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T configuration dual excitation (488, 561) microscope

Yong's Projects at the 2014 EMBO LSM Practical Course

Project A: Image Zebrafish Brain via Ptf1a-GFP

Pancreas transcription factor 1 subunit alpha (Ptf1a) is a protein that in humans is encoded by the PTF1A gene. This gene encodes a protein that is a component of the pancreas transcription factor 1 complex (PTF1) and is known to have a role in mammalian pancreatic development. Ptf1a is also shown to play an important role in the neurogenesis of different central nervous system structures. In particular, Ptf1a is important for the generation of many inhibitory (primarily γ-aminobutyric acid (GABA)-ergic) interneurons in different areas, such as the spinal cord and cerebellum. In this project, we imaged a transgenic zebrafish line expressing fluorescent Ptf1a-GFP fused proteins, with an emphasize on the structures in the brain.

Large FOV

Small FOV

Overview of zebrafishes expressing Ptf1a-GFP.

The major locations of cells expressing Ptf1a-GFP are at the regions of hindbrain, retina, and pancreas.

Imaging of a zebrafish expressing Ptf1a-GFP by Zeiss Lightsheet Z.1.

Intensity map for estimating the expression level of Ptf1a-GFP in the hindbrain of zebrafish.

Multi-view Reconstruction (7 angles)

3D distribution of the expression level of Ptf1a-GFP in the head of zebrafish.

Timelapse of the hindbrain development (Huisken SPIM3)

Project B: Image Zebrafish Brain via HuC-GFP

Imaging of a zebrafish expressing HuC-GFP by Zeiss Lightsheet Z.1.

Marketa's Project

Task: In vivo realtime imaging of primordium development.

mDSLM

Objective: 40x/0.75W.

Movie:

Movie:

Zeiss Lightsheet Z.1

Z projection of older primordium.

Emese's Project

Clearing and imaging primate cortical tissue

Clearing technique

For clearing the brains I used the DBE technique. It worked very well on the primate cortical tissue. I tried only this method.

Microscopes used in the course

• LSM Z1

On this microscope you allowed to image only uncleared tissue and the size of your sample is limited.

Question1: How deep can the lightsheet penetrate in the uncleared tissue?

Sample: Cortical tissue with somatostatin labeled interneurons, embedded in 1% agarose.

We used 20x objective and tried single and dual sided illumination too. The penetration of the lightsheet was around 60 um.

Question2: How deep can the antibodies penetrate? What can we achieve with thinner samples?

Sample: 100 um thick cortical tissue labeled with 3 interneuron markers, embedded in 1% agarose.

We used 20x objective and dual sided illumination. All of the antibodies penetrated through the sample. This is a nice way to investigate the presence and distribution of these interneurons in the cortex.

• Ultramicroscope

This technique was developed for imaging big samples. The highest magnification we could get was 5x objective with 2x zoom.

Question: What can you see in the cleared tissue? Sample: DBE cleared cortical tissue. With this technique we imaged a big (2x2,5x1 mm) DBE cleared cortical tissue. We found only a few labeled cells. The poor staining may have caused by the antibody penetration or/and the clearing.