1 September 2008 Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy
Author Affiliations +
J. of Biomedical Optics, 13(5), 050502 (2008). doi:10.1117/1.2981817
The fruit fly Drosophila melanogaster is one of the most valuable organisms in studying genetics and developmental biology. To gain insight into Drosophila development, we successfully acquired label-free, in vivo images of both developing muscles and internal organs in a stage 2 larva using the minimally invasive imaging modality of multiphoton autofuorescence (MAF) and second harmonic generation (SHG) microscopy. We found that although MAF is useful in identifying structures such as the digestive system, trachea, and intestinal track, it is the SHG signal that allowed the investigation of the muscular architecture within the developing larva. Our results suggest that multiphoton microscopy is a powerful in vivo, label-free imaging technique to examine Drosophila physiology and may be used for developmental studies.
Lin, Hovhannisyan, Wu, Lin, Chen, Lin, and Dong: Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy



Near-infrared (NIR) multiphoton microscopy has become the preferred tool of choice for microscopic imaging of biological specimens. A number of unique applications, such as biomedical diagnostics, initiation of photochemical reactions, and nanoprocessing within living cells/tissues, can be achieved with NIR illumination.1 In biomedical imaging, the noninvasive and highly penetrative multiphoton microscopy with spatial resolution of <500nm has the potential of offering new insight into morphological and developmental studies in vivo. Specifically, multiphoton imaging allows submicrometer three-dimensional (3-D) resolution, millimeter-penetration depth, and minimally invasive nature.2, 3

Drosophila melanogaster is one of the most studied model organisms in biological research, particularly in genetics and developmental biology. Moreover, 61% of known human disease genes have recognizable orthologs in the Drosophila genome, and 50% of fly protein sequences have mammalian analogs.4 Because of the similarity between human and fly proteome, Drosophila is a useful organism for studying mechanisms underlying immunity disorders, diabetes, cancer, and even psychiatry problems, such as drug abuse. In the past, multiphoton microscopy has been successfully applied in the imaging of Caernorhabditis elegans and other important biological systems.5, 6, 7, 8 Specifically, the nonlinear imaging modalities of fluorescence, second harmonic generation (SHG), and third harmonic generation (THG) imaging have been used to study physiological processes in Drosophila.9, 10, 11, 12 However, to the best of our knowledge, the combination of multiphoton auto fluorescence (MAF) and SHG imaging for the structural exploration of Drosophila larval organelles on the whole-body scale has not been demonstrated. In this study, we attempt to achieve label-free imaging of living stage 2 D. melanogaster larva, and to test the feasibility of using this imaging modality for investigating the development and other significant physiological phenomena in Drosophila.


Materials and Methods


Sample Preparation

Stage 2 larvae of the D. melanogaster strain w1118 was used in this study. The larvae were anesthetized by exposure to ether fumes for about 45min . The anesthetized second larva was then mounted into a phosphate-buffered saline observation chamber made of coverslips and spacers. After imaging, the anesthetized larva was retrieved from the observation chamber and placed on a grape agar plate with wet nutritional yeast.


Multiphoton Microscope Setup

A femtosecond, titanium-sapphire (Ti–Sa) laser was used as the excitation source. The Ti–Sa laser (Tsunami, Spectra Physics, Mountain View, California) was tuned to 780nm with 5570mW output at the objective. The Ti–Sa laser has a 80-MHz repetition rate and 150-fs pulse duration. The nonlinear optical imaging system used in this experiment was based on Zeiss Meta LSM510 with a Fluar 40X 1.3NA oil-immersion objective (Zeiss) as the imaging objective. The detection bandwidths of the broadband MAF and narrowband SHG signals are approximately 435–700 and 380400nm , respectively.


Results and Discussion

Depth-resolved, MAF and SHG images of a stage 2 Drosophila larva are shown in Fig. 1 . Because both the muscular architecture and internal organs have evolved at this point of development, studying a stage 2 Drosophila larva is significant. To demonstrate that we can image throughout the thickness of the larva, we conducted multiphoton imaging at different depths in the second larvae stage. Images acquired at the depths of 0, 15, 30, 45, 60, 75, 90, and lateral 15μm are shown in Fig. 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h. Shown in Fig. 2 is the SHG image of the larva and the MAF image is shown in Fig. 3 . From our results, a number of significant observations can be made. First, the muscular architecture can be imaged by the second harmonic signal. In addition, we found that the trachea system can also be visualized by SHG imaging. Furthermore, MAF can be used to image the outer surface and various internal organs, such as the digestive system, trachea, and intestinal track. Our label-free images are structurally consistent with the known internal architecture of the Drosophila larva.12 In addition, we did not observe visible structural alteration from the femtosecond laser illumination, suggesting that the photodamage caused by multiphoton imaging is minimal.

Fig. 1

(a–h) In vivo, depth-resolved multiphoton imaging of the stage 2 larva. The imaging depths are 0, 15, 30, 45, 60, 75, 90, and lateral 15μm (Green: autofluorescence, Red: SHG).


Fig. 2

(A) Drosophila stage 2 larva SHG imaging. (B) is the enlarged SHG image of a selected region of interest. Note that both the muscular architecture and the trachea system can be imaged by the SHG signal. Scale bar is 200μm .


Fig. 3

Large area and detailed MAF images (A–H) of the stage 2 Drosophila larva. Shown in details are (A, D) digestive system, (B) trachea, and (C, E) intestinal track of the developing larva.


In this work, we demonstrated label-free multiphoton in vivo imaging in a stage 2 Drosophila larva. Although Drosophila is one of the important models in developmental biology, this study shows that the combination of MAF and SHG microscopy is capable of imaging different organelles of stage 2 Drosophila larva and that this approach may be used for the detailed investigation of developmental and other significant physiological processes in Drosophila in the future.


This work was supported by the National Science Council of Taiwan and was completed in the Optical Molecular Imaging Microscopy Core Facility of Taiwan’s National Research Program for Genomic Medicine (NRPGM).


1.  K. Konig, “Multiphoton microscopy in life science,” J. Microsc.0022-2720 10.1046/j.1365-2818.2000.00738.x 200, 83–104 (2000). Google Scholar

2.  J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol.1087-0156 10.1038/11698 17, 763–767 (1999). Google Scholar

3.  C. L. Phillips, L. J. Arend, A. J. Filson, D. J. Kojetin, J. L. Clendenon, S. Fang, and K. W. Dunn, “Three-dimensional imaging of embryonic mouse kidney by two-photon microscopy,” Am. J. Pathol.0002-9440 158, 49–55 (2001). Google Scholar

4.  G. M. Rubin, “Comparative genomics of the eukaryotes,” Science0036-8075 287(5461), 2204-2215 (2000). Google Scholar

5.  G. Filippidis, C. Kouloumentas, G. Voglis, F. Zacharopoulou, T. G. Papazoglou, and N. Tavernarakis, “Imaging of Caenorhabditis elegans neurons by second-harmonic generation and two-photon excitation fluorescence,” J. Biomed. Opt.1083-3668 10.1117/1.1886729 10(2), 024015 (2005). Google Scholar

6.  J. A. Palero, H. S. de Bruijn, A. V. van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues,” Biophys. J.0006-3495 10.1529/biophysj.106.099457 93(3), 992–1007 (2007). Google Scholar

7.  W. R. Zipfel, R. M. Williams, R. Christie, R. A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A.0027-8424 10.1073/pnas.0832308100 100, 7075–7080 (2003). Google Scholar

8.  A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. U.S.A.0027-8424 10.1073/pnas.172368799 99(17), 11014–11019 (2002). Google Scholar

9.  W. Supatto, D. Debarre, B. Moulia, E. Brouzes, J. L. Martin, E. Farge, and E. Beaurepaire, “In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses,” Proc. Natl. Acad. Sci. U.S.A.0027-8424 10.1073/pnas.0405316102 102, 1047–1052 (2005). Google Scholar

10.  C. Greenhalgh, N. Prent, C. Green, R. Cisek, A. Major, B. Stewart, and V. Barzda, “Influence of semicrystalline order on the second-harmonic generation efficiency in the anisotropic bands of myocytes,” Appl. Opt.0003-6935 10.1364/AO.46.001852 46, 1852–1859 (2007). Google Scholar

11.  D. Debarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods1548-7091 3, 47–53 (2006). Google Scholar

12.  V. Hartenstein, Atlas of Drosphila Development, Cold Spring Harbor Laboratory Press, Woodbury, NY (1993). Google Scholar

Chiao-Ying Lin, Vladimir A. Hovhannisyan, June-Tai Wu, Chii-Wann Lin, Jyh-Horng Chen, Sung-Jan Lin, Chen-Yuan Dong, "Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy," Journal of Biomedical Optics 13(5), 050502 (1 September 2008). http://dx.doi.org/10.1117/1.2981817

Second-harmonic generation

Multiphoton microscopy

In vivo imaging

Imaging systems

Harmonic generation


Auto-fluorescence imaging

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