1.

Introduction

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 $<500\mathrm{nm}$ 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, $\sim 61\%$ 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.

2.

Materials and Methods

3.

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 $15\mu \mathrm{m}$ 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 $15\mu \mathrm{m}$ (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 $200\mu \mathrm{m}$ .

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.