Journal of Photochemistry and Photobiology B: Biology 115 (2012) 42–50

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Pulse duration and energy dependence of photodamage and lethality induced by femtosecond near infrared laser pulses in Drosophila melanogaster Ilyas Saytashev a,1, Sergey N. Arkhipov a,1, Nelson Winkler a, Kristen Zuraski a, Vadim V. Lozovoy a, Marcos Dantus a,b,c,⇑ a b c

Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA Department of Physics and Astronomy, Michigan State University, East Lansing, MI 48824, USA Biophotonic Solutions Inc., 1401 E. Lansing Drive, Suite 112, East Lansing, MI 48823, USA

a r t i c l e

i n f o

Article history: Received 16 March 2012 Received in revised form 22 June 2012 Accepted 25 June 2012 Available online 4 July 2012 Keywords: Ultrafast Multiphoton Phototoxicity DNA damage SHG THG

a b s t r a c t The potential application of nonlinear optical imaging diagnosis and treatment using femtosecond laser pulses in humans accentuates the need for studies carried out in whole organisms instead of single cells or cell cultures. While there is a general consensus that in order to minimize the level of photodamage the excitation power has to be kept as low as possible, it has yet to be determined if shorter pulses have greater benefit than longer pulses. Here we evaluate the rate of death in Drosophila melanogaster as the integral parameter related to photodamage resulting from femtosecond near infrared (NIR) laser irradiation under conditions comparable to those used in two-photon excited fluorescence (TPEF) microscopy. We found that the lethality (resulting from photodamage) as a function of laser energy fluence fits a 3region dose–response curve. The lethality was accompanied with development of necrosis and apoptosis in irradiated tissues. Quantitative analysis showed that the damage has a mostly linear character on energy fluence per pulse, and for a given TPEF signal, shorter (37 fs) pulse duration results in lower lethality than longer (100 fs) pulse duration. These results have important implications for the use of femtosecond NIR laser pulses in microscopy as well as in vivo medical imaging. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Photodamage is known to be a major concern in laser scanning imaging of living biological samples via multiphoton microscopy [1,2]. Multiphoton imaging systems are capable of directly imaging tissues through nonlinearly excited fluorescence and second harmonic signals [3]. The use of near-infrared (NIR) femtosecond pulses can provide high resolution images of tissue morphology even without the use of contrast reagents [4]. Using focused ultrafast laser pulses in the NIR also makes it is possible to reach deeper tissue layers compared to UV light irradiation [5]. However, the photodamage that results from using laser irradiation has been an outstanding concern in regard to sample perturbation for in vivo imaging, especially for purposes of medical diagnostics [6,7]. There are a number of nonlinear multiphoton optical process contributors that may lead to phototoxicity in cells [2,8,9], including the generation of reactive oxygen species [9,10] and free radicals [11,12], direct DNA damage [12–14], and plasma formation

⇑ Corresponding author at: Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA. Tel.: +1 517 355 9715x314; fax: +1 517 353 3023. E-mail address: [email protected] (M. Dantus). 1 These authors contributed equally to this work. 1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2012.06.009

[11,15]. Linear photodamage from heating due to infrared laser irradiation [16–18] is a problem that is particularly detrimental to pigment-rich and highly-vascularized tissues [7,18]. The availability of a wide variety of commercial femtosecond laser sources prompts the question of optimal energy parameters for safer imaging of living tissues. Due to the complexity of evaluating the short and long term effects of laser irradiation on living tissues, the ideal approach for determination of safer in vivo imaging conditions is still in debate. Adequate and reliable detection of laserinduced photodamage is an important part of the solution to this problem and numerous evaluation methods have been developed to target such damage in living tissues. Most of these methods are mechanism-specific techniques and include: tracking DNA damage by detecting DNA strand breaks [11,12,19,20] and DNA repair proteins [12,14], level of apoptosis [21], mutagenicity risks [4], etc.; observing oxidative stress and mitochondrial dysfunction thru monitoring of reactive oxygen species [4], H2O2 [22], or NADH [17,23,24]; and checking for changes in functional activity of the cells by scoring the resting [Ca2+] level [2,23] or cell viability (cloning efficiency) [6,9,25]. It is also known that a small increase in local temperature of even a few degrees centigrade may induce significant damage in living tissues. For example, viability of human adipocytes dropped from 89% to 20% when subcutaneous temperature increased from

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45 to 50 °C during a 1 min exposure, while only 40% of viability was lost during three minutes of exposure at 45 °C [26]. This thermal damage consists of two stages, both affecting cell injury/necrosis. The first stage is due to heat-induced protein denaturation and cell death, and the second is the result of an intense inflammatory reaction, which, due to increased vascular permeability, causes burn shock [27–29]. This can eventually lead to a disruption of intracellular ion homeostasis and cell/nuclear membranes, cell swelling, and release of injurious lysosomal enzymes and inflammatory messenger molecules. Endogenous self-adjuvant (also termed as damage-associated molecular patterns) released during necrotic cell death could also induce sterile inflammation and immune responses [30]. The second major mechanism of photodamage induced by the laser pulses is DNA damage [12,13,31,32]. Apoptosis is a prominent route of cell death following the introduction of DNA damage [33,34], and it has been shown that DNA strand breaks may trigger apoptosis [33]. Various photochemical processes create free radicals and in particular reactive oxygen species (ROS) [34-38]. DNA damage may occur directly through three-photon excitation [39] or through reaction with ROS [12]. Apoptosis, therefore, is a result of direct and indirect DNA damage. Another important aspect in the optimization of in vivo imaging is determining the physical parameters for laser irradiation using a dose–response relationship with photodamage. In experiments on Chinese hamster ovarian (CHO) cells by König et al. [9], it was shown that damage to the cell (cell vitality) from NIR excitation radiation depends on pulse duration (P2/s dependence, where P is a mean power) in the range from 120 fs (170 fs at the sample) to 1000 fs. Here we explore the incidence of death in developing Drosophila melanogaster (irradiated in the larva stage) as a result of photodamage and determine its dependence on laser parameters for 100 fs and 37 fs pulses with a Gaussian shape spectrum of 26 nm bandwidth centered at 800 nm. The body of Drosophila larva (insect) consists of various tissues presenting in mammalian organisms. It has been known that NAD(P)H and flavin compounds within the skeletal muscle tissue can be simultaneously excited with the laser pulses at 800 nm [40]. Similarly, lipoamide dehydrogenase (LiDH) and flavin adenine dinucleotide (FAD) can be efficiently excited at the same wavelength by one and two-photon mechanisms [41]. We score the levels of necrosis and apoptosis induced by the laser in the larvae as additional parameters to determine possible mechanisms involved in the observed deaths. Mathematical modeling is used to analyze the experimental data. A sigmoidal dose–response function is found to fit our data and allows us to determine three regions. The first region, where there is no statistical difference between irradiated larva and control (not irradiated); the second region where death increases with laser energy, and a third region where the majority of the larvae die. We identify the second region as the region of interest for nonlinear optical imaging, because greater pulse energy causes much brighter images. The goal of our project, within the context of the second region, is to determine if longer or shorter pulses are better (brighter images for a given amount of photodamage) for nonlinear optical microscopy such as TPEF.

2. Materials and methods 2.1. Drosophila culture A wild type (WT) strain of D. melanogaster was used in this study. The flies were grown at room temperature in culture vials with instant Drosophila fly culture media (Carolina Biological Supply Company, Burlington, NC) and prepared as described in the

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manufacturer’s manual. Experiments were performed on third instar larvae collected from the upper part of the medium in the vial and rinsed with distillated water.

2.2. Irradiation of living larvae and scoring lethality Sample chambers made of silicon polymer were created in order to irradiate the larvae. These were made by combining a curing agent and Elastomer Base of SylgardÒ 184 Silicone Elastomer Kit (Dow Corning Corp, Midland, MI) in a 1:10 ratio and then thoroughly mixing. This solution was poured over a 35  10 mm Petri dish (Becton Dickinson, Franklin, NJ), containing a metal cylinder with a 1.9 mm diameter and a height of 9.2 mm placed at the center, and allowed to crystallize for 48 h. The silicone slab was then extracted from the Petri dish using a plastic or wooden stick and the metal cylinder was removed, creating a precise cylindrical channel. A schematic diagram of the chamber is shown in Fig. 1. Experiments were carried out on three groups of larvae, according to laser exposure (short pulses, long pulses, and no irradiation). To expose the groups to the laser pulses, larvae were extracted from the vials in small amounts of media and gently rinsed with distilled water. Using a 7-mL transfer pipette (USA Scientific, Ocala, FL), two of the freshly harvested larvae, in a drop of water, were then transferred into the silicon chamber that laid on a folded piece of KimwipesÒ paper (Kimberly-Clark Inc., Roswell, GA). Once transferred, the chamber with the two larvae was placed onto the lid of the Petri dish, and the excess water in the chamber’s channel was drained off with a twisted piece of Kimwipe. The chamber with the larvae was then covered with cover glass No. 1 (Corning Inc., Corning, NY) and used for irradiation under the laser beam for a given pulse duration and energy fluence. The larvae were irradiated for 10 min in the setup shown in Fig. 1. To ensure homogeneous irradiation of the entire larva body, laser parameters were adjusted to mimic the total energy fluence and peak intensity (several PW/m2) that is used for typical twophoton fluorescence imaging (20, 25). The relevant parameters are given in Table 1. Our setup used a regenerative amplified femtosecond laser system with Ti:Sapphire oscillator (KapteynMurnane Laboratories, Inc., Boulder, CO) and Spitfire amplifier (Spectra-Physics, Newport corp., Irvine, CA) that was compressed and shaped by a MIIPS Box 640 (Biophotonic Solutions Inc., East Lansing, MI). The laser pulses, centered at 800 nm, with a Gaussian shape spectrum of 26 nm bandwidth were corrected to produce maximum output power with 1 kHz repetition rate. The MIIPS software was used to obtain either transform-limited pulses with time duration of 37 fs or linearly chirped pulses (1290 fs2) with pulse duration of 100 fs. No difference was found for positive or negative linear chirp. In order to adjust the energy fluence of the pulses, the beam diameter was changed by moving the focusing lens (See Fig. 1, L2). In all cases, the beam diameter was greater than the irradiation chamber, ensuring that the entire larvae were fully bathed by the laser beam without the need for scanning. After irradiation the larvae were gently flushed out of the chamber into a Petri dish with a pipette and transferred into a vial with freshly prepared fly culture media. This procedure was repeated 4 more times (five runs in total) to obtain 10 larvae irradiated in the same conditions for a given day. The larvae were put in the same vial (which was incubated at a temperature of 21–22 °C) and were counted for survival every other day for a total of 15 days. In order to obtain each individual experimental group of larvae, this irradiation procedure was performed over several days (n = 50–70 in 5–7 experiments). Only groups exposed to the laser pulses with high energy fluence resulted in a high early death rate (up to 100%) which reduced the number of surviving larvae (n = 20) for the higher energy group. Control experiments for larva that were not

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Fig. 1. Schematic diagram for irradiating larvae M1 is a flip mirror to change the beam path between the characterization arm and irradiation of the sample; M2 is a mirror used to direct the laser vertically onto the sample and through a hole in the table; L1 and L2 are F = 1000 mm lenses; C.S. is a cover slip; F1 is a blue filter; and F2 is a neutral density filter. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1 Comparison of parameters of two different laser setups used in experiments for irradiation and multiphoton imaging for TUNEL. Setup parameters

Amplified setup

Laser scanning microscopy

Peak intensity Number of pulses at dwell, N Total fluence of laser energy Expected two-photon signal per pulse, E2/s Total two-photon signal, N  E2/s

1.12  1015 W/m2 6.0  105 2.6  107 J/m2 4.91  1016 s  W2/m4 2.95  1022 s  W2/m4

20.33  1015 W/m2 9.2  103 3.4  106 J/m2 7.44  1018 s  W2/ m4 6.81  1022 s  W2/ m4

irradiated were also performed and scored (n = 140, 14 experiments). Three parameters of lethality were calculated based on the data obtained from these experiments. Total lethality was determined by calculating the difference between the number of larvae before irradiation and the number of flies that developed, divided by the number of larvae before irradiation. In contrast, early (larva) lethality was considered to be the number of larvae before irradiation minus the number of pupae that survived, divided by number of larvae before irradiation. This parameter was used to characterize the percentage of deaths during the third instar larvae stage of development that occurred prior to the development of pupae. The delayed deaths of pupae were also calculated by taking the difference between the number of pupae and flies, divided by the number of pupae that survived. This measurement was used to evaluate the deaths that resulted during the pupae stage. To score the relative impact of the early (larva) and delayed (pupa) development stages on total lethality in population, we calculated the contribution of each stages’ lethality (%) to the total lethality observed for each experimental and control group. Averages with standard errors for all parameters were also calculated for each group. Finally, the significance of difference (P < 0.05) between experimental and control groups were scored using a two-tailed Mann–Whitney U-test.

2.4. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) The ability of laser pulses with various durations and intensities to induce apoptosis in larvae was scored by measuring the total brightness of TUNEL-positive cells in salivary glands superficially located in the larva’s body. Six or more experiments for each laser treatment condition were done over several days. The laser treatment was performed as described previously, and the larvae were incubated in Drosophila fly media (Carolina Biological Supply Company, Burlington, NC) for 3 h after irradiation. After incubation the salivary glands and imaginal tissues were collected from dissected larvae then fixed and stained for DNA fragmentation (TUNEL-assay) according to the protocol for visualization of apoptosis [42] with the modifications given in the supplemental material. 2.5. Imaging of irradiated tissues fixed and stained with TUNEL The images of the fixed, TUNEL-stained salivary glands and imaginal discs were acquired using an inverted Eclipse TE-2000 (Nikon, Japan) microscope equipped for multiphoton imaging with a water-immersion LD C-Apochromat 40/1.1W Corr Objective (Carl Zeiss, Germany), Ti:Sapphire Laser (Kapteyn-Murnane Laboratories, Inc., Boulder, CO), pulse shaper with MIIPS adaptive pulse compression, galvanic scanner QuantumDrive-1500 (Nutfield Technology, Inc.), dichroic mirror, shortpass emission filter (both from Chroma Technology Corp.), prism compensator, and photomultiplier HC120-05MOD Hamamatsu. The average power of the excitation beam was measured with a FieldMaxII-TOP power meter (Coherent, Santa Clara, CA) to be 20 mW after the objective; prior to imaging, pulses were compressed to be close to transform limited and pulse duration was measured 19 fs after the objective using the pulse shaper. LabVIEW 7.1 software (National Instruments) developed in our lab was used to acquire images of the peripheral regions from the salivary glands, 512 pixels  512 pixels in size, with a horizontal resolution of 0.25 lm/pixel. The final images produced two signals: (1) Autofluorescence from all of the tissue and (2) a signal from the fluorescent antibody label in specific binding sites.

2.3. Imaging of irradiated larvae 2.6. Image processing for TUNEL-assay A digital camera Canon t2i with a Canon MP-E65 mm f/2.8 1–5 Macro-lens set at 65 mm and attached flash was used to image some of the irradiated larvae in order to document the visible changes in appearance following irradiation. The larvae were anesthetized with FlyNap (Carolina Biological Supply Company, Burlington, NC) for 10–15 min prior to imaging. The images were acquired with an exposure time of 5 ms, an aperture of f/16.0, and an ISO value of 400 approximately 5–20 min after irradiation as well as 3 h later.

In total, 80 SHG-images for each one-section view were averaged and transformed into 8-bit format to generate each final image using the program ImageJ (National Institute of Mental Health, Bethesda, MD). Adobe Photoshop 12.04 (Adobe Systems Inc.) was then used for further adjustments of the brightness in the final images. Those images containing fluorescent antibody labeling for DNA fragmentation were used for apoptosis analysis. The portion of cells with DNA fragmentation (TUNEL-positive cells, which

I. Saytashev et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 42–50

are much brighter than other cells) in the images was calculated from a ratio of the area of these cells to the total area of the salivary gland in the image by selecting and measuring these regions using ImageJ. With the same software, we are able to measure the average fluorescence (brightness) of any selected region of the image. Brightness characterizes the average level of fluorescence signal from an imaged area collected on the photomultiplier tube and expressed in arbitrary units. Relative brightness of TUNEL-positive cells was calculated as a ratio of the average fluorescence signal of these cells to the average fluorescence of the surrounding TUNEL-negative cells. To characterize the intensity of apoptosis in the samples we calculated a parameter (apoptosis intensity), which was the product of a portion of the TUNEL-positive cells and their relative brightness. To score the significance of differences between the groups a two-tailed Mann–Whitney U-test was applied, where a probability of P < 0.05 was considered as significant. Values of the presented parameters are given as the mean ± standard error of mean. 3. Mathematical modeling of lethality and n-photon signal relationship (Theory) 3.1. Lethality as a function of n-photon signal Lethality as a function of energy fluence per pulse and pulse duration can be described by the dose–response relationship [43] as modified for our case:

LðEp ; sÞ ¼ C þ

1C 1 þ 10 

ð1Þ

Ep Em ðsÞ E s ðsÞ

where L is the lethality fraction, Ep is the energy fluence per pulse (J/ m2), s is the pulse duration, C is the lethality fraction of the control group, Em is the energy dose required to kill half the population proportional to the equation, ((1  C)/2 + C), and Es is the energy sensitivity characteristic of the organism. The normalized nonlinear (average order n) signal, S(n), can be expressed as,

SðnÞ ¼ Enp =sn1

ð2Þ

The lethality proportional signal can be derived by combining Eqs. (1) and (2). When we limit the equation to two-photon induced emission, we obtain the relation,

LðSð2Þ ; sÞ ¼ C þ

1C pffiffiffiffiffiffiffiffiffiffi ð2Þ m ðsÞ 1 þ 10  S Es ðssE Þ

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excited with an NIR femtosecond laser at 800 nm can produce two-photon fluorescence signal [40,41]. Using a laser scanning microscopy setup and NIR femtosecond laser pulses on Drosophila larvae (unpublished data) we found that the maximum emission signal from spectrally resolved images corresponded to autofluorescence in the 430–520 nm range, while emission at 400 nm (where SHG signal would be found) was minimal. 4. Results 4.1. Observation of necrotic changes in irradiated tissues When using high energy fluence (115 J/m2), necrotic changes in the irradiated larvae appeared in the same or next day in addition to swelling of the body, dark brown/black spots alongside the larva, dark ‘‘lines’’ inside the body on nerves and vessels, and partial darkening of the trachea and surrounding tissues (Figs. 2 and 3). The necrotic spots became darker and wider over the course of 3 days (maximum life remaining for necrotic larvae) for the larvae that did not die on the first day. Fig. 4 may reflect a possible difference between 37 and 100 fs pulses in their ability to induce necrosis. Abnormalities of the necrotic larva’s mobility that prevented them from moving and feeding indicates that the damage may have affected the central and peripheral nervous systems and (or) the neuromuscular apparatus in these larvae. This damage is considered necrotic due to the character of visible changes inside of the affected larvae, which accompanied the impaired mobility and unusual behavior. 4.2. Apoptosis in populations of irradiated and control larvae The ability of femtosecond NIR laser pulses to induce apoptosis was scored by measuring a portion of TUNEL-positive salivary gland cells and apoptosis intensity in the larvae irradiated with 42 J/m2 (37 fs and 100 fs pulses), 86 J/m2 (37 fs pulses) and 115 J/ m2 (100 fs pulses). The tissues were imaged by multiphoton microscopy; a portion of the measurements is shown in Fig. 5, together with a plot of the results. When the non-irradiated (control) samples (n = 17) were analyzed, we found 7.9 ± 0.6% of cells to be

ð3Þ

In the intermediate region of the dose–response curve, we assume an l-order process is the primary cause of death. By repeating the experiment for two pulse durations, we obtain:

El1 =sl1 ¼ El2 =sl1 1 2

ð4Þ

We solve for l in this equation by taking the natural logarithm to obtain:

l¼

lnðs2 =s1 Þ   ln EE12 ss21

ð5Þ

Using Eqs. (2) and (4) (calculations are given in supplemental material): ðnÞ

n

S ðsÞ ¼ ðEðsÞÞ =s

n1

¼

E1

s1 l1l

!n n

=s l 1

ð6Þ

Eq. (6) gives the dependence of signal as a function of energy fluence and pulse duration for an n-photon process. Both two-photon and SHG signal have an n = 2 dependence. Living tissues

Fig. 2. An example of necrotic changes (tracheal darkening) induced by femtosecond laser pulses in Drosophila larva. Photographs of the control larva and the larvae irradiated for a duration of 10 min with 100 fs NIR pulses with energy fluence 115 J/ m2. The image of the irradiated larva was acquired 5 min after treatment with laser pulses. The presented treated larva has necrotic darkening of one trachea (identified by white arrows), which appeared after the laser treatment. The permanently contracted body of the irradiated larva lost its natural glitter and transparence.

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Fig. 3. Necrotic spots induced by femtosecond laser pulses in Drosophila larva. Two photographs of another larva irradiated for a duration of 10 min with 100 fs NIR pulses with energy fluence 115 J/m2. The first (top) image was acquired 18 min after treatment, the second (bottom) – 3 h after irradiation. Several small necrotic (brown and black) spots, which appeared after laser treatment and became more visible with time, are indicated by white arrows. The irradiated larva also lost transparence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Percentage of larvae population with necrotic changes after irradiation with 37 fs (red scale bars) and 100 fs (blue scale bars) pulses. 60–70 larvae were used for each group. No necrotic effects were seen in the controls or the larvae irradiated with NIR femtosecond pulses with energy fluence of 27 J/m2 (37 fs). Comparison of the level of deaths with the numbers of larvae exhibiting necrotic changes displays that the level of necrosis had higher growth with increasing energy fluence for shorter pulses. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

apoptotic, and the apoptosis intensity was 0.59 ± 0.10 a.u. Larvae treated with laser pulses of relatively low energies (42 J/m2) had similar amounts of TUNEL-stained cells: 8.8 ± 1.0% (37 fs) and 9.7 ± 0.7% (100 fs) and the similar apoptosis intensity: 0.63 ± 0.10 a.u. and 0.76 ± 0.10 a.u., respectively. These values did not differ significantly from the control larvae in a two-tailed U-test (P > 0.05). Irradiation with 37 fs- and 100 fs pulses with higher energy fluence (86 J/m2 and 115 J/m2, respectively) increased the number of apoptotic cells in the salivary glands by 13.5 ± 0.9% and 14.1 ± 1.4%, respectively, (P < 0.0001 for both) and the apoptosis intensity (1.4 ± 0.3 a.u., P < 0.01 and 2.30 ± 0.4 a.u., P < 0.0001, two-tailed U-test), with values significantly greater than observed in the control. 4.3. Quantitative analysis of lethality as a function of energy fluence The use of mathematical modeling allowed us to analyze the dependence of total lethality in populations of larvae irradiated

Fig. 5. TUNEL-analysis of irradiated and control larvae. (A) Microphotographs of the salivary glands from Drosophila larvae exposed for 10 min to laser femtosecond pulses (37 and 100 fs) and various energy fluence (42, 86 and 115 J/m2). Irradiated larvae were allowed to develop an apoptotic response over the course of 3 h and were then dissected to obtain salivary glands, which were processed for the TUNELassay. Control larvae were treated in the same manner but were not exposed any laser pulses. Microphotographs were obtained using the multiphoton imaging system, which provided autofluorescence signal from unstained regions of imaged tissues (mostly cellular membranes) as well as a TUNEL-specific signal from positive cells (cytoplasm and nuclei). Each row consists of 3 representative singlesection images of salivary glands from larvae treated under the same conditions. The greatest intensity of TUNEL-staining was observed in cells obtained from larvae irradiated with 100 fs 115 J/m2 pulses. In samples from larvae irradiated with 37 and 100 fs pulses with energy fluence 42 J/m2 the apoptosis intensity (a result of multiplication of the portion of TUNEL-positive cells by their relative brightness) was similar to the level in the control samples. White scale bar is 20 lm. (B) Bars represent apoptotic intensities in the groups of control and irradiated larvae. Values are mean ± SE of at least six independent experiments. The P values for experimental groups were obtained by comparison with the control group: P < 0.01,   P < 0.0001 (two-tailed U-Test).

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with shorter and longer femtosecond pulses as a function of energy fluence (Fig. 6). Experimental data are fit using the dose–response function as given in Eq. (1). The fitting parameters for the two different pulse durations are given in Table 2. For both curves the adjusted R-squared coefficients are close to 1.0. This shows that the dose–response function is adequate for modeling the experimental data on the dependence of lethality on energy fluence. Three regions can be identified on the curves. In the first region (energy density < 50 J/m2), the experimentally observed total lethality after irradiation (<20%) is not significantly higher than that observed in the control. In the second region (50–120 J/m2), the deaths increase from 27–30% to 70–73%, and in the third region (>120 J/m2) lethality approaches 95–100%. We note that the two functions describing the dependence of lethality on the energy fluence for shorter and longer femtosecond pulses are very similar. The value for Em, or energy required to kill half of the population, is (76 ± 1.5) J/m2 and (94 ± 2.1) J/m2 for shorter and longer pulses respectively. Laser parameters used for irradiation are chosen to be as similar as possible to conditions used during typical multiphoton imaging (see Table 1). For this purpose we have developed a theoretical model of imaging based on using amplified femtosecond pulses (see Supplemental materials). This model includes the theoretical analysis and calculation of the necessary parameters for 1 kHz amplified laser pulsed irradiation in order to match the two-photon emission yield (or imaging signal) of conventional two-photon microscopy. To estimate the possible photodamage during multiphoton imaging due to illumination with shorter and longer femtosecond pulses, we compare the measured photodamage (estimated from observed lethality) with the calculated two-photon signal resulting from the same energy fluence. As seen on Fig. 7, irradiation with shorter (37 fs) and longer (100 fs) pulses of the same energy fluence causes different levels of photodamage (calculated based on the fitting parameters in Table 2). The same photodamage (lethality) results from treatment with shorter or longer pulses of slightly different levels of energy (lower and higher respectively) (Fig. 7A). However, a much higher two-photon signal for imaging is realized by illumination with shorter pulses in comparison with longer pulses of the same or higher energy fluence (Fig. 7B).

Fig. 6. Total lethality as a function of energy fluence per pulse for 37 fs and 100 fs pulses. Lethality (mean ± SE) was scored in 6–7 independent experiments of each energy condition for 37 fs pulses (red triangles) and 100 fs pulses (blue squares). Results of fitting for 37 fs and 100 fs pulses are outlined with red solid and blue dash lines, respectively. In both cases, the lethality/energy dependence fits a sigmoid-shape, 3-region curve with a dose–response relationship. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 2 Parameters of logistic functions for fitting of experimental data. Pulse duration

C

Em (J/m2)

Es (J/m2)

Adj. R2

37 fs 100 fs

0.08571 0.08571

75.9 93.5

38.7 52.9

0.99917 0.99934

The difference between longer and shorter pulses in terms of expected TPEF signal and lethality becomes clearer by plotting lethality versus two-photon signal, as shown in Fig. 8. It shows that for a given two-photon signal, the shorter (37 fs) pulses cause less damage (lethality) than the longer (100 fs) pulses. This indicates that shorter pulses are a better choice for use in multiphoton imaging than longer pulses. This conclusion is based on the range of energies studied here, the wavelength of the excitation pulses (800 nm) and the fact that whole organisms were irradiated. Relying on our experimental data for laser-induced lethality (considered as a result of photodamage) in Region II (Fig. 6) and using Eq. (5), we estimated the nonlinearity order of the process. We based our calculation on the fitting parameters used to model our experimental data. The average calculated nonlinearity coefficient, l, was 1.28 ± 0.05, while a calculation based on our experimental data without fitting gave l = 1.19 ± 0.2. Values of l < 2 imply that shorter pulses are better, while values of l > 2 would imply that longer pulses are preferable for two-photon microscopy. For third-harmonic generation or three photon excitation microscopy, shorter pulses are always better, given the higher nonlinearity of the excitation process. Using Eq. (6), we estimated two-photon signals for a constant level of total lethality (54%) with different possible orders of

Fig. 7. Comparison of two photon signals for different pulse durations. For a 54% lethality level, the two-photon signal is shown as bars. Conventions are as in the previous figure. (A) The result of fitting the experimental data on lethality. (B) Calculated two-photon signal for two different pulse durations. As seen from the figure, for the same given lethality level two-photon signal is greater for shorter pulses.

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Fig. 8. Total lethality as a function of two-photon signal. Conventions are as in the previous figures.

photodamage processes: 1, 1.28, 2 and 2.5 (Fig. 9). As previously stated, l = 1.28 is closest to the order found from our experimental data on lethality within region II. In the same graph, a one-photon linear process of photodamage with l = 1; one with l = 2 and a final one with l = 2.5 are plotted. As seen in Fig. 9, if photodamage has a (l = 2) order, then for a fixed photodamage level of (54%) one observes that the expected two-photon signal becomes independent of pulse duration. This is because pulse fluence would need to be adjusted in order to maintain the fixed photodamage level. On the other hand, for nonlinear processes with (l < 2) two-photon signal is inversely proportionality to pulse duration. This indicates that shorter femtosecond pulses, in comparison with longer pulses, provide greater signal (for a fixed acceptable level of photodamage). The situation reversed if the damaging mechanism is highly nonlinear, for example three-photon DNA damage. For (l > 2) processes, longer pulses create more signal than shorter pulses (based on a fixed acceptable level of photodamage). Fig. 9 includes two bars that correspond to our observed experimental results. 5. Discussion This study uses D. melanogaster as a model organism to estimate the photodamage caused by femtosecond NIR laser pulses with

Fig. 9. Two-photon signal as a function of pulse duration for a total lethality equal to 54%. Total lethality in a population of Drosophila melanogaster larvae is modeled as 1.28 photon process (red solid curve); one-photon process (black dashed curve) and 2.5 photon process (green dotted curve). Two-photon signal for the pulse duration 37 fs is normalized to 1. For 37 fs and 100 fs data, estimated two-photon signal is shown as bars. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

energy fluence ranging from 20 to 240 J/m2. Numerical analysis of the total percentage of deaths in the larva populations induced by laser radiation allows us to make conclusions about the dynamics of photodamage. We found that observed photodamage can be fit to a sigmoid-shape, 3-region curve with a dose–response relationship as a function of energy fluence. We consider the necrotic changes accompanying the early deaths in our experiments to be the result of primary and secondary necrotic processes induced by thermal injury from the femtosecond NIR laser irradiation. Counting the number of necrotized larvae in the population showed that the contribution of necrosis on survival had positive dynamics. In the middle region of applied energy fluence (50–120 J/m2), 37 fs pulses produced more larvae with visible necrosis than 100 fs pulses (Fig. 4). This difference disappeared when the total number of deaths as well as the level of necrosis in population approached terminal levels (100%). In contrast, the delayed (pupa) deaths are considered to be the result of mostly secondary necrotic processes that follow the thermal or mechanical injuries triggered by the laser treatment. Based on the nature of secondary necrosis, the possible mechanism of delayed death can be due to induction of acute inflammation and immune response to denatured proteins in necrotic tissue. This would be resultant of the overproduction of inflammatory cytokines/ mediators, which provide toxic/damaging effects on the body. These delayed deaths are less likely to be associated with DNA damage after the laser treatment, however, as non-reparable DNA damage in the genome should prevent survival of the irradiated larvae and continuation to the next stage in development. The use of the TUNEL-assay for scoring apoptosis in the cells of salivary glands from larvae exposed to the laser provides evidence that pulses within our evaluated range of duration and energy may induce apoptosis in the irradiated tissues. We found a significant increase in the number of apoptotic cells in those larvae irradiated with energy fluence near 100 J/m2, while the observed level of apoptosis was not significantly different from that of the control when the applied energy fluence was <50 J/m2. Comparison between the percentage of deaths and the number of larva having necrotic changes provides evidence as to the sensitivity and accuracy of the two methods performed. It was determined that scoring the necrosis by counting the necrotic larvae in population was less effective than scoring the number of deaths in each stage. It is possible that the level of necrosis was underestimated when analyzing the larva, especially when low energy fluence pulses were used for irradiation. When relying on the approximate scoring of necrosis for 115 J/m2 (100 fs) and 86 J/m2 (37 fs) pulses, we estimate the contribution of necrosis to the total number of deaths in the population to be as much as 40% and 46% respectively. However, again, this level could be significantly underestimated. To improve this analysis, the use of a more sensitive cellular assay for detection, such as nuclear staining of highmobility group box 1 (HMGB1) [44] would be suitable and more accurate for future experiments. Another aspect of our study is the relationship between necrosis and apoptosis in damaged tissues. Using mammalian models of acute dermal burn injury, it has been found that thermal damage generally inhibits apoptosis [44,45], while intensity of necrosis increases [44]. Due to cell death (apoptosis, necrosis) and inflammation/immune response being inextricably linked with their effectors modulating other processes [30], extrapolation of this mechanism on invertebrate organisms is possible. We suggest that this effect could exist in the irradiated larvae. Indeed, just a small increase in the number of apoptotic cells in our experiments could be a result of such inhibition. It should be considered that the apoptosis scored by us here could be partially from the primary necrotic process induced by the irradiation. However, due to the activity of mediators seen during the development of primary

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and secondary necrosis, we assume the presence of secondary apoptosis in the irradiated tissues observed later to be a potential contributor of the process leading to the delayed deaths. Our study differs from others in the literature in that the time scale of irradiation was 10 min in our experiments versus 10– 40 ls performed in previous studies [ref], however the two-photon signal level is comparable to that of typical two-photon microscopy experiments. Also notable is that in our study we tracked lethality for 14 days after irradiation; therefore being sensitive to damage that was not evident during the first minutes following laser exposure. Combining our experimental data with mathematical modeling showed evidence that for a given two-photon signal the lethality proportional to photodamage with 100 fs pulses is higher than that from 37 fs pulses. Moreover, calculations based on the dose–response fitting of experimental data and the comparison of experimental points gave similar levels of average nonlinearity values to be 1.2–1.3. This shows that damage manifested as death in the population has mostly linear character. The observation implies that most of the damage is thermal (l = 1) with some small fraction having a higher order (possible l = 2 or 3). Our result seemengly contradicts previous studies concerning damaging effects of femtosecond pulses [2], where it was found that damage caused by femtosecond pulses scales as a 2.5 order process. The difference in the nonlinear exponent found in this study versus the previous finding is likely associated with using different biological models, parameters, and observation time to study the effects of photodamage. Our results are in agreement with the study by Rockwell’s group on the effects of laser pulse duration on damaging the retina, which were instrumental in the determination of laser safety standards [46]. Our conclusion regarding the near linearity of photodamage has been indirectly confirmed by another study carried out on a skin tissue [47]. Masters and co-workers showed that decreasing average laser power while maintaining same peak power decreases thermal mechanical damage on skin tissue. Our findings can be placed in the context of imaging using different excitation wavelengths. Pulses with shorter wavelengths (500–800 nm) will cause greater linear absorption in endogenous compounds such as melanin and hemoglobin and hence cause a greater frequency of cell death. Longer wavelengths, in the 1–2 lm range, which have the advantage of greatly reduced scattering, fall in a spectral regions where water absorption becomes significant. Newer microscopy modalities such as third harmonic generation and three-photon excitation fluorescence typically involve the use of longer wavelength pulses. In these cases, signal scales as the inverse pulse duration squared, and therefore a reduction in pulse duration from 100 fs to 10 fs results in two-orders of magnitude greater signal.

6. Conclusion The whole-organism model of photodamage used has several advantages over cell culture models. One advantage being that it allows us to evaluate the effects of laser pulses in a model that more closely reproduces the irradiation effects that may occur during nonlinear optical imaging being developed for in vivo diagnosis in humans. Our results are well represented by a dose–response function. We found that the middle region, where gains in image brightness result in photodamage, shows a linear dependence of lethality as a function of energy fluence. The observed lethality is a result of cumulative photodamage at the level of the whole organism. The major result of our current work provides evidence that for a given two-photon signal intensity, the lethality produced by irradiation with longer pulses is higher than the lethality

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induced by irradiation with shorter pulses. For a given pulse energy shorter pulses lead to brighter images. Our results imply that thermal damage is a major concern in nonlinear imaging applications; therefore, shorter pulses will lead to lower thermal damage and brighter nonlinear signals independent of excitation wavelength. Acknowledgments We graciously thank Dr. Chuck Elzinga from Michigan State University for his donation of the D. melanogaster flies. We are grateful to Christine Kalcic for critically reading the manuscript. This research was made possible by Grant Number R21EB8843 from the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health.

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