2.1.

Multiphoton Imaging System

The multiphoton imaging system DermaInspect (JenLab, Jena, Germany), which was designed for deep-tissue optical tomography, consisted of a titanium-sapphire laser (Mai Tai, Spectra Physics, Darmstadt, Germany) with 80-MHz pulse frequency, 75-fs pulse duration, and a tuning range of 710 to $920\mathrm{nm}$ ; a scanning module with a motorized shutter; a beam attenuator; and a two-axis galvanometric scanner. A beamsplitter (Chroma 640 DCSPXR, AHF Analysentechnik AG, Tübingen) and a short-pass filter (BG39, Schott, Mainz, Germany) separated the excitation irradiation from the emission. Fluorescence light was detected in the wavelength range between 350 and $600\mathrm{nm}$ by a photon-counting photomultiplier tube module (PMH 100, Becker & Hickl, Berlin, Germany). High spatial resolution was provided by a high NA oil-immersion objective (Plan-Neofluar $40\times$ , 1.3 oil, Zeiss, Göttingen, Germany) with 140-mm working distance.

The single-photon count rate and the arrival times of the individual photons with respect to the excitation pulse were recorded at each spot in the sample by a PC-based, time-correlated single-photon counting (TCSPC) system (SPC-830, Becker & Hickl, Berlin, Germany).¹² From the measured photon arrival times, fluorescence lifetimes were calculated by a special software (SPCImage, Becker & Hickl, Berlin, Germany), which analyzes and visualizes the fluorescence lifetime data. Curve fitting with a single exponential decay curve to the measured data was used to calculate the fluorescence lifetime for each pixel, which was subsequently displayed in color-coded images.

Evaluation of the fluorescence lifetimes was based either on fitting a single exponential decay curve to the data or by multiexponential fitting with the calculation of a mean decay time.

2.2.

Cell Culture and Sample Preparation

The human lymphoma cell line Karpas-299 was kindly provided by Research Center Borstel, Borstel, Germany. Cells were routinely grown in suspension culture in RPMI 1640 $(1\times )$ medium with hepes and L-glutamine supplemented with 10% fetal calf serum, antibiotic, and antijmycotic solution (all cell culture media, PAA Laboratories, Pasching, Austria) in a $37°\mathrm{C}$ incubator (5% ${\mathrm{CO}}_2$ , 95% air).

Immunogold NPs were made of gold NPs with 30-nm size (British Biocell International, United Kingdom) and ACT-1 antibodies (kindly provided by Professor J. Gerdes, Research Center Borstel, Germany) according to the protocol of the manufacturer. ACT-1 is a monoclonal mouse antibody against CD25, the interleukin-2 receptor (IL-2-R).¹³ The fluorescent dye Alexa Fluor 488 (Invitrogen GmbH, Karlsruhe, Germany) was conjugated to the antibodies as recommended by the manufacturer.

Cell suspensions were centrifuged at $1400\mathrm{rpm}$ for $5\min$ at $20°\mathrm{C}$ and then resuspended in phosphate-buffered saline (PBS, pH 7.2, containing 1% bovine serum albumin) with cell densities of ${10}^6{\mathrm{ml}}^{-1}$ . Then, either NP-ACT-1 or A488-ACT-1 conjugates was added to the cell suspension at certain ratios between conjugate and cell number. After incubation for $20\min$ at $37°\mathrm{C}$ , the cells were centrifuged and washed twice and then resuspended in PBS.

Hoechst 33258 (Invitrogen GmbH, Karlsruhe, Germany), which is a fluorescent nucleic acid stain for labeling DNA, was diluted to a concentration of $1\mu \mathrm{g}∕\mathrm{ml}$ with PBS. After the cell suspension was centrifuged and resuspended in PBS, diluted H258 solution was added, and the mixture was incubated for $1\mathrm{h}$ at $37°\mathrm{C}$ . Then, the cells were centrifuged, and the staining solution was replaced by PBS buffer.

2.3.

Temperature Calculations

The temperatures inside and surrounding the particles were calculated by an analytical solution of the differential equation of heat diffusion for a single spherical particle that has different thermal properties than the surrounding medium.¹⁴ A series of rectangular 100-fs broad laser pulses with 80-MHz repetition rate was assumed. Since the equations for heat diffusion are linear in time, a superposition of the solutions for a single pulse with appropriate scaling and time delays provided the solution for a pulse train.^{15, 16} Individual particle absorption was calculated from data that were provided by the manufacturer of the particles for a wavelength of $520\mathrm{nm}$ (absorption of $5{\mathrm{cm}}^{-1}$ at a particle concentration of $7.8\times {10}^{11}{\mathrm{ml}}^{-1}$ ). By absorption measurements with a scanning photometer (Lambda 14, PerkinElmer, Überlingen, Germany) the absorption cross section was scaled to a wavelength of $750\mathrm{nm}$ . From this calculation, an absorption cross section of 5% of the geometrical cross section was calculated for the 30-nm particles. The focal diameter of the $1.3\mathrm{NA}$ object was calculated by Abbe’s formula¹⁷ to be $700\mathrm{nm}$ .

3.1.

Imaging of Karpas-299 Cells Labeled with NP-ACT-1 Conjugates

The special linear and nonlinear optical properties of gold NPs have been the subject of recent studies. Multiphoton absorption has been shown to produce strong luminescence of gold NPs.¹⁸ For these experiments, we used gold NPs of two different diameters ( $15\mathrm{nm}$ and $30\mathrm{nm}$ ) with the same concentration for multiphoton imaging and compared their luminescence efficiency. Figure 1 shows the intensity images and false-color fluorescence lifetime images of gold NPs with 15-nm (a) and 30-nm (b) diam. A broad range of emission intensities is observed for gold NPs with 15-nm diam in Fig. 1c. Although it is difficult to quantify their differences precisely due to the range of efficiencies present for gold NPs of the same average size, we observed that gold NPs with 30-nm diam exhibited similar luminescence to 15-nm-diam gold. Only 30-nm-diam gold NPs were chosen for the following cell labeling experiments.

Fig. 1

Intensity images and corresponding false-color FLIM images for gold NPs with different diameters: (a) $15\mathrm{nm}$ ; (b) $30\mathrm{nm}$ . NP (Excitation wavelength: $750\mathrm{nm}$ ; radiant flux: $20\mathrm{mW}$ ; collection time: $60\mathrm{s}$ ; imaged field: $135\mu \mathrm{m}$ ). A central region ( $32\times 32$ pixels) of the image in (a) was represented as contour intensity plot, which is shown in (c).

Intensity images and FLIM images of Karpas-299 cells, and cells incubated with NP-ACT-1 conjugates are shown in Fig. 2 . According to the different lifetimes of cells and gold NPs, there are some distinct differences between cells incubated without and with gold NPs.

Fig. 2

Intensity images, corresponding false-color FLIM images of Karpas-299 cells (a), and cells incubated with NP-ACT-1 conjugates (b). Excitation wavelength was $750\mathrm{nm}$ , radiant flux was $20\mathrm{mW}$ , collection time was $60\mathrm{s}$ , and imaged field was $90\mu \mathrm{m}$ . Histograms of the lifetime distribution of the images are also shown.

Multiphoton excited fluorescent microscopy of living cells demonstrated individual cellular components at subcellular resolution. An intensity image of the autofluorescent signal delineated cytoplasm and cellular nucleus structure [Fig. 2(a1)]. The corresponding time-resolved fluorescence lifetime measurements demonstrated different lifetimes for different cell compartments [Fig. 2(a2)]. The fluorescence lifetime of Karpas-299 at 750-nm, 20-mW laser excitation measured from the decay curves were $1800\mathrm{ps}$ for cytoplasmic areas and about $1200\mathrm{ps}$ for the cellular nucleus.

ACT-1 is a specific antibody to the cell membrane receptor of interleukin-2 (CD25). Due to the binding of ACT-1 to CD25 on the cell surface, gold NPs are only distributed on the surface of cell membrane of Karpas-299 [Fig. 2b]. In contrast to the image in Fig. 2a, binding areas of gold NPs to cell membrane are seen with an additional very short lifetime. Due to the two lifetimes contained in these areas, two or more component fits were needed in these particular areas. For a selected region, the fit curve yields two components with a contribution of 90% of the 195-ps and 10% of an 1800-ps component, which results in a mean fluorescence lifetime of $355\mathrm{ps}$ . As depicted in the histogram, the fluorescence lifetime of gold NPs is distributed in the red area, whereas the fluorescence lifetime of cellular autofluorescence is typically on the order of 1.0 to $2.0\mathrm{ns}$ . In fact, the short lifetime of gold NPs was not resolved, because the time resolution was limited by the transit time spread of the detector to about $180\mathrm{ps}$ .

3.2.

Comparison of Cells Labeled with NP-ACT-1 and A488-ACT-1 Conjugates

The optical properties of the fluorescent dye A488 were studied through conjugating it to ACT-1 antibodies and incubating Karpas-299 cells with the conjugates. As a relatively new molecular probe, dye A488 is brighter and more resistant to photobleaching than FITC or some other popular dyes.¹⁹

As expected, the intensity image and false-color FLIM image showed a distinct staining pattern throughout the surface of the cells [Fig. 3a ]. The corresponding histogram of the lifetime distribution of photons of the image is shown in the right column. The blue color is caused by the dye A488 and corresponds to a mean lifetime of $3.5\mathrm{ns}$ .

Fig. 3

Intensity image (a1); false-color FLIM image (a2) of Karpas-299 cells incubated with A488-ACT-1 conjugates (excitation wavelength: $750\mathrm{nm}$ ; radiant flux: $25\mathrm{mW}$ ; collection time: $60\mathrm{s}$ ; imaged field: $70\mu \mathrm{m}$ ); and corresponding histogram of lifetime distribution of the image in (a). (b) Intensity image, discrete false-color FLIM image of cells incubated with A488-ACT-1 and NP-ACT-1 conjugates, and histogram of lifetime distribution in middle column.

In order to compare the relevant optical properties of gold NPs with fluorescent dye, cell suspension incubated for $20\min$ with Au-ACT-1 conjugates and A488-ACT-1 conjugates were mixed and prepared as a sample for a direct comparison.

Figure 3b shows the intensity image and corresponding discrete false-color FLIM image. According to their different fluorescence lifetimes, gold NPs and A488 can be distinguished easily. Under the same conditions of laser excitation, the emission intensity of gold NPs is much stronger than that of A488. The emission intensity of gold NPs was photostable for hours of continuous irradiation time.

3.3.

Imaging of Cells Labeled with NP-ACT-1 and Dye H258

As an example for an intracellular stain, we combined gold NPs with the fluorescent probe H258 to characterize their individual optical properties under fluorescence lifetime imaging microscopy. H258 is an important staining molecule for cellular DNA. Prior to the imaging experiment, Karpas-299 cell suspensions were incubated with NP-ACT-1 conjugates and H258.

Figure 4 displays the images obtained from these cells at 750-nm, 15-mW laser excitation. Figure 4a shows the intensity image and FLIM image of the sample. A corresponding histogram of the lifetime distribution of the image is shown in Fig. 4b. When the fluorescence lifetime was analyzed for specific areas of interest, a long lifetime was found in the nuclear area that was stained by H258 (d), and a short one was found in the membrane area labeled by gold NPs (c). The blue-green color shows regions stained with H258 with lifetime of $2.5\mathrm{ns}$ , which is comparable to that found in spectroscopic time-resolved measurements,²⁰ the yellow color shows gold NPs.

Fig. 4

Multiphoton microscopy of Karpas-299 cells labeled with gold NPs and dyes H258. (a) Intensity image, and corresponding false-color fluorescence lifetime image. (b) Histogram of lifetime distribution of photons within image frame (excitation wavelength: $750\mathrm{nm}$ , radiant flux: $16\mathrm{mW}$ ; collection time: $60\mathrm{s}$ ). (c) and (d) Analysis for specific area of interest in image delivers the fluorescence lifetime of gold NPs and H258 (imaged field: $90\mu \mathrm{m}$ ).

3.4.

Cell Damage

Under laser irradiation of NP-ACT-1 incubated cell with more than 20 mW cell damage with bleb formation was observed.²¹ In contrast to a fluorescent dye, gold NPs have a high-absorption cross section and a low-fluorescence quantum yield. Additionally, there is still a few percent residual linear absorption at $750\mathrm{nm}$ . Temperature calculations show that this leads to a significant temperature increase inside the particles of $30\mathrm{K}∕\mathrm{mW}$ irradiation power at the end of each pulse [Fig. 5a ]. Under conditions of cell damage, with an average radiant flux above $20\mathrm{mW}$ , the temperature increase is larger than $600\mathrm{K}$ . However, at the beginning of the next pulse, which follows after $12\mathrm{ns}$ , the temperature is cooled down below $2\mathrm{K}$ . Within $100\mathrm{ns}$ , an average background temperature of $15\mathrm{K}$ is established, on which 600-K particle heating of the individual laser pulses occurs. The temperature increase is highly confined to the particle. At the end of the first femtosecond laser pulse, the temperature increase outside of the particle is virtually zero [Fig. 5b]. Even after a steady state is established, the elevated temperature is still confined to a few tens of nanometers.

Fig. 5

Calculation of the temperature in and around single gold particles during laser irradiation. (a) Temperature course inside a 30–nm NP during the irradiation with 1-mW radiant flux focused to a spot of $700\mathrm{nm}$ (solid black line). Rectangular pulses with 100-fs width and 12-nm temporal separation were assumed. Additionally, the temperature course after one pulse (dashed line) and after true CW irradiation with the same average radiant flux (dotted line) are shown (b) Spatial temperature distribution at the end of the first pulse (solid line) and after $10\mu \mathrm{s}$ irradiation (dashed line).