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1 September 2008 Two-photon autofluorescence and second-harmonic imaging of adult stem cells
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Human and animal stem cells (rat and human adult pancreatic stem cells, salivary gland stem cells, and human dental pulp stem cells) are investigated by femtosecond laser 5-D two-photon microscopy. Autofluorescence and second-harmonic generation (SHG) are imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. In particular, the reduced coenzyme nicotinamide adenine (phosphorylated) dinucleotide [NAD(P)H] and flavoprotein fluorescence is detected in stem cell monolayers and stem cell spheroids. Major emission peaks at 460 and 530 nm with typical long fluorescence lifetimes (τ2) of 1.8 and 2.0 ns, respectively, are measured using spectral imaging and time-correlated single photon counting. Differentiated stem cells produce the extra cellular matrix (ECM) protein collagen, detected by SHG signals at 435 nm. Multiphoton microscopes may become novel noninvasive tools for marker-free optical stem cell characterization and for on-line monitoring of differentiation within a 3-D microenvironment.



In stem cell research, there is a high demand on techniques for the noninvasive, marker-free observation of growth, proliferation, differentiation, and stability of living stem cells under physiological conditions. The use of exogenous markers may alter the metabolic balance, influences the cell life, and avoid further therapeutic application. Noninvasive multiphoton microscopes with near-infrared (NIR) femtosecond laser sources1, 2, 3, 4 have been applied to image living single cells and different tissues with a high spatial resolution without any staining. Two-photon autofluorescence is obtained due to intrinsic fluorophores such as nicotinamide adenine (phosphorylated) dinucleotide [NAD(P)H], flavins, porphyrins, elastin, and melanin. In addition, second-harmonic generation (SHG) is induced by certain biomolecule structures such as collagen.5, 6, 7, 8, 9

In addition to the measurement of the fluorescence/SHG intensity by optical sectioning (3-D imaging), fluorescence lifetime imaging (FLIM, 4-D) and spectral imaging (5-D) can be performed. In particular, the arrival times of the fluorescence photons with respect to the excitation of the molecule and the particular location (pixel) can be determined by time-correlated single photon counting (TCSPC) and the use of photomultipliers with as short rise time. When using a photomultiplier tube (PMT) array in combination with a polychromator, the “color” of the emitted photon per pixel can be also determined (spectral imaging).

Multiphoton imaging is very suitable for long term analysis of living cells due to the absence of out of focus photostress. The excitation volume is limited to a subfemtoliter focal volume. NIR femtosecond lasers enable deep light penetration into tissues and provide the possibility of optical sectioning of 3-D biological objects. Nondestructive imaging of living specimens is restricted to a certain optical window determined by the spectral range and the light intensities.10 Intrinsic cellular fluorophores deliver information on the cell structure as well as on cell activities. Authors could distinguish certain cancer cells from normal cells,11, 12 even malignant from benign tumors,13 by optical properties of native fluorescent molecules. Many studies on the intrinsic metabolic state show that innate cellular fluorescence has the potential to discriminate proliferating and nonproliferation cell population,14 and also metabolic changes of self-renewing and differentiating cells.15, 16 The differentiation of human mesenchymal stem cells into an adipogenic pathway (formation of lipid droplets, change of morphology and autofluorescence) has been investigated recently by two-photon microscopy.17

A unique feature of stem cells is the extended self-renewal of the cell population and their differentiation potency. The aim of the present study was to investigate the two-photon excited autofluorescence of human stem cells and the onset of collagen production of differentiated cells by the detection of SHG signals. An interesting question arises as to whether it is possible to select differentiated cells from undifferentiated cells without interfering with the native environment and without the use of external markers.

Here we report on the application of a 5-D two-photon microscope for imaging different human and animal stem cell lines by autofluorescence and SHG in monolayers and spheroids.


Material and Methods


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 used18, 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 4.5gL glucose, L-glutamine, and supplemented with 15% fetal calf serum Gold (FCS-Gold) (PAA Laboratories GmbH, Germany), 100Uml penicillin, and 100mgml streptomycin. hDPSC were cultured in α-MEM with 15% FCS, 100μM L-ascorbic acid 2-phosphate, 2mM L-glutamine, 100Uml penicillin, and 100μgml streptomycin. Cultures were maintained at 37°C in a 5% CO2 humidified atmosphere.

For imaging, cells were transferred (confluence 80 to 90%) into sterile miniaturized cell chambers with two 0.17mm 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.


Pellet Culture and Chondrogenic Differentiation

For chondrogenic differentiation, a pellet culture was used. Approximately 200,000 cells were placed in a 15ml polypropylene tube and centrifuged to a pellet for 5min (1000×min) . The flattened pellet at the bottom of the tube was cultured at 37°C with 5% CO2 in the culture medium for 5to6days until a spherical form (spheroid) was formed. For the differentiation, the spheroids were cultured in 1ml of the chondrogenic medium DMEM (GIBCO, Germany) supplemented with 10mgml transforming growth factor (TGF)-β3 , 100nM dexamethasone, 50mgml ascorbic acid-phosphate, 4mgml L-proline, 100mgml natrium piruvate, 15% FCS-Gold, 100Uml penicillin, and 100mgml streptomycin. The medium was replaced every 3to4days for a period of 4to5weeks . For microscopy, the pellets were transferred to dishes with 0.17mm thick glass coverslips.


Instrumentation and Measurements

A laser scanning microscope (LSM 510 Meta NLO, Zeiss, Germany) equipped with a tunable 80MHz 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 900nm ( 140fs laser output) at 5mW mean power were used. The beamsplitter HFT KP 650 and the short pass filter KP 685 were employed. The cells were scanned in a 512×512pixel field at 25.6μspixel dwell time. Fluorescence emission was recorded through a Plan-Neofluar 40×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 435nm bandpass filter at an excitation wavelength of 870nm .

3-D two-photon autofluorescence and SHG images were obtained by optical sectioning with z -intervals of 5to10μm . 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 10nm per channel. Typically, the spectral range of 382to714nm 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.





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 900nm ) to separate different native fluorescence molecules. When using 750nm , NAD(P)H as well flavins/flavoproteins can be excited by a two-photon process. However, when changing to 850 or even 900nm , 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 460to470nm when excited with 750nm light, which is consistent with the emission behavior free and protein bound NADH. The maximum shifted to 530to535nm when using 900nm 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 750nm . Fluorescence emission was recorded through a Plan-Neofluar 40×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 382to714nm . Excitation wavelength was at 750nm .


Fig. 5

Spectral measurement of hSGSC stem cells.



Fluorescence Lifetime Imaging Measurement and Analysis

Fluorescence decay curves have been obtained by exciting at 750 and 900nm . Figure 6 presents a typical 750nm 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 χ2=1.00 reveals two components. A fast one with a short lifetime (τ1) of 0.17ns and an amplitude of a1=72% , and a second one with τ2=1.8ns and the amplitude a2=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 0.202ns [standard deviation (SD) 0.019, amplitude 70.5%] was found in this particular cell. The long component had an average value of 2.014ns (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.

τ1 (ns) τ2 (ns) τm (ns) a1 (%) χ2

When calculating the mean fluorescence lifetime τ2 of the long-lived component within this frame of about ten cells in Fig. 6, we obtained a major value of 1.87ns , as depicted in the histogram. When analyzing ten frames of about 100 cells in total of the same cell line, we obtained a mean τ2 value of (1.82±0.02)ns . In contrast, when changing the excitation wavelength to 900nm , an average value τ2 of 100 cells of 2.00±0.03ns was obtained.

When calculating the mean lifetime τm=(a1τ1+a2τ2)(a1+a2) , per pixel we obtained an average value of 0.7ns for the image in Fig. 6. Considering 100 cells within ten frames, the mean value was found (0.680±0.04)ns . The employment of the 900nm excitation wavelength resulted in a longer value of (0.820±0.05)ns .

Interestingly, when analyzing the short fluorescent components within these FLIM images (frames), we obtained up to three maxima in the τ1 -histogram (Fig. 7 ). The first one at around 0.07ns 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±0.015)ns and (0.660±0.05)ns at 750nm excitation wavelength and shifted to (0.200± 0.015)ns and (0.800±0.05)ns , respectively, at 900nm . 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 0.2ns , whereas seven out of ten cells possessed two short components with lifetimes of 0.2 and 0.7ns , respectively.

Fig. 7

Histograms for the distribution of the fluorescent components (a) τ1 , (b) τ2 , and (c) τm at 750 and 900nm 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 0.783ns , respectively. The fluorophores with a lifetime in the range of 0to0.5ns are depicted red, and the fluorophores with a lifetime of 0.5to1.4ns are shown green in the false color t1 image. (b) The measured instrumental response function with a full width half maximum of 0.247ns 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.

cell τ1a (ns) τ1b (ns) τ2 (ns) τm (ns) a1a (%) a1b (%)ratio a1a:a2b χ2


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 5weeks . The spheroids had dimensions of 0.2to2mm in diameter. The two-photon autofluorescence and SHG images of spheroids were obtained by optical sectioning in z -steps of 5to10μm . The occurrence of the first SHG signal was detected after eight days after the introduction of the stimulating agent TGF-β3 [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 4355 (mainly SHG signals).




Detected two-photon autofluorescence with emission maxima at 460to470nm and 530to535nm , and the long fluorescence lifetimes of 1.8 and 2.0ns , respectively, prove the presence of the biomolecules NAD(P)H and flavins/flavoproteins such as FAD in stem cells and stem cell clusters. The occurrence of some extremely bright cells shows the heterogeneity within a cell population of the same culture dish.

Interestingly, the analysis of the short-lived component of human salivary gland stem cells was found to be even more complex. Some cells exhibited only one short-lived fluorophore with a fluorescence lifetime of about 0.2ns only (excellent biexponential fit with χ2 of 1.00, Fig. 6), whereas the majority of cells revealed an additional short-lived component with 0.7ns fluorescence lifetime.

The interpretation of these two short fluorophores is complicated. Free NAD(P)H has a short lifetime around 0.200to0.300ns , whereas a variety of flavoproteins have short picosecond lifetimes. The free flavin mononucleotide (FMN) has a typical lifetime of 4.7to5.2ns ,23, 24 while flavin adenine dinucleotide (FAD) has a lifetime of 2.3to2.8ns .24, 25, 26 When binding to a variety of proteins, the fluorescence lifetime of NAD(P)H in a variety of cells and solutions shifts to higher values of about 2ns .23 Chia 27 reported on the presence of three NAD(P)H fluorophores in rat brain tissue. A very short one of 0.48ns for the free (nonbound) coenzyme, a second short one of 0.77ns and a long lifetime of 3to6ns for bound NAD(P)H.

Future work needs to be done for the interpretation of the cellular FLIM data and spectra with regard to the specific cellular metabolism and differentiation value. For that purpose, the particular imaged cell of interest should also be characterized with selective biochemical means, such as antibody staining of the cell as well as the surrounding cells.

Very interestingly, SHG can be used to detect the biosynthesis of collagen as a result of the differentiation process.28, 29 We performed long-term studies of up to 35days on 3-D stem cell spheroids and were able to monitor the expression of the extracellular matrix protein. The possibility of nondestructive marker-free imaging allows the study of the organization and development of the ECM structure, and of feedback mechanisms. Recently it was shown that collagen influences the differentiation of stem cells.30

Femtosecond laser microscopes with their capability of nondestructive two- and three-photon excited autofluorescence and SHG/THG imaging may become novel noninvasive multidimensional tools for marker-free optical stem cell characterization and for on-line monitoring of differentiation of living cells in a 3-D environment, including ECM components.


The authors would like to thank Katja Schenke-Layland [University of California, Los Angeles (UCLA)], and Alexander Ehlers for helpful discussions and comments, Daniel Sauer for technical support, and Erwin Gorjup (Fraunhofer IBMT, Sankt Ingbert, Germany) for stem cell characterization.



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© (2008) Society of Photo-Optical Instrumentation Engineers (SPIE)
Aisada A. Uchugonova and Karsten König "Two-photon autofluorescence and second-harmonic imaging of adult stem cells," Journal of Biomedical Optics 13(5), 054068 (1 September 2008).
Published: 1 September 2008

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