Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy

Abstract

Abstract. 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 ͑MAF͒ and second harmonic generation ͑SHG͒ 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.

<em>In vivo</em> Imaging of Intact <em>Drosophila</em> Larvae at Sub-cellular Resolution

“…In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution [1][2][3] . While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described 4,5,6,7,8 , the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm 1 , (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days 2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter 1 in stable transgenic lines and the exon trap line FasII-GFP , the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.…”

“…While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described 4,5,6,7,8 , the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm 1 , (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days 2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter 1 in stable transgenic lines and the exon trap line FasII-GFP 1 .…”

Visualization of Mouse Neuronal Ganglia Infected by Herpes Simplex Virus 1 (HSV-1) Using Multimodal Non-Linear Optical Microscopy

“…Although there has been much written about the potential of tissue imaging with multimodal multiphoton microscopy [20], [42], to the best of our knowledge, this study constitutes the first report of combining CARS microscopy and two-photon fluorescence microscopy to study viral infections deep within whole tissue mounts. Combining the two techniques enabled us to visualize distinct subanatomical structures within the TG, both in infected and uninfected regions of interest (ROI).…”

“…This type of microscopy has already been exploited to visualize lipid droplets induced by the Hepatitis C virus (HCV) in cell culture [18]. In whole tissue mount, multiphoton fluorescence microscopy can be used to identify different structures by their autofluorescence, and it affords great sectioning capability [19], [20]; however, autofluorescent properties of the tissue under study can be problematic if the emission spectra overlap with that of the fluorescent protein used for tracking of cells [21]. Optimization of the experimental conditions for each imaging modality, as well as their compatibility, is crucial if one is to combine their respective strengths to achieve multimodal high spatial resolution imaging deep in tissue.…”

Signal enhancement in multiphoton imaging by the use of coated glass substrates

“…Other applications in multiphoton imaging of thinner specimens are significant. Such examples include drosophila embryo, engineered tissue specimens fluorescence correlation spectroscopy (FCS) and twophoton uncaging experiments [6][7][8][9]. In order to optimally enhance signal, different strategies have been developed to optimally enhance the collection of signal.…”