2.1.

Mouse Model

Female Balb/c mice ($n=5$), and C57BL/6 mice ($n=5$) 8 to 10 weeks of age, were purchased from Charles River Laboratories (Sulzfeld, Germany) and kept under standard conditions with free access to food and water. Animals were anesthetized with a combination of Fentanyl (Bayer, Leverkusen Germany), Midazolam (Curamed, Karlsruhe, Germany), and Medetomidin (Pfizer, Karlsruhe, Germany) injected intraperitoneally. After assuring adequate anesthesia, the abdominal cavity was opened and an isolated ileal loop was gently protruded without disturbing the blood supply. The loop was glued (3M Vetbond TissueAdhesive, St. Paul, Minnesota) on a heated metallic block and sliced carefully so that the mucosa could be gently fixed to a microscopic cover slip to dampen motion artifacts, while still maintaining tissue viability.^3,6 During all procedures, the small intestine was constantly moisturized with saline, and the core body temperature was maintained at 37°C using a homeothermic table. All animal experiments were approved by the state government (Ministerium für Umwelt, Naturschutz und Landwirtschaft Schleswig-Holstein V742-72241.122, TV-No. 23/p/04, V312-72241.122-1 (4-1/10), TV-No. 23/1f/10).

2.2.

Autofluorescence 2-Photon Microscopy

Imaging with both lasers was compared at the same instrument, the multiphoton tomograph Derma Inspect 101 (JenLab, Jena, Germany). A tunable femtosecond Ti:sapphire laser (Mai Tai Spectra Physics, Mountain View, California) with a pulse width below 100 fs and 80 MHz repetition frequency was used for imaging at the excitation wavelengths 730 and 800 nm. As is usually done, the group delay dispersion (GDD) of the microscope and objective was not compensated, which increased the pulse width from below 100 fs to approximately 220 fs. This value was calculated from the measured pulse width of the Mai Tai and the GDD which was determined separately for the microscope objective and the scanning optics. A transform limited output of the Mai Tai was assumed. Since both, laser pulse width and GDD dropped between 730 nm and 800 nm, the pulse width changed less than 10% in this range.

In addition, sub-10 fs broadband Ti:sapphire laser (Integral Pro, Femtolasers Produktions GmbH) with a 85-MHz repetition was additionally coupled to the Derma Inspect using a flipping mirror [Fig. 1(a)]. A dielectric mirror based prechirp unit (Mosaic Pro V, Femtolasers Produktions GmbH) compensated second-order dispersion of the optical components. The overall GDD in the light path was determined to be $7950{\mathrm{fs}}^2$ for the 800 nm wavelength (Table 1). This corresponded to the maximum negative GDD of the prechirp unit and could thus be compensated. Pulse duration was measured in the focal volume by an autocorrelator (Femtometer, Femtolasers Produktions GmbH). The autocorrelation traces showed that the pulses are not transform limited. Though the autocorrelator software determined a pulse width of around 10 fs from the central peak, the increased asymptotic level and subpulses at 80 fs indicate considerably longer pulses, probably above 15 fs [Fig. 2(b)]. A pulse duration below 10 fs, which is supported by a spectral width of 150 nm, is only reached when the higher-order GDD is compensated, which was beyond our technical means.

Fig. 1

(a) Schematic drawing of the 2-photon microscope with fluorescence detection in four spectral channels and (b) detection probability of the spectral channels including the collection yield of the objective, the transmission of all optical elements, and the quantum efficiency of the detectors.

Table 1

Calculated efficiency of 2-photon excitation of NAD(P)H and flavin adenine dinucleotide (FAD) with tunable and broadband pulses.

Fig. 2

(a) Spectral dependence of the 2-photon action cross sections of NAD(P)H⁷ and FAD.²² For comparison, the spectrum of the Spectra Physics Mai Tai (220 fs pulse) and the Femtolasers Integral Pro (10 fs pulse) are also shown. (b) Autocorrelation trace of the pulses from Femtolasers Integral Pro measured with a second-harmonic generation (SHG) signal inside the sample volume.

The small intestine was imaged by A2P microscopy using a $40\times /1.2$ water immersion objective (Carl Zeiss Microscopy GmbH, Jena, Germany). Emission between 380 and 680 nm was detected in four spectral channels. Detection efficiencies are shown in Fig. 1(b). Channel 4 was not included in data evaluation because a significant amount of scattered excitation light was picked up by the detector and the fluorescence signal in that channel was very weak.

The relation between gray values registered by the PMTs and the corresponding photon numbers was established by calibration measurement. For this purpose, images of homogeneously fluorescing plastic slides (Chroma Chroma Technology GmbH, Olching, Germany) were acquired with different excitation intensities, and the mean intensity and variance of fluorescence emission values were evaluated. For Poisson distributed noise, there is a linear correlation between variance and mean fluorescence intensity values. Their ratio was used as the conversion factor between the gray value and number of detected photons.²³ Using these factors, fluorescence intensities of the autofluorescence images were converted into photon numbers per pixel.

2.3.

Comparison of the Photodamage Potential of Both Laser Systems

The time-averaged rate of photon detection $⟨F(t)⟩$ for 2-photon excitation depends on the 2-photon action cross-section ${\sigma }_{2P}$, concentration $c$ of the excited fluorophores, time-averaged radiant flux $⟨P(r,t)⟩$, wavelength $\lambda$, pulse duration $\tau$, and repetition rate $f$ of the excitation:²⁴

For a quantitative comparison of the damage potential of both lasers, image sequences were taken at different excitation fluxes in the apical and basal part of the villus epithelium of the mouse intestine. Imaged areas covered a field of $120\times 120\mu {\mathrm{m}}^2$ at a pixel size of $0.23\times 0.23\mu {\mathrm{m}}^2$.

Time-lapse series of 100 A2P images were recorded at intervals of 7.4 s. Each series covered a time of 14 min. In order to evaluate the average brightness of each fluorescence image in the three spectral channels, the mean number of photons from 10 regions of interest (ROI) in the cytoplasm was determined (Fig. 3).

Fig. 3

Autofluorescence images of a plane through the apical cytoplasm of villus epithelium. Images in (a) and (b) were acquired using 220 fs pulses with excitation wavelengths of 730 and 800 nm, respectively. The image in (c) was recorded with ultra-broadband pulses. In each image, regions of interest (ROIs) were used for the determination of image brightness. (d) Fluorescence in the three spectral channels divided by the squared excitation flux. For comparison, calculated normalized fluorescence from Table 1 is shown in arbitrary units.

The time for the onset of hyperfluorescence was measured during irradiation of the apical cytoplasm. When hyperfluorescence started, image acquisition was stopped after a few more images, as the hyperfluorescent lesions grew fast, destroyed large tissue areas, and led to saturation of the detector signal (Fig. 5). The total number of fluorescence photons before the onset of hyperfluorescence was calculated by multiplying the mean photon number per pixel with the number of images that could be acquired before hyperfluorescence started (Fig. 6).

The immigration of inflammatory cells into the irradiated tissue was evaluated during and after irradiation of the villus epithelium as a sensitive physiological marker of tissue damage. In the irradiation series of the basal part of the epithelium, polymorphonuclear leucocytes (PMNLs) could occasionally be observed migrating into the irradiated area. When the apical part of the villus epithelium was imaged, no inflammatory cells were observed, as inflammatory cells can probably move only in the basal part of the epithelium due to tight junctions and desmosomes in the apical part of the cells. In order to quantify the immune response, image stacks of the irradiated villi were acquired up to one hour after the irradiation to screen the tissue for immunological reactions. The fraction of irradiation sequences taken at the basal cytoplasm and the fraction of image stacks of the villi, in which the immigration of immune cells was observed, was counted for assessing the tissue damage (Fig. 9).

2.4.

Image Analysis

The image brightness in the cellular compartments and ROIs was evaluated with the software ImageJ.²⁵ Pixel values were corrected for background signal by subtraction of a dark image. The resulting intensities were converted into detected photon numbers as described in Sec. 2.2.

Stacks and time series were processed using the software Imaris version 7.2.0 (Bitplane, Zurich, Switzerland). For cell tracking, image series were transformed into volume-rendered movies, and spot analysis was used for manual tracking of cell motility. The calculations of the cell velocity, straightness, track length, and displacement were performed using the algorithms provided by Imaris.

3.1.

Efficiency of Autofluorescence Excitation

The 2-photon action cross-section ${\sigma }_{2P}$ of NAD(P)H provides the main contribution to the cellular autofluorescence of epithelial enterocytes in the small intestine.⁷ Between 730 and 800 nm, the 2-photon excitation of NAD(P)H drops by more than an order of magnitude (Fig. 2). In contrast, for the same spectral shift of excitation, the second relevant chromophore FAD looses only a little more than 50% of its absorption. Table 1 compares the expected efficiency of the 2-photon excitation of NAD(P)H and FAD with tunable 220 fs pulses at 730 and 800 nm to the excitation efficiency with ultra-broadband pulses if they were compressed to 10 fs. The 2-photon action-cross-section ${\sigma }_{2P}$ for these pulses was calculated based on the fluorophore spectra and the laser emission spectrum shown in Fig. 2. Excitation-normalized values of the expected fluorescence were obtained by relating ${\sigma }_{2P}$ to the pulse duration, repetition rate, and wavelength (center wavelength for the ultra-broadband pulses). In order to facilitate the comparison between the different excitation modalities, all (${\sigma }_{2P}/\lambda f\tau$) values were, in turn, normalized by the value for $730\mathrm{nm}/220\mathrm{fs}$ excitation.

According to the data in Table 1, excitation of NAD(P)H should be eight times more efficient with 10 fs ultra-broadband pulses than with 220 fs pulses at a wavelength of 730 nm. The higher irradiance of 10 fs pulses should more than compensate the lower spectral overlap between the 2-photon absorption and excitation. Excitation of NAD(P)H at 800 nm should be 40 times less efficient compared to 730 nm and even 300 times less efficient than with broadband pulses compressed to 10 fs. Excitation of FAD is expected to be only 14 times more efficient than with $730\mathrm{nm}/220\mathrm{fs}$ pulses.

These predictions were experimentally tested by in vivo imaging of the apical villus epithelium (Fig. 3). As expected, fluorescence was drastically reduced when the excitation wavelength of 220 fs pulses was tuned from 730 to 800 nm. Twice the excitation flux had to be used to get an acceptable image quality. Enterocytes were visible at both wavelengths, while lysosomes appeared more pronounced at the 800 nm excitation. Since a pulse compression to 10 fs was not achieved, the use of ultra-broadband pulses did not lower the required excitation flux compared to $730\mathrm{nm}/220\mathrm{fs}$ excitation. Thus, at comparable image quality, a similar light exposure (see Fig. 3) was needed.

For quantitative evaluation of the fluorescence intensities, 10 ROIs inside the apical cytoplasm of an enterocyte were evaluated for each excitation condition. The mean photon number per pixel was calculated [Fig. 3(d)]. Instead of a 7 to 15-fold increase of excitation-normalized fluorescence, which is expected for a 10 fs pulse width, the ultra-broadband excitation with our not fully compressed pulses generated for the same excitation power slightly less fluorescence than 220 fs pulses at 730 nm. Compared to 800 nm excitation, the normalized fluorescence excited by ultra-broadband pulses increased only 4-fold instead of the 300 times calculated for 10 fs pulse width.

This discrepancy corroborates the incomplete GDD compensation seen in the autocorrelation traces [Fig. 2(b)]. Generation of pulse widths below 10 fs (with a spectral width of over 150 nm) in the microscope focus requires an exact compensation of the GDD introduced by the highly dispersive microscope objectives. This is only achieved if higher-order dispersion compensation is employed. Advanced control of the spectral phase of broad pulse using, for example, a multiphoton intrapulse interference phase scan (MIIPS) would be needed to reach the 2-photon excited fluorescence and SHG levels that are predicted by Eqs. (1) and (2) at these bandwidths. At a spectral width of 30 nm, a deviation from the linear increase of the detected emission with spectral width or the inverse pulse duration was already observed.²⁰ Since a five times broader spectral width was used in these experiments with first-order GDD compensation, fluorescence levels are significantly lower than expected for bandwidth-limited pulses. Increased pulse width and intensity in the side lobes did not optimally contribute to fluorescence excitation. Since the excitation efficiency depends in a complex manner on the pulse chirp,²⁶ an exact calculation of the excitation efficiency would have to incorporate the spectral phase, which was not measured. The short-wavelength spectral components, which have the highest deviation from a constant GDD and also the highest absorption by the fluorophores, are not optimally corrected and will, therefore, contribute much less to excitation than assumed in Table 1. Additionally longitudinal chromatic aberration, which was approximately $1\mu \mathrm{m}$ over the wavelength range from 700 to 900 nm for the objective used, also prevents a perfect overlap of all wavelength components in the sample and may further reduce the excitation efficiency.

In conclusion, the differences between the calculated and measured excitations of cellular autofluorescence reflect deficiencies in the setup. Increased technical efforts are needed for optimal compensations of GDD and chromatic aberrations. If successfully done, a reduction of the excitation flux by one order of magnitude can be expected. However, as will be shown below, the speed and quality of imaging are not only limited by the excitation power, but also by photodamage. Hence, no efforts were made to increase the excitation efficiency in our setup. In contrast, in a clinical setting, where A2P microscopy with fixed optics will be used, specifically tailored dispersive mirrors could be used to exactly compensate higher-order GDD and attain optimum excitation efficiency.²⁷

A series of images were taken at different depths within the small intestinal mucosa from the apical cytoplasm of the epithelium down to the lamina propria to compare $220\mathrm{fs}/730\mathrm{nm}$ excitation with ultra-broadband excitation (Fig. 4, Video 1 and Video 2). Mitochondria containing NAD(P)H as the main fluorophore are equally visible in both cases. FAD in lysosomes and antigen presenting cells, which has a red-shifted absorption,³ was considerably more visible with ultra-broadband excitation due to the good overlap with the emission spectrum. Hence, the ultra-broadband pulses revealed the combined information of 730 and 800 nm excitation in just one scan. SHG by collagen is efficient above 800 nm and should also be visible with ultra-broadband excitation. However, it was not observed in the present experiments due to a lack of SHG active type I collagen in the small intestine.

Fig. 4

(a, d) Autofluorescence 2-photon (A2P) images of the apical cytoplasm. Autofluorescence was mainly from NAD(P)H in mitochondria of enterocytes. Mucus of goblet cells (arrows) appears dark. (b, c) The basal part of the epithelial cells. The cells nuclei (asterisks) are surrounded by a small rim of bright cytoplasm. (c, f) Epithelium and the underlying lamina propria with many different cells. The increased spectral bandwidth of the sub-10 fs Integral Pro laser shows lysosomes, macrophages, and other FAD-containing structures with good contrast (scale bar, $20\mu \mathrm{m}$) (Video 1, MPEG, 3.91 MB; Video 2, MPEG, 4.15 MB) [URL: http://dx.doi.org/10.1117/1.JBO.20.11.116001.1]. [URL: http://dx.doi.org/10.1117/1.JBO.20.11.116001.2].

3.2.

Hyperfluorescence and Photodamage

For different wavelengths and pulse durations, not only the amount of tissue damage but also the damage mechanism may differ. We investigated the tissue damage under different excitation conditions by acquiring image sequences of intestinal villi with the focal plane in the apical or basal cytoplasm of the epithelium. Hyperfluorescence, a strong increase of fluorescence intensity, is one obvious and well documented limit for A2P microscopy.^6,19

Here, it was generally observed during the acquisition of image sequences of up to 100 frames at a higher excitation flux (Fig. 5). Bright hyperfluorescence appeared first in small areas which could often be assigned to lysosomes [Fig. 5(b)]. Cells that developed hyperfluorescence were immediately destroyed and the affected area spread fast during further irradiation [Figs. 5(c) and 5(d)]. Only at a lower radiant flux could images be taken over a long time without the development of hyperfluorescence.

The total number of fluorescence photons before the onset of hyperfluorescence was determined as a function of the mean excitation flux. Figure 6 shows measurement values for 10 ROI. Each spot represents one irradiation series similar to that shown in Figure 5. The onset of hyperfluorescence was defined by a rise of the emission signal to a level two times above the starting fluorescence intensity. Green spots in the diagram represent an irradiation series with 300 photons detected without hyperfluorescence. These series set a lower limit for the amount of detectable light.

The onset of hyperfluorescence followed a strongly nonlinear relation. In contrast to linear photochemistry, in which a fixed photon budget of incident light is available for imaging, here higher fluorescence rates drastically decrease the total detectable fluorescence. We observed that 220 fs pulses tuned to a wavelength, where NAD(P)H excitation is efficient (730 nm), could generate more fluorescence photons than ultra-broadband pulses. With 730-nm excitation, 20 photons per pixel and frame could be measured without observing hyperfluorescence [Fig. 6(a)], whereas with ultra-broadband pulses, the detection rate was limited to 10 photons per pixel [Fig. 6(b)]. When irradiation was conducted with 220 fs at 800 nm, hyperfluorescence was observed after less than 100 photons even with a reduced detection rate of only a few photons per pixel [red squares in Fig. 6(a)].

Hyperfluorescence severely limits A2P microscopy. The strong dependence on the excitation rate actually results in a threshold-like behavior, which limits the image quality in each subsequent frame. With $220\mathrm{fs}/730\mathrm{nm}$ excitation, a slightly higher signal-to-noise ratio (SNR) ($\sqrt{20}=4.4$) is achieved than with ultra-broadband pulses (SNR: $\sqrt{10}=3.1$). The lower threshold for hyperfluorescence generation could be caused by the shorter pulse duration and/or by a different spectral overlap of excitation and 2-photon absorption spectra.

Comparison with 220 fs excitation at 800 nm demonstrates the imminent importance of efficient NAD(P)H excitation. Here, the SNR was merely $\sqrt{5}=2.2$. Apparently, longer wavelengths around 800 nm contribute more strongly to the formation of hyperfluorescence than to autofluorescence. Spectral overlap has a much stronger influence than pulse width. The low damage threshold for ultra-broadband pulses is probably caused by the poor spectral overlap with NAD(P)H absorption rather than by the higher peak irradiance. As a consequence, it is not expected that the onset of photodamage can be mitigated simply by increasing the pulse width. It may be better to decrease the long-wavelength content of the excitation, even at the cost of reduced excitation of red-absorbing flavines and lipopigments. We conclude that increasing the fluorescence-to-damage ratio requires optimization of the excitation spectrum.

Fig. 5

Four frames from an imaging series, during which a hyperfluorescent lesion in the apical cytoplasm is formed. Tissue was irradiated at 800 nm with 220 fs pulses. Total radiant exposure for each frame was $1.7\mathrm{kJ}/{\mathrm{cm}}^2$. (a) First frame of the imaging sequence without any visible tissue damage. Lysosomes are visible as small bright spots inside the apical cytoplasm of enterocytes, exhibiting a fluorescence red-shift with respect to cytoplasmatic NADH fluorescence. (b) Onset of hyperfluorescence (white arrow) in frame 15 (radiant exposure $26\mathrm{kJ}/{\mathrm{cm}}^2$) starting from a position where a lysosome was detected before (white arrow). (c) After 45 frames ($77\mathrm{kJ}/{\mathrm{cm}}^2$), hyperfluorescence (white arrows) has started at multiple sites in the epithelium. (d) Hyperfluorescent lesion after the acquisition of 60 frames ($102\mathrm{kJ}/{\mathrm{cm}}^2$). Existing lesions had grown in size and more lesions had developed during continued imaging.

Fig. 6

Total number of detected photons before onset of hyperfluorescence in relation to the fluorescence detection rate for (a) excitation with 220 fs pulses and (b) ultra-broadband pulses. Image sequences of the apical part of the villus epithelium were acquired at different irradiances. The number of detected photons was calculated by multiplying the number of images before the onset of hyperfluorescence with the mean number of photons per pixel. The number of detected photons decreased strongly as fluorescence rate, that is, photons per pixel and frame, increased. Green data points indicate irradiation series, where a total of 300 photons were detected without observation of hyperfluorescence. The inset shows regions of interest in the apical cytoplasm of the villus epithelium that were used to determine the mean fluorescence intensity. The standard deviation shown is the variance between results in the 10 ROIs.

3.3.

Recruitment of Immune Cells into the Epithelium as a Sign of Photodamage

Already at laser powers below the onset of hyperfluorescence, immunological reactions were observed as a first sign of photodamage in the basal part of the epithelium in intestinal villi. Under physiological conditions, the epithelium of small intestinal mucosa contains intraepithelial lymphocytes⁶ but no PMNLs, which include eosinophil and neutrophil granulocytes. Intraepithelial lymphocytes usually reside in the basal part of the epithelium right above the basement membrane and appear dark between the bright enterocytes. This is because lymphocytes have a prominent nucleus which gives no fluorescent signal and only a small rim of cytoplasm.⁶ They move with an approximate speed of $8\mu \mathrm{m}{\min }^{-1}$ and have an average diameter of $6\mu \mathrm{m}$ (Fig. 7, Video 3). Intraepithelial lymphocytes probably play a major protective role against invasion and systemic dissemination of enteric pathogens and commensal bacteria.²⁸ In contrast, PMNLs in the epithelium are only expected under pathological conditions. They are attracted after severe tissue damage, such as that caused by laser-induced cavitation,²⁹ and can be identified in A2P microcopy by size, speed, and the special shape of their nucleus.²⁹ PMNLs, as a sign of sterile inflammation, appeared within the epithelium in the long-time imaging series of the basal part of the epithelium (Figs. 8, 11, Video 4, Video 5, and Video 6) even at radiant flux values below the threshold of hyperfluorescence formation (corresponding to the green spots in Fig. 6). Most of PMNLs show multilobular nuclei and thus probably resemble eosinophil granulocytes, which are abundant in the small intestinal lamina propria beneath the epithelium. They have diameters of $12–15\mu \mathrm{m}$ and move with average speed of $22\mu \mathrm{m}/\min$ between the enterocytes (Figs. 8, 11, Video 4, Video 5, and Video 6). We attribute their appearance to light-induced tissue damage and the release of pro-inflammatory cytokines and damage-associated molecular pattern molecules (DAMPs) by the affected cells.

Fig. 7

Time series of the basal cytoplasm of the epithelium with 220 fs laser pulses covering 14 min. Intraepithelial lymphocytes (red outline) move between the enterocytes (Video 3, MPEG, 3.5 MB) [URL: http://dx.doi.org/10.1117/1.JBO.20.11.116001.3].

Fig. 8

Time series of the basal cytoplasm of the epithelium with 220 fs laser pulses. Within the first 14 min, (a–c) no tissue damage can be detected. After 28 min, (d) polymorphonuclear leucocytes (PMNLs) (red outline) can be detected in the epithelium. (d–f) Many of them show multilobular nuclei (arrow) (d–f). Photodamage of the tissue causes also a loss of autofluorescence and blurred cell borders. The yellow line in the inset in (a) shows the imaged plane for the series (scale bar, $15\mu \mathrm{m}$). (d–f) Video 4 shows the time series presented (Video 4, MPEG, 7.46 MB) [URL: http://dx.doi.org/10.1117/1.JBO.20.11.116001.4].

Recruitment of PMNLs was far less pronounced when the basal part of the small intestinal epithelium was imaged with $220\mathrm{fs}/730\mathrm{nm}$ pulses. In a 100-frame time series of over 14 min, only the movement of intraepithelial lymphocytes above the basement membrane was observed in most of the imaged villi (Fig. 7, Video 3). Only in 17% of the imaged villi was the invasion of PMNLs visible (Fig. 9). Using ultra-broadband pulses for acquiring images of equal quality, PMNLs appeared in 62% of the image series (Fig. 9).

In addition to the 14-min time-lapse series, further image stacks of the irradiated villi were taken at single time points after the end of the image series. Not surprisingly, the PMNLs’ incidence rate further increased with time. Within 1 h after the start of imaging, in all of the volumes recorded with ultra-broadband pulses immigrated granulocytes were visible (Fig. 9). In contrast, when using 220 fs pulses, PMNLs were only observed for 40% of the villi (Fig. 9). Irradiation of the apical cytoplasm of the epithelium seems to less affect the cells.

Here, no PMNLs at all were observed with 200 fs excitation, and they appeared only in 33% of the villi imaged with ultra-broadband pulses (Fig. 9).

When long imaging series in the basal cytoplasm of the epithelium were recorded with ultra-broadband pulses, PMNLs were recruited much faster into the epithelium. They could be observed already moving between the enterocytes after 6 to 8 min of continuous imaging (Fig. 11, Video 5). After 16 min of consistent scanning with ultra-broadband laser pulses, considerably more PMNLs were seen in the epithelium than with 200 fs laser pulses (Figs. 8 and 11). Fluorescence increased when these cells probably degranulated and died (Fig. 11 Video 6). The faster appearance and higher incidence of PMNLs for images with ultra-broadband pulses is consistent with a higher degree of other typical signs of tissue damage, such as decreased autofluorecence, blurring of cell borders, and contraction of villi (Figs. 10 and 12).

Fig. 9

Ratio between the number of image sets containing granulocytes and total number of datasets. Imaging with ultra-broadband pulses triggered the appearance of polymorphonuclear leucocytes (PMNLs) more frequently compared to imaging with 220 fs pulses at the 730 nm excitation wavelength.

Fig. 10

(a, d, g) Stack of optical sections through a villus imaged with 220 fs laser pulses at 730 nm showing the apical cytoplasm, (b, e, h) the section of enterocyte nuclei, and (c, f, i) the lamina propria beneath the epithelium. Within the first 6 min (a–c), the tissue remains visually unaffected by the scanning procedure. After 20 min (d–f), the tissue shows the first signs of photodamage. Epithelial cells show loss of autofluorescence as seen by the darkening of the cells (red outline in a, b, d, e). After 69 min (g–i), the tissue shows massive signs of photodamage, as seen by further darkening of the tissue and blurred cell borders. The villus responds to photodamage with contraction (arrow in i) (scale bar, $15\mu \mathrm{m}$).

While PMNLs are not present in the small intestinal epithelium under physiological conditions, they may invade the tissue in response to several chemical mediators. Tissue damage and cellular necrosis lead to the release of “immune triggers” that are typically hidden in quiescent states. In contrast, the regular frequent cell shedding in the villus epithelium is the result of an orchestrated cell death program and is immunologically silent.

Various mechanisms produce chemical mediators that can recruit granulocytes to inflammatory sites. Intravital imaging with 2-photon microscopy can lead to the formation of singlet oxygen and reactive oxygen species (ROS) when tissue endogenous fluorophores, such as NAD(P)H and FAD, act as photosensitizers in photo-oxidative processes.^30,31 ROS are oxygen-containing molecules with unpaired electrons in their outer orbit, that is, metabolites of oxygen that possess strong oxidizing capabilities. In a healthy organism, they are mainly produced by the mitochondrial respiratory chain. At low concentrations, the ROS function as signaling molecules that regulate cell growth, the adhesion of cells toward other cells, differentiation, and apoptosis.³² An enhanced production of ROS can pose a severe threat to cells and, if not controlled by sophisticated antioxidant machinery, ultimately leads to cell death. Necrotic cell death is followed by the liberation of a plethora of DAMPs which act as chemical mediators and lead to chemotaxis of eosinophil and neutrophil granulocytes to the site of lesion.^33,34 For imaging at the same fluorescence brightness, production of ROS seems to be more pronounced with the ultra-broadband pulses. Many more PMNLs were recruited into the epithelium compared to imaging with 220 fs pulses. Granulocytes are among the first blood-borne cells that travel into damaged organs in which they phagocytose particulate material, such as cell debris. During phagocytosis, however, they also generate ROS. This functional response, termed an oxidative burst, contributes to the host’s defense, but it can also result in collateral damage of host tissue. Thus, these cells are central to both repair and destruction of tissues following sterile injury.^35,36 Degranulation of granulocytes, as seen in A2P microscopy with the ultra-broadband laser pulses, resulted in the generation of highly fluorescent material within the tissue, quiescent PMNLs, and destruction of these immune cells (Figs. 11, 12, Video 6). These highly fluorescent areas are not identical with the described hyperfluorescence, as these areas did not spread during further irradiation and hyperfluorescence was only detected at higher excitation flux.

Invasion of PMNLs into the epithelium has also be observed after UV-A laser nanosurgery of intestinal mucosa, involving bubble formation and localized tissue destruction.²⁹ However, just as during long imaging series with 220 fs laser pulses, no degranulation of PMNLs was observed.

Previous experiments in cell culture with longer pulse widths ranging from 120 to 2000 fs showed no dependence of fluorescence yield before cell damage on excitation pulse length.¹⁶ The results of the present study indicate that this may not hold for ultra-broadband pulses, which excite the autofluorescence in the range from 700 to 900 nm.

Fig. 11

Time series of the basal cytoplasm of the epithelium with ultra-broadband pulses. After 6 to 8 min of repeated scanning, the first PMNLs (red outline) appear in the epithelium (b). Many of them show multilobular nuclei (arrow) (b, Video 5; Video 6) Some PMNLs show very strong fluorescence and seem to degranulate (arrowheads). Invasion of leucocytes and degranulation leads to tissue damage, seen by the loss of autofluorescence in epithelial cells. The yellow line in the inset in (a) shows the imaged plane (scale bar, $15\mu \mathrm{m}$). (a–c) Video 5 shows the time series presented. (d–f) Video 6 shows the time series presented (Video 5, MPEG, 9.21 MB; Video 6, MPEG, 10.8 MB) [URL: http://dx.doi.org/10.1117/1.JBO.20.11.116001.5]. [URL: http://dx.doi.org/10.1117/1.JBO.20.11.116001.6].

Fig. 12

(a, d) Stack of optical sections through a villus with ultra-broadband excitation showing the apical cytoplasm, (b, e) the section of enterocyte nuclei, and (c, f) the lamina propria beneath the epithelium. (a–c) Within the first 6 min, the tissue remains visually unaffected by the scanning procedure. (d–f) After 45 min, the tissue shows massive signs of photodamage. Highly fluorescent material (arrowheads) is visible above the villus (d) and also in the basal cytoplasm, (e) where the time series (Fig. 11) were done. (d, e) Epithelial cells also show loss of autofluorescence as seen by darkening of the cells. The villus responds to photodamage with contraction (arrow in f) (scale bar, $15\mu \mathrm{m}$).