapping of nucleic acid (NA) and protein for five commonly cultured and three primary cell types. The dry mass distribution at submicron resolution was determined on a single-cell basis for 250–500 cells from each of these types. Since the method carries a direct reference to a spectrophotometric standard (molar extinction coefficient), we are able to calibrate the absolute weight distributions both on a cell-to-cell basis within each type and across types. We also provide a calibration in absolute mass units for fluorescence-based measurements (flow cytometry and fluorescence microscopy). As might be expected the cultured cell lines show a high concentration of nucleic acids in the nuclear compartment, much larger than the genomic 2C number even in the G1 stage. The whole-cell nucleic-acid/protein ratio was found to be a characteristic of cell lines that persists independent of cell cycle and, as a result, this ratio has some value for phenotyping. Primary chicken red blood cells (cRBC), often used as a cytometry standard, were determined to have a nuclear-isolated nucleic acid content much closer to the genomic number than the cultured cell lines (cRBC: 3.00 pg total NA, 2.30 pg DNA, and 0.70 pg RNA). The individual blastomeres (n = 54) from mouse embryos at eight-cell stage were measured and found to vary by more than a factor or two in total protein and nucleic acid content (0.8–2.3 ng total protein, 70–150 pg total NA). The ratio of nucleic acid to protein was more nearly constant for each blastomere from a particular embryo and this ratio was found to be an identifying characteristic that varies from embryo to embryo obtained from a single flushing of a mouse. © 2013 International Society for Advancement of Cytometry

Historically, the absolute measurement of protein and nucleic acid content of biological cells has been primarily by using purified samples pooled from a population of interest; information at the level of individual cells is lost. There is growing understanding that, even in “synchronized” cells, biological state is far from uniform across most populations. A current interest is to explore the true nature of heterogeneity in the context of the overlapping concepts of phenotype and differentiation.

Single-cell measurements by indirect transfer using stains and fluorescent dyes are widely used in microscopy and flow cytometry (1–4). However stain transfer methods, when analyzed carefully, are known to be highly variable and unstable (5, 6). Stain uptake depends strongly on chromatin packaging, secondary structure for nucleic acids, and efflux pumping, all with which affect dye access and stoichiometric binding. Alternatively, intercalating DNA stains are used on extracted nuclei to more closely attach stoichiometry at the level of base-pair content. However, the process of isolating nuclei, and more generally of preparing cells for flow cytometry, hinders cell-resolved measurements of other parameters such as cell morphology and intracellular protein distribution. Therefore, while fluorescence methods will certainly continue to have a dominant role in the exploration of biological heterogeneity, deep ultraviolet (UV) mass mapping (7, 8) has the potential to provide cell-resolved measurement of nucleic acid and protein mass, to preserve cell morphology, and to avoid utilizing fluorescence channels that can otherwise be dedicated to tracking specific proteins of interest.

The classical spectrophotometric capability of wavelengths shorter than 300 nm, as applied to single cells, has been known for some time (9–11). We have shown (7, 8) that an updated implementation with modern sources and detectors greatly increases throughput and, through practical extension to shorter wavelengths, can substantially increase the measurement accuracy for determination of intracellular protein and nucleic acids. In the current study, we have applied these methods to establish a baseline of absolute measurements for a small array of cell lines that are commonly cultured in the laboratory. We have also characterized two terminally differentiated primary cell types, namely human and chicken red blood cells (RBC's) and undifferentiated mouse blastomeres in eight-cell embryos. An important implication of UV mass mapping is the ability to measure the nucleic acid (substantially RNA) content of cells within the cytoplasm on a cell-by-cell basis. By the UV method, total nucleic acid can be measured within the nucleus and in the subcompartment of the nucleolus and, through enzymatic treatment, the RNA and DNA contributions can be separated.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Literature Cited

UV Microscope

Deep-UV mass mapping is a form of quantitative transmission imaging. Our instrument is based on an upright Zeiss Axioskop microscope fitted with Zeiss Ultrafluar 100X/numerical aperture 1.2 and 32X/numerical aperture 0.6 glycerin-immersion objectives. The resolution is determined by the diffraction-limit (<120 nm at 100X) combined with the image magnification at the camera (80 nm pixel at 100X, 278 nm pixel at 32X). We retain most of the ability of the standard optical system for transmission and epifluorescent imaging on the visible light channels. The UV/visible source is a laser-induced plasma point source (EQ-99, Energetiq, Woburn, MA), which produces a smooth continuum from 170–750 nm at a near constant radiance of 10 mW mm-2 sr-1 from a source size of 100 × 100 × 200 μm3. At 250 nm wavelength this is ∼500x brighter than a deuterium lamp. To select for deep UV wavelengths, three 10-nm-wide bandpass filters were used, with nominal peak transmission at 280, 265, and 220 nm (Omega Optical, Brattleboro, VT). The plasma output is coupled into a solarization-resistant fiber (450 μm core, 0.22 NA, #QP450-1-XSR, Ocean Optics, Dunedin, FL), directed onto a collimator, passed through a filter wheel and focused onto the specimen by a UV-Kond (0.8 NA) condenser (Carl Zeiss AG, Oberkochen, Germany.) Fluorescence imaging was conducted in a trans-illumination configuration. Images were acquired with an EMCCD camera (PhotonMax 512, Princeton Instruments, Trenton, NJ) and WinView software package (Princeton Instruments) and stored for off-line analysis.

Cell Culture and Sample Preparation

All procedures were conducted in accordance with Boston University institutional guidelines. CHO-K1 (Chinese Hamster Ovary cells), Mel-10 (human derived melanoma), and Swiss 3T3 (fibroblast) cells were grown in 25-ml flasks in complete HyClone Dulbecco's Modified Eagles Medium (#SH30285, Fisher Scientific, Pittsburgh, PA), with 10% fetal bovine serum (#10082139, Invitrogen, San Diego, CA), 2 mM Glutamax (#35050-079, Invitrogen), and 100 I.U. penicillin/100 μg/ml streptomycin (#30-002-CI, Cellgro, Herndon, VA) at 37°C in 5% CO2 and were passaged twice a week. QGY-7703 (human derived hepatocellular carcinoma) cells were cultured a similar medium, but without penicillin and streptomycin. Jurkat cells were grown in 25-ml flasks in C-10 medium: Advanced RPMI 1640 (#12633012, Invitrogen), 10% FBS, 10 mM Hepes (#H0887, Sigma-Aldrich, St. Louis, MO), 100 I.U. penicillin and 100 μg/ml streptomycin, 55 μM 2-mercaptoethanol (#21985-023, Invitrogen), and 2 mM Glutamax.

For imaging experiments, cells were transferred to a fused-silica coverslip during passage and incubated overnight in complete medium. Cells were then washed with filtered (0.22 μm, #430758, Corning, Corning, NY) PBS, fixed with 3.7% formaldehyde (1:10 dilution of 37% formaldehyde; #252549-500ML, Sigma-Aldrich), and stained with 10 μg/ml (16 μM) of Hoechst 33342 (#S3570, Invitrogen). Coverslips were washed again with filtered PBS and then mounted onto fused-silica slides, in 3 mM sodium citrate, and sealed with nail polish. In recycling fused-silica slides, some care is taken not to leave toxic residues. Treatments with commercial glass cleaners that contain ammonia are avoided. We have found that thin layers of growth matrix such as fibronectin do not perturb the method and are automatically compensated by our data reduction without artifacts.

Mouse 8-cell embryos were collected from CFW (Crl:CFW) females. The zona pellucida was removed with 0.1% pronase (Sigma). Blastomeres were fixed with 3.7% formaldehyde and stained with Hoechst 33342 (10 μg/ml) and then mounted on slides.

White Bovan, male, cRBCs were purchased from Innovative Research (#IC05-0810; Novi, MI). Upon delivery, cRBCs were isolated and suspended in 0.1 M PBS (pH 7.4) and stored for up to 1 week at 4°C. cRBCs were fixed in 3.7% formaldehyde for 10 min, washed and rinsed, mounted on quartz slide that had been treated with 1 mg/ml concanavalin A (#C5275-5MG, Sigma), and sealed with nail polish. For extraction of nuclei, cRBCs were treated with 0.1% Triton X-100 for 2 min, washed, suspended in 1 mg/ml RNase A (#12091-021, Invitrogen) solution for 20 min, washed, and resuspended in PBS. Nuclei were mounted in PBS, on concanavalin treated quartz slides, before sealing with nail polish.

Image Analysis

Analysis of images was performed in MatLab using custom-written software that solves the coupled system of equations that describe the Beer's law absorption by nucleic acid and protein at two wavelengths of measurement, as described in detail previously (7, 8).

The total optical density (OD wavelength n) at wavelength "n" is the linear sum of contributing species:

  • equation image(1)

where ε is the extinction coefficient, l is the path length, and c is the concentration. Since we do not resolve volume elements in the “z” direction (focus), we report results as the product of l and c, mass per unit area. Optical density is measured by taking the ratio of the transmission in a field with the specimen and an empty background field, that is, log10 (Ibackground/Ispecimen). Since the extinction coefficient is wavelength dependent, we can generate two independent relations by imaging at two deep-UV wavelengths, then solve Eq. (1) as a couplet at the two wavelengths to find both the product term of path-length and concentration (for each pixel):

  • equation image(2)

The model does not attempt to isolate minority absorption from lipids or flavins, which for the samples and wavelengths below, are expected to contribute less than 1% to the OD on a whole-cell basis (12, 13). However lipid absorption rises rapidly near 200 nm. Generalization of the method with a third (e.g., 200 nm) wavelength might be sufficient to also separate out these components. As is, lipid vacuoles are assigned as (miscalibrated) protein, and appear as holes in the protein and NA mass maps. Lipids should make no appreciable distortions to the whole-cell measurements of protein and nucleic acid in the experiments below.

Whole-cell segmentation was performed manually. For determination of nuclear-isolated mass, an automated image segmentation algorithm was used (morphological watershed; disk-shaped structuring element (radius = 3), with a mask generated by Hoechst 33342 fluorescence. Objects smaller or larger than 2 standard deviation of mean nucleus size were excluded from analysis. Each region of interest (ROI) was then used to read out pixel values. Scatter plots, linear regression, histograms and distribution fits were calculated in MatLab.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Literature Cited

Mass Maps of Five Cell Types

The mass mapping method generates images from native contrast based on the wavelength-dependent absorption properties of nucleic acid and protein. At 220 nm, protein absorption is over 100-fold greater than nucleic acid; at 260 nm, that ratio drops to fivefold. Using optical density images at two different wavelengths allows for the separation of the protein and NA contributions (see Methods). However there is insufficient spectral difference between RNA and DNA, so an enzymatic step is needed to isolate these components.

In our previous study (8), we showed that data reduction using the 220 nm/260 nm couplet provided lower noise mass measurements when compared to previous measurements at the 260 nm/280 nm couplet used for "classical" spectrophotometric protein measurement in solution. This is because of the low optical density of single cells, which leads to noise in the measurement. The 220 nm measurement has appreciably higher OD, particularly for protein, and therefore has a better noise characteristic. In addition there is extremely good separation of the nucleic acid and protein components; at 220 nm the ratio of molar extinction coefficients of averaged intracellular protein and nucleic acid is over 100:1. At the wavelengths 260 nm and 280 nm the same ratio is much smaller (∼ 5:1 and ∼15:1 respectively).

Figure 1 shows representative dry-mass maps and classical fluorescence images of the nucleus (Hoechst 33342) for five cell lines: CHOK1, Jurkat E6, QGY-7703, melanoma-10, and Swiss 3T3 cells. The mass maps were generated using the 220 nm/260 nm image pairs.

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Figure 1. Mass maps of five cell lines. Cell lines CHO-K1, QGY-7703, Mel-10, Swiss 3T3, and Jurkat E6 mounted on fused silica slides. Mass maps by 220 nm/260 nm absorption at intermediate magnification, 32X objective (278 × 278 nm/px; 0.077 μm2/px). Nucleic acid (left) and protein (middle) are shown in false color, for each cell type, with the corresponding Hoechst 33342 fluorescence image (right). Pixel values of the mass maps converted from grayscale values into mass per pixel.

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In all cell types nucleic acid (DNA and RNA) is distributed broadly in the nucleus, in the perinuclear region, and, in many cases, in a clearly identified nucleolus. The Hoechst fluorescence images coincide well with the total nucleic acid mass map on the large scale length of the nucleus, and do a good job delineating the nucleus, but, in accord with the known sensitively of the nuclear stains to secondary structure, the mass map shows fine nuclear structure that is not seen with the classical stains. Notice, for example, how much new fine structure (not apparent with Hoechst) is visible in the CHOK1 and QGY-7703 mass maps. A combination of DNA and RNA stains will create a fluorescence map that better approximates the NA mass map, but we found no simple combination of DNA and RNA stains that reproduces the full distributions of intracellular nucleic acid (8). There is a good, not perfect, correspondence between nuclear-isolated nucleic acid content and the total Hoechst signal (8). In the perinuclear region, the NA component can be assumed to be almost entirely RNA; the total contribution of DNA from mitochondria should be less than 0.1 pg (14).

In general the protein mass maps show less structure with nearly an equal total protein content across the cell, both in the nucleus and the cytoplasm. But the broad features of the protein distributions are quite different in the different cells types. For S3T3, CHO, and QGY-7703 cells, nucleic acid drops to near zero in a ring outside of the perinuclear region, whereas protein remains high in the same regions. Mel-10 cells have numerous small NA bodies in the cytoplasm but have nucleic acid filling the entire space up to the plasma membrane. For most of the cell types there are numerous punctuate nucleic acid structures outside the nucleus at several size scales, accounting for much of the fine structure in the vicinity of the cell membrane.

Measurement of the Intracellular Mass in a Statistical Population of Several Common Cell Lines

Calibration of the accuracy and intrinsic noise of mass measurements

To fine-tune the absolute mass calibrations and to reveal the intrinsic noise of our methods, we first made a series of measurements on red blood cells (chicken and human). These cells are commonly used cytometry standards, in large part because they have undergone terminal differentiation, so state differences are minimized and this leads to a more nearly homogeneous sample. The standard deviations of our measurements these RBCs should be more nearly limited by the technical noise of our method.

We prepared fresh blood smears from a human subject on quartz and mounted in 50% glycerol, then collected images at 280, 260, and 220 nm. For intracellular protein in mammalian somatic cells we developed the 220 nm/260 nm algorithm as a low noise method that benefits from the high molar extinction of all proteins at 220 nm. We had selected human red blood cells as a validation marker for deep UV imaging because their dry mass is comprised of 95% hemoglobin (12). An effective extinction coefficient for protein can be calculated as a weighted sum of the epsilons for hemoglobin (ε-280 nm = 118,872 M−1 cm−1; ε-260 nm = 116,376 M−1 cm−1) and an "average" protein (ε-280 nm = 54,129 M−1 cm−1; ε-260 nm = 36,057 M−1 cm−1). For the specialized case of hRBC, total protein is dominated by hemoglobin: εeffective 280 nm = 115,634 M−1 cm−1 and εeffective 260 nm = 112,360 M−1 cm−1.

A representative protein mass map is shown in Figure 2. The protein map shows a narrow distribution of nearly uniform discs. Using the protein map to generate an automated segmentation mask, we obtained a total protein mass of 26.6 pg, assuming a protein distribution of 95% hemoglobin (Fig. 2B, mean ± s.d., 27.0 ± 8.0 pg).

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Figure 2. Human red blood cells (hRBCs). A: Protein (substantially hemoglobin) mass map. Histograms (n = 1034) of (B) total protein mass by 260 nm/280 nm absorption (see text), and (C) total protein mass by 220 nm/260 nm absorption.

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These numbers are consistent with the hemoglobin quantities (10–30 pg) measured using classical hematology methods and by bulk protein determinations in lysates [i.e. by lysing RBCs, reacting hemoglobin with cyanide, and measuring absorption at 540 nm (15)]. The CV of the protein histogram is 0.30, which provides an upper bound on the noise for the mass mapping technique when using the classical 260 nm/280 nm wavelength pair. This is comparable to good imaging cytometry with fluorescent markers—a respectable result, particularly since mass mapping provides absolute quantitative measurement and is based on native contrast.

Some further improvement in the measurement is possible through the new 220 nm/260 nm data reduction. Although we could not find good literature values for the molar extinction coefficient of hemoglobin at 220 nm (ε-220), we derived this number, as previously (8) using our measurements with the 260 nm/280 nm literature values as calibration and requiring overlay of the 260 nm/280 nm and 220 nm/260 nm histograms. This resulted in an empirical ε-220 nm for protein of 600,000 M−1 cm−1. We cross-checked this empirically derived epsilon by measuring the UV absorption spectra for hemoglobin solutions (1.0 mg/ml, 0.1 mg/ml, 0.01 mg/ml, fused-silica cuvette, 1-cm path length), yielding an εeffective 220nm of 50,5620 M−1 cm−1. However, RBCs in solution showed significant differences in absorption at deep UV wavelengths, compared to hemoglobin solutions (16). Interpolation of RBC absorption at 220 nm and 260 nm yielded an εeffective 220nm of 606,000 M−1 cm−1.

Using the now-calibrated 220 nm/260 nm method, we calculated peak protein content at 26.4 pg (Fig. 2C), with the CV contracted to 0.17 (mean ± s.d., 27.5 ± 4.6 pg). This is a reduction by a factor of nearly two in standard deviation compared to the 260 nm/280 nm method.

We next addressed calibration to a known nucleic acid standard using chicken RBC nuclei, a common calibration standard for genomic content in flow cytometry (17–20). As an approximation, we assumed the same effective epsilons for protein as for hRBCs.

Our results show that whole cRBCs (nuclei shown in Fig. 3A) contain peak nucleic acid (Fig. 3B) content of 8.5 pg (n = 376 cRBCs; mean ± s.d., 8.5 ± 1.6 pg) and peak protein (Fig. 3C) content of 24.0 pg (mean ± s.d., 27.0 ± 6.1 pg). The CVs are nearly as good as the simpler case of hRBCs (Fig. 2C), for both nucleic acid (CV= 0.18) and protein (CV = 0.23). For the nuclear compartment we found a nucleic acid peak of 3.00 pg (mean ± s.d., 3.2 ± 0.8 pg), from 468 nuclei (Fig. 3D). This represents the total nucleic acid, RNA and DNA, in the cRBC nucleus.

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Figure 3. Distribution of nucleic acid and protein mass for chicken red blood cells. A: Nucleic acid mass map in extracted, RNase-treated cRBC nuclei. Histograms showing distribution of total nucleic acid (B) and protein (C) mass for 376 whole cRBCs. D: Histogram of nucleic acid in isolated nuclei (n = 468). E: Histogram of DNA mass from RNase-treated cRBC nuclei (n = 1170).

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To directly measure the genomic DNA content of cRBCs, we treated extracted cRBC nuclei with RNase and isolated nuclei automated segmentation (Figs. 3A and 3E). The extracted nuclei showed a peak DNA mass of 2.30 pg (n = 1170 nuclei, mean ± s.d., 2.2 ± 0.4 pg). In these same nuclei, the CV in Hoechst fluorescence is respectable but appreciably higher (0.38) compared to the NA measurement by UV (Fig. 3E, CV = 0.18): that is we were able to achieve better signal-to-noise ratio with the UV quantitation method than wide field fluorescence. Our measurement for a White Bovan male falls at the low end of the range of the literature values for various other breeds of male and female chickens (2.33–2.54 pg, Ref.18–25).

By subtracting mean genomic DNA (2.30 pg) from the mean nucleus-isolated total NA (3.00 pg), we estimated nuclear RNA to be ∼0.70 pg for cRBCs. Thus in cRBC, the nucleus has markedly higher DNA than RNA, with the ratio typically being closer to 1:1 for somatic mammalian nuclei (see text for Fig. 5, Ref.3). In addition, the whole cell (total) nucleic acid was measured to be 8.5 pg, and with 3.0 pg (total NA) in the nucleus, this leaves ∼5.5 pg (total NA) localized to the cRBC cytoplasm.

An important broader result is that the calibrations in RBCs have proven that the mass mapping method can determine population distributions and compartmental localization of NA and protein with good overlay to previous hematology methods. Furthermore, in populations of 500–1000 cells we achieve CV values of 0.15–0.23, which is equivalent or better than imaging cytometry using indirectly calibrated fluorescence.

Whole cell protein and nucleic acid

We then proceeded to build histogram distributions of five cell lines under conditions of exponential expansion. Figure 4 shows the distributions for whole cell nucleic acid and protein in CHOK1, Jurkat, QGY-7703, S3T3 and Mel-10 cell lines. We also extended the population analysis to mouse embryos extracted at the eight-cell stage (50 hours post-fertilization), shown in the same figure.

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Figure 4. Histograms of whole cell nucleic acid and protein masses. Histograms for six cell types are shown (CHO, Jurkat, QGY-7703, Swiss3T3, Melanoma-10, and mouse embryo) for nucleic acid (top) and protein (bottom). All mass maps were calculated using 220 nm/260 nm image pairs. Manual segmentation was used to delineate regions of interest (ROIs) for individual cells. Histograms were fitted using the ‘polyfit’ function in MatLab. The peak was found for that fitted curve; mean and standard deviation were calculated across all objects of a given cell type.

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Our first objective was to provide absolute anchor points for fluorescence-based cytometry. Generally, the peaks of the fitted curves (MatLab “polyfit”) lie close to the mean of the distributions. The cells can be ranked (Table 1) in order of most probable nucleic acid content (G1 peak): Jurkat (17.2 pg), CHO (31.6 pg), QGY-7703 (39.6 pg), S3T3 (73.1 pg), Mel-10 (31.6 pg). If we assume that the genomic DNA component of this total is near the 2C diploid cell at G1, then the whole-cell RNA content is 3X to 15X the genomic content. The total whole-cell protein distributions have qualitative similarities to total nucleic acid, (Fig. 4). The same ordering of most probable mass in the G1 peak is obtained for the cell lines with total protein values: Jurkat (32.1 pg), CHO (82.4 pg), QGY-7703 (251 pg), S3T3 (207.7 pg), and Mel-10 (182.6 pg).

Table 1. Nucleic acid and protein masses for cell lines (ranked by NA)
  nSize (μm2)Nucleic acid (pg)Protein (pg)
PeakMean ± s.d.PeakMean ± s.d.
Whole cell
 Jurkat46674.1 ± 23.617.220.0 ± 7.132.139.4 ± 15.7
 CHO368216 ± 10626.028.2 ± 9.282.490.6 ± 36.4
 QGY-7703489638 ± 25439.644.1 ± 16.7251303 ± 93.2
 Swiss 3T3441690 ± 51573.189.8 ± 45.9207.7305.2 ± 198.6
 Melanoma337665 ± 67931.692.4 ± 97.6182.6336.6 ± 300.2
Nucleus
 Jurkat49344 ± 259.311.3 ± 4.520.923.6 ± 10.7
 Melanoma269104 ± 5015.616.4 ± 7.366.268.5 ± 36.6
 CHO32557 ± 2912.617.3 ± 11.637.644.8 ± 31.2
 QGY-7703509116 ± 4514.818.2 ± 4.799.3101 ± 33.8
 Swiss 3T3306165 ± 5928.337.8 ± 15.879.997.6 ± 46.8
Mass in isolated nuclei

We then used the overlay of Hoechst stain to automate a segmentation and separate mass components in the nuclear and cytoplasmic compartments. In general we found less than half the total nucleic acid in the nucleus (Fig. 5). Arranged in ascending most probable (peak) mass (Table 1), the sequence is Jurkat (9.3 pg), CHO (12.6 pg), QGY-7703 (14.8 pg), Mel-10 (15.6 pg), and S3T3 (28.3 pg). In this case the S3T3 line is highly exceptional, with a much greater total NA in the nucleus – even when compared to the melanoma (Mel-10) line or the mouse blastomere (23.2 pg, Table 2). It must be quickly added that the Mel-10 line shows a highly distorted NA distribution in the nucleus with both a low-mass (7.4%, 20/269 cells) and a high-mass (14.1%, 38/269 cells) shoulder. The high-masses are easily assigned to the cells at high ploidy number, however identity of the low-mass shoulder population is less obvious. If the peak values in the histograms are assigned to the G1 phase with nominal 2C genomic complement, then CHO, Jurkat, QGY-7703 and Mel-10 cells all would have a RNA:DNA ratio of between 1:1 or 1.5:1 in the nucleus. The S3T3 line would appear to have more than twice as much RNA as DNA, and the mouse blastomere, at the eight-cell stage, has a nuclear RNA:DNA ratio of nearly 4:1.

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Figure 5. Histograms for nucleus-isolated, nucleic acid, and protein masses. Histograms for six cell types are shown (CHOK1, Jurkat E6, QGY-7703, Swiss3T3, Melanoma-10, and mouse embryo) for nucleic acid (top) and protein (bottom), in the nucleus. All mass maps were calculated using 220 nmn/260 nm absorption images. Corresponding Hoechst fluorescence images were used as masks in an automated segmentation algorithm. Histograms were fitted using the ‘polyfit’ function in MatLab. The peak was found for that fitted curve; mean and standard deviation were calculated across all objects of a given cell type.

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Table 2. Nucleic acid and protein masses, in picograms, for primary cells
  nNucleic acid (pg)Protein (pg)
PeakMean ± s.d.PeakMean ± s.d.
Whole cell
 Chicken RBC3768.58.5 ± 1.624.027.0 ± 6.1
 Human RBC1034−0.4−0.2 ± 0.526.427.5 ± 4.6
 Mouse embryo54104.0104.3 ± 22.81267.81409.9 ± 329.2
Nucleus
 Chicken RBC4683.03.2 ± 0.83.05.3 ± 3.8
 Mouse embryo5423.223.2 ± 3.2247.3253.3 ± 67.7
Nucleolus
 Mouse embryo541.71.8 ± 0.417.219.9 ± 6.4
Extracted nuclei
 Chicken RBC11702.32.2 ± 0.43.83.8 ± 1.2

When we compared the nucleus to cytoplasmic NA, the nucleus contains approximately half of the whole cell total. We confirmed this when we plotted cytoplasmic and nuclear quantities across CHO, QGY-7703, Jurkat, and Mel-10 cell types (n = 1420 matched nucleus-cytoplasm pairs; R = 0.50; slope = 1.05). In contrast the eight-cell mouse blastomere sequesters only one quarter of its total NA in the nucleus. Note that the there is a tight CV (0.14) for the total NA content in the nuclear compartment of the blastomeres, even when compared across multiple embryos (Table 2).

Figure 5 also shows the distributions of protein in the nuclear compartment. For CHO, QGY-7703, and S3T3 fully 40%–45% of the total cell protein is found in the nucleus. This increases to more than 65% for Jurkat (a lymphocyte) and, as would be expected, falls to 20% of the total protein in the (much larger) blastomere. We observed the following sequence, in order of increasing peak protein mass in the nucleus: Jurkat (20.9 pg), CHO (37.6 pg), Mel-10 (66.2 pg), S3T3 (79.9 pg), QGY-7703 (99.3 pg), and mouse embryos (247.3 pg).

For most cells that we measured there is ∼3X more protein than nucleic acid in the whole cell (Fig. 6A). It is of interest, however, that a significant minority of Mel-10, Swiss 3T3 and QGY-7703 cells have anomalously high protein content. The whole-cell protein:NA ratio for the different cell types is plotted in Figure 6B. The different cell types separate according to absolute protein mass much as would be expected based on their 2D image area (Table 1). However there is larger variability in NA content after normalization to image area. For example, QGY-7703 cells have half the total NA content as Mel-10 and Swiss 3T3 cells, despite having nearly the same 2D area and protein mass. Somewhat surprisingly, the boxplots show that a simple, protein:NA ratio is sufficiently distinct to differentiate between cell lines. This suggests a possibility of phenotyping based on this ratio and hints at a potential role for deep-UV mass mapping in histology and diagnostics. The data also underscore the obvious but often ignored fact that, in wide-field microscopy, cell area is not a good proxy for cell mass.

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Figure 6. Nucleic acid and protein scale proportionally, although protein:NA ratios are specific for cell type. A: Scatter plot showing paired nucleic acid and protein mass across six cell types: Jurkat E6(orange), CHOK1 (green), S3T3 (purple), QGY-7703 (brown), blastomere (light blue) and Mel-10 (red). Regression line (red) was calculated across all plotted cells (n = 1896 cells). B: Protein to NA ratios were calculated for individual cells of each cell type, with their distributions summarized into boxplots. The edges of the box denotes 25% and 75% percentile bounds for the sample; dashed line denotes minimum and maximum range of values not considered “outliers”. The number of cells is listed below each cell type.

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Nucleic acid and protein variability in mouse blastomeres.

We analyzed eight-cell mouse embryos with the idea of testing the homogeneity of an ideally synchronized system that had progressed only three divisions after fertilization. It is fairly routine to remove one of the eight blastomeres for testing at this stage during in vitro fertilization with no serious consequences to the fetus. Therefore these cells should be a perfect model of functional equivalence.

Figure 7 presents mass maps for the blastocysts taken from seven embryos from the same dam after removal of the zona pellucidas. The embryonic cells are much larger than the somatic cells, with total protein masses of 1–2 ng and have a much higher protein to NA ratio (∼10X). There is a fair amount of well-defined structure in the nucleus, including nucleolar structure in both the NA and protein. This includes several ring structures around the nucleolar regions evident in both the NA and protein mass maps.

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Figure 7. Detail of eight cell mouse embryos. A total of 54 individual blastomeres were imaged, from 8 embryos, zona pellucida removed. A representative field of view is shown for nucleic acid (A) and protein (B) mass maps. C: Scatter plot of whole cell nucleic acid and protein masses for individual blastomeres. Whole cell ROIs were manually generated. Cells from each of the eight embryos are coded with a specific color. D: Paired nucleus-isolated, NA, and protein masses were measured and plotted, using automated segmentation from matching Hoechst 33342 images. E: Paired nucleolus-isolated NA and protein masses are plotted. Nucleoli were determined by visual inspection as the low DNA regions within nuclei defined by Hoechst staining.

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When pooled across seven embryos, the masses of individual blastomeres showed a standard deviation of 22% of the mean. For small, relatively homogeneous cells such as cRBCs (above), we have confirmed an approximate upper bound on the technical noise of our system (i.e. CV = 0.15). However, for the much larger mouse blastomeres (presented as nearly flat samples trapped under cover glasses), the expected errors are much lower—in our estimation on the order of 5%. We believe the data of Figure 7 reflect the true variance of the distribution with only a small component of technical noise (Table 2 for aggregate data summary). Blastomeres from an individual embryo (coded the same color in Fig. 7) have whole-cell NA and protein values dispersed over nearly a factor of two. The nuclear compartments of the eight blastomeres in each embryo have a tighter distribution, but NA and protein values also span a 1.5-fold variation in the full nucleus. Finally, the nucleolus exhibits a twofold variation in mass within blastomeres from the same embryo. There is a higher protein content in the nucleolus, approaching the same ratio as is in the cytoplasm (∼10 times more protein (peak: 17.2 pg; mean ± s.d.: 19.9 ± 6.4 pg) than NA (1.7 pg; 1.8 ± 0.4 pg) mass, within the nucleolus).

It is of considerable interest that the cells of each embryo are grouped by NA:protein ratio. This can be proven formally by forcing a regression fit through the origin in the plot of NA versus protein for the cells grouped by embryo. But the trend is seen by eye from the apparent linear distribution that separates each embryo (data of a particular color) from the aggregate data presented in Figures 7C–7E. NA:protein ratio seems to be an identifying characteristic that separates each embryo from the litter average even at this very early stage of development. It would be interesting to explore the epigenetic and genetic origins of this early differentiation.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Literature Cited

Single-cell analysis is essential if biological mechanism is to be detected beneath the natural cell-to-cell heterogeneity that accompanies metabolic factors, cell cycle, microenvironment, and circadian rhythm. In this study, we used deep-UV mass mapping and compared cellular content across five common cell lines and three primary cell types. The method, particularly after our extension to the shorter 220-nm wavelength, yields precise localization of total NA and protein with structure to the level of single image pixels (∼100 nm) and, since it is based on molecular absorption, images are calibrated in absolute mass units without the calibration errors and instability of fluorescence.

We see detailed images of condensed NA structure in the nucleus and the cytoplasm, some of which is not seen with common combinations of nuclear stains. In the case of hRBCs, we observed a protein content that is well matched to hematology standards. We also determined the precision of mass measurement by cross checking distributions of human and chicken RBCs for their dominant NA and protein components. We were able to confirm CV's between 0.17 and 0.23, somewhat better than the variance reported in the same samples, as imaged by Hoechst fluorescence, wide-field microscopy. We do note that CV's reported via flow cytometry for DNA quantitation (RNase treated, extracted nuclei and stained with an intercalating dye such as propidium iodide) are often 5% or less. However, in our hands the UV mass mapping appears to have less noise than wide-field Hoechst quantification of DNA.

The measurement technique does not replicate the exact information gathered by flow cytometry using nuclear stains since it does not distinguish between RNA and DNA contributions. This leads to a greater continuous range of whole-cell NA than is seen with flow cytometry where distinct phases of cell cycle are easily resolved with specific DNA markers. However the correspondence between the flow cytometry data and the UV mass histograms is easily established through enzymatic treatment that allows quantification of the separate RNA and DNA components. In this study, we found a value for the diploid genomic DNA content for a male chicken of 2.30 pg, which compares well to previous literature values and simultaneously provides a new value of 0.70 pg for RNA in the cRBC nucleus.

The mass maps for the five cell lines provide a basis for absolute calibration of protein and NA mass and anchor points for fluorescence-based methods. There is a fair amount of variation across the human and rodent cell types tested but also some similarities across types. In broad terms half the total NA in these mammalian cells was found in the nucleus. In the cell line analyzed most closely, CHO, (8), we found that 50% of the total NA in the nucleus is RNA (8). But certain lines, particularly S3T3 cells, have a much larger total NA component in the nucleus (nearly 5X the presumptive genomic complement), and Jurkat can have no more than half as much RNA as DNA in the nucleus.

The current study concentrated solely on mass measurements in fixed cells. For live cell measurements the technique must contend with the cell-toxic properties of the deep UV. However, since cells have standard machinery to repair inevitable mutations, all such effects are dose-rate dependent. By minimizing the dose rate with a sensitive camera, we were previously able to make time lapse movies of live macrophages and HeLa cells showing normal motility and proliferation for up to several hours (7). More recently we have extended live-cell movies, with temperature control, but no CO2 control, for up to 6 hours with CHO and Swiss 3T3 without seeing the obvious indicators of apoptosis. Therefore considerable measurement in live cells is possible, particularly in an early window and at low dose rate.

It is appropriate to make some comments on the practicality for wide adaptation of UV mass mapping. In our system glass optical elements, slides, and cover glasses are UV-grade fused silica or calcium fluoride. This adds some expense, so we recycle a supply of slides and cover glasses using good cleaning practice with care not to leave toxic residues on the growth surfaces. Single-element tube lenses are readily available at low cost and deep UV objectives are available from major microscope suppliers. We prefer the high-brightness Energetiq Co. light source but a conventional UV arc or a deuterium lamp is an acceptable broad-band source. We have also used low-cost UV LED sources with some success (7). With these changes, UV mass mapping merges naturally into the work-flow of traditional fluorescence microscopy and can be performed on the same platform. The most obvious direction to explore is the use of the deep-UV mass maps as reference images for immunofluorescence microscopy with more specific markers.

Furthermore there is an opportunity for significant improvements in the UV apparatus. Deep ultraviolet lenses, sources, and metrology have all taken a large step forward in the last decade as a result of the sudden commercial importance of semiconductor manufacturing at these wavelengths. This has greatly increased industry capability to design optics at wavelengths needed for mass mapping. Most importantly a new generation of microscope objectives is now possible with significantly improved transmission at shorter wavelengths down into the near edge of the vacuum ultraviolet (180 nm). With the older Zeiss objectives, the transmission may be as low a few percent at the highest magnifications and shortest wavelengths used in the current study. The shorter wavelengths that should now be feasible (180–220 nm) would open up a capability to resolve thinner samples and to distinguish particular proteins, carbohydrates and fats. In a recent example we were able to achieve absolute protein density measurements, for single fibronectin fibers (26). Mass mapping with shorter wavelengths and higher OD might allow quantitation of small organelles and single vesicles.

Also, in our experiments, we have not yet pushed to the extreme limits in optical resolution in either transmission mode or fluorescence. Most of the methods of super-resolution microscopy and polarization techniques should be viable in the deep UV. Therefore an overlay of these techniques on the intrinsic factor-of-two improvement in the diffraction limit (from wavelength scaling) should provide up to twice greater resolution if the appropriate fluorophors can be found. By this reasoning, by scaling non-linear microscopies into the deep UV, it is likely possible to set new records in the absolute resolution of optical microscopy and, for example, perhaps move optical methods into the domain of imaging chromatin.

Literature Cited

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Literature Cited