2.1.

Stem Cell Lines

The stem cell lines human salivary gland stem cells (hSGSC), human pancreatic stem cells (hPSC), rat pancreatic stem cells (rPSC), and human dental pulp stem cells (hDPSC) were used^{18, 19, 20, 21} for two-photon autofluorescence imaging. FLIM measurements and spectral imaging were performed on hSGSC stem cells. The first three cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Germany) containing $4.5\mathrm{g}∕\mathrm{L}$ glucose, L-glutamine, and supplemented with 15% fetal calf serum Gold (FCS-Gold) (PAA Laboratories GmbH, Germany), $100\mathrm{U}∕\mathrm{ml}$ penicillin, and $100\mathrm{mg}∕\mathrm{ml}$ streptomycin. hDPSC were cultured in $\alpha \text{-}\mathrm{MEM}$ with 15% FCS, $100\mu \mathrm{M}$ L-ascorbic acid 2-phosphate, $2\mathrm{mM}$ L-glutamine, $100\mathrm{U}∕\mathrm{ml}$ penicillin, and $100\mu \mathrm{g}∕\mathrm{ml}$ streptomycin. Cultures were maintained at $37°\mathrm{C}$ in a 5% $\mathrm{C}{\mathrm{O}}_2$ humidified atmosphere.

For imaging, cells were transferred (confluence 80 to 90%) into sterile miniaturized cell chambers with two $0.17\mathrm{mm}$ thick glass windows with etched grids (MiniCeM-grid, JenLab GmbH, Jena) and additionally incubated for another day until they were attached to the glass surface.

2.2.

Pellet Culture and Chondrogenic Differentiation

For chondrogenic differentiation, a pellet culture was used. Approximately 200,000 cells were placed in a $15\mathrm{ml}$ polypropylene tube and centrifuged to a pellet for $5\min$ $(1000\times ∕\min )$ . The flattened pellet at the bottom of the tube was cultured at $37°\mathrm{C}$ with 5% $\mathrm{C}{\mathrm{O}}_2$ in the culture medium for $5\text{to}6\text{days}$ until a spherical form (spheroid) was formed. For the differentiation, the spheroids were cultured in $1\mathrm{ml}$ of the chondrogenic medium DMEM (GIBCO, Germany) supplemented with $10\mathrm{mg}∕\mathrm{ml}$ transforming growth factor $\left(\mathrm{TGF}\right)\text{-}\beta 3$ , $100\mathrm{nM}$ dexamethasone, $50\mathrm{mg}∕\mathrm{ml}$ ascorbic acid-phosphate, $4\mathrm{mg}∕\mathrm{ml}$ L-proline, $100\mathrm{mg}∕\mathrm{ml}$ natrium piruvate, 15% FCS-Gold, $100\mathrm{U}∕\mathrm{ml}$ penicillin, and $100\mathrm{mg}∕\mathrm{ml}$ streptomycin. The medium was replaced every $3\text{to}4\text{days}$ for a period of $4\text{to}5\text{weeks}$ . For microscopy, the pellets were transferred to dishes with $0.17\mathrm{mm}$ thick glass coverslips.

2.3.

Instrumentation and Measurements

A laser scanning microscope (LSM 510 Meta NLO, Zeiss, Germany) equipped with a tunable $80\mathrm{MHz}$ Ti:sapphire laser source (Chameleon, Coherent Inc., Santa Clara, California) was used to study autofluorescence and SHG of stem cells. For autofluorescence imaging, laser beams at 750, 850, and $900\mathrm{nm}$ ( $140\mathrm{fs}$ laser output) at $5\mathrm{mW}$ mean power were used. The beamsplitter HFT KP 650 and the short pass filter KP 685 were employed. The cells were scanned in a $512\times 512\text{pixel}$ field at $25.6\mu \mathrm{s}\text{pixel}$ dwell time. Fluorescence emission was recorded through a Plan-Neofluar $40\times ∕\mathrm{NA}$ 1.3 oil objective. The experimental parameters such as scanning time and the settings for contrast and brightness were identical for all autofluorescence images obtained from different cell lines for comparison purposes.

For SHG detection, the external PMT-GaAsP (H7422P-40, Hamamatsu, Japan) was mounted to the microscope in a forward direction. A SP 610 filter was installed in front of the detector to prevent scattered laser light onto the detector. SHG signals were collected through a $435\mathrm{nm}$ bandpass filter at an excitation wavelength of $870\mathrm{nm}$ .

3-D two-photon autofluorescence and SHG images were obtained by optical sectioning with $z$ -intervals of $5\text{to}10\mu \mathrm{m}$ . Fluorescence lifetime imaging (FLIM) was performed by single-photon counting (TCSPS) using the board SPC-830 (Becker and Hickl GmbH, Germany) and the fast detector PMC-100 with an SP 610 filter.

The instrumental function (IRF) of the optical system was measured using the second-harmonic generated signal from collagen II (Sigma-Aldrich, Germany). SPCImage software was used to analyze the fluorescence lifetime decay curves as well as the calculation of histograms (Becker and Hickl GmbH, Germany). Spectral imaging was performed with the 32 channel PMT array (META, Hamamatsu). The grating in the polychromator provided a resolution of $10\mathrm{nm}$ per channel. Typically, the spectral range of $382\text{to}714\mathrm{nm}$ was imaged. Figure 1 shows the experimental setup.

Fig. 1

Experimental setup. PMT is the photomultiplier tube, NDD is the nondescanned detector, DBS is the dichroic beam splitter, BP is the bandpass filter, SP is the short pass filter, AOTF is the acousto optical tunable filter, and GaAsP is gallium arsenide phosphide.

3.1.

Two-Photon Autoflourescence Imaging and Spectral Analysis

The two-photon measurements showed that different stem cell lines have an autofluorescence with different intensity. hDPSC, hPSC, and hSGSC human stem cells exhibited an intense autofluorescence compared to rPSC rat cells. Figure 2 demonstrates the differences in the fluorescence intensity between human and rat pancreatic cells. Also, we noted significant fluorescence intensity variations between cells of the same line (Fig. 3 ). Intrinsic fluorescence from living cells was detected at different NIR excitation wavelengths (750, 850, and $900\mathrm{nm}$ ) to separate different native fluorescence molecules. When using $750\mathrm{nm}$ , NAD(P)H as well flavins/flavoproteins can be excited by a two-photon process. However, when changing to 850 or even $900\mathrm{nm}$ , flavins/flavoproteins (e.g., FAD) only and not NAD(P)H can be efficiently excited.²² Spectral measurements (Fig. 4 ) showed that the detected fluorescence light was intense in the blue/green spectral region. The maximum of the fluorescence was found to be at $460\text{to}470\mathrm{nm}$ when excited with $750\mathrm{nm}$ light, which is consistent with the emission behavior free and protein bound NADH. The maximum shifted to $530\text{to}535\mathrm{nm}$ when using $900\mathrm{nm}$ light, which is consistent with flavin emission (Fig. 5 ). We have proven that the presence of the culture medium did not add significant background to the autofluorescence signals from the cells.

Fig. 2

Autofluorescence images of (a) rat pancreatic stem cells (rPSC), (b) human pancreatic stem cells (hPSC), (c) dental pulpa stem cells (DPSC), and (d) salivary gland stem cells (hSGSC). Excitation wavelength was at $750\mathrm{nm}$ . Fluorescence emission was recorded through a Plan-Neofluar $40\times ∕\mathrm{NA}$ 1.3 oil objective.

Fig. 3

Autofluorescence image of rPSC cells. Note that some cells $(<5\%)$ exhibited a very intense fluorescence.

Fig. 4

Spectral analysis of hSGSC stem cells obtained from 32 images in the spectral range of $382\text{to}714\mathrm{nm}$ . Excitation wavelength was at $750\mathrm{nm}$ .

Fig. 5

Spectral measurement of hSGSC stem cells.

3.2.

Fluorescence Lifetime Imaging Measurement and Analysis

Fluorescence decay curves have been obtained by exciting at 750 and $900\mathrm{nm}$ . Figure 6 presents a typical $750\mathrm{nm}$ excited FLIM image, a histogram, and a fluorescence decay curve of hSGSC stem cells. The decay curve reflects the autofluorescence of one mitochondrium in a specific cell. 6740 photons were detected for this curve. The biexponential fit with the fitting parameter ${\chi }^2=1.00$ reveals two components. A fast one with a short lifetime $\left({\tau }_1\right)$ of $0.17\mathrm{ns}$ and an amplitude of $a_1=72\%$ , and a second one with ${\tau }_2=1.8\mathrm{ns}$ and the amplitude $a_2=28\%$ .

Fig. 6

Data analysis window of the SPCImage software.

To get a statistical information on the variance of intracellular decay curves, we analyzed the curves of ten different perinuclear fluorescence regions of the bright cell on the right side within Fig. 6. The data are depicted in Table 1 . Interestingly, only one major short component with an average value of $0.202\mathrm{ns}$ [standard deviation (SD) 0.019, amplitude 70.5%] was found in this particular cell. The long component had an average value of $2.014\mathrm{ns}$ (SD 0.077).

Table 1

Intracellular FLIM data obtained from ten decay curves within the cytoplasm of the bright cell on the right in Fig. 6.

When calculating the mean fluorescence lifetime ${\tau }_2$ of the long-lived component within this frame of about ten cells in Fig. 6, we obtained a major value of $1.87\mathrm{ns}$ , as depicted in the histogram. When analyzing ten frames of about 100 cells in total of the same cell line, we obtained a mean ${\tau }_2$ value of $(1.82\pm 0.02)\mathrm{ns}$ . In contrast, when changing the excitation wavelength to $900\mathrm{nm}$ , an average value ${\tau }_2$ of 100 cells of $2.00\pm 0.03\mathrm{ns}$ was obtained.

When calculating the mean lifetime ${\tau }_m=(a_1{\tau }_1+a_2{\tau }_2)∕(a_1+a_2)$ , per pixel we obtained an average value of $0.7\mathrm{ns}$ for the image in Fig. 6. Considering 100 cells within ten frames, the mean value was found $(0.680\pm 0.04)\mathrm{ns}$ . The employment of the $900\mathrm{nm}$ excitation wavelength resulted in a longer value of $(0.820\pm 0.05)\mathrm{ns}$ .

Interestingly, when analyzing the short fluorescent components within these FLIM images (frames), we obtained up to three maxima in the ${\tau }_1$ -histogram (Fig. 7 ). The first one at around $0.07\mathrm{ns}$ reflects the influence of backscattered laser light, which was able to transmit through the beamsplitter and the short pass filter (SP 610). When adding a second short pass filter, this peak nearly disappeared. The other two peaks occurred around $(0.170\pm 0.015)\mathrm{ns}$ and $(0.660\pm 0.05)\mathrm{ns}$ at $750\mathrm{nm}$ excitation wavelength and shifted to $(0.200\pm 0.015)\mathrm{ns}$ and $(0.800\pm 0.05)\mathrm{ns}$ , respectively, at $900\mathrm{nm}$ . Control measurements were done in culture medium as well as after washing the cells with phosphate buffer saline (PBS). The bands were therefore not background signals from the system such as autofluorescence of optics, etc., but of biological origin. We analyzed these two short components in more detail. For that purpose, we performed a statistical analysis of the histograms (Table 2 ) on ten bright fluorescent cells out of Fig. 6 and 8 . Rectangular regions of interest (ROI) were created as depicted in Fig. 8. Each ROI covered one cell. According to the table, just three cells exhibited only one short fluorescence lifetime of about $0.2\mathrm{ns}$ , whereas seven out of ten cells possessed two short components with lifetimes of 0.2 and $0.7\mathrm{ns}$ , respectively.

Fig. 7

Histograms for the distribution of the fluorescent components (a) ${\tau }_1$ , (b) ${\tau }_2$ , and (c) ${\tau }_m$ at 750 and $900\mathrm{nm}$ of hSGSC stem cells.

Fig. 8

(a) Average FLIM data of a particular cell were obtained from a region of interest (white rectangle).The histogram reveals two short-lived components with two fluorescence lifetime maxima at 0.238 and $0.783\mathrm{ns}$ , respectively. The fluorophores with a lifetime in the range of $0\text{to}0.5\mathrm{ns}$ are depicted red, and the fluorophores with a lifetime of $0.5\text{to}1.4\mathrm{ns}$ are shown green in the false color $t_1$ image. (b) The measured instrumental response function with a full width half maximum of $0.247\mathrm{ns}$ was obtained as SHG signal from collagen II.

Table 2

Average FLIM data calculated from ten regions of interest covering one single cell in each region. “x” means no component was detected.

3.3.

Second-Harmonic Generation Detection

We monitored the biosynthesis of collagen in 3-D salivary gland and pancreatic stem/progenitor cell cultures by monitoring the occurrence of SHG signals for a long time period up to $5\text{weeks}$ . The spheroids had dimensions of $0.2\text{to}2\mathrm{mm}$ in diameter. The two-photon autofluorescence and SHG images of spheroids were obtained by optical sectioning in $z$ -steps of $5\text{to}10\mu \mathrm{m}$ . The occurrence of the first SHG signal was detected after eight days after the introduction of the stimulating agent $\mathrm{TGF}\text{-}\beta 3$ [Figs. 9a and 9b ].

Fig. 9

Multiphoton autofluorescence and SHG images of hSGSC stem cells after chondrogenic differentiation. (a) Image taken with the short-pass filter SP 610 autofluorescence (green) and SHG (red-yellow fibrils), and (b) imaging with the bandpass filter BP $435∕5$ (mainly SHG signals).