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Team members

File:Pernisova.jpg|Markéta Pernisová File:Palfi.jpg|Emese Palfi File:Wang.jpg|Yong Wang File:Burri.jpg|Olivier Burri File: WP_20140823_003.jpg|First official group photo

Local Guides

File:Schmied.jpg|Christopher Schmied File:Schindelin.jpg|Johannes Schindelin

Building our openSPIM

T configuration dual excitation (488, 561) microscope

Workflow

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
Video showing heart-beating of live zebrafish sample

align="left" | Overview of zebrafishes expressing Ptf1a-GFP.

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

Reconstructed whole fish (stitching) Zoom-in at the hindbrain (20X)
Zoom-in at one side of the hindbrain (50X)

align="left" | Imaging of a zebrafish expressing Ptf1a-GFP by Zeiss Lightsheet Z.1.

Hindbrain (20X) Hindbrain (50X)

align="left" | Intensity map for estimating the expression level of Ptf1a-GFP in the hindbrain of zebrafish.

Multi-view

align="left" | Multi-view Reconstruction (7 angles)

Intensity distribution

align="left" | 3D distribution of the expression level of Ptf1a-GFP in the head of zebrafish.

View A View B View C

align="left" | Timeplase of the hindbrain development (Huisken SPIM3)

Project B: Image Zebrafish Brain via HuC-GFP

Reconstructed whole fish Head
Expression level

align="left" | 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.

4 cells primordium. Color coding shows the
depth.

Movie:

Primordium_mDSLM_4cells.avi

2 days old primordium. Color coding shows the
depth.

Movie:

PrimordiummDSLMolder.avi

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.

 Uncleared and cleared (DBE) corical tissue

Microscopes used at the course

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.

 Somatostatin labeled interneurons in the primate cortex. Single sided
illumination. 20x  Somatostatin labeled interneurons in the primate cortex. Dual sided
illumination. 20x

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 internurons in the cortex.

 Parvalbumin labeled interenurons in the primate cortex. Color coding
shows the depth. 20x  Parvalbumin (green), calretinin (purple) and somatostatin (blue)
labeled interenurons in the primate cortex. 20x

This technique was develpoed 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.

 One overview plane of the cortical tissue. 10x  2 parvalbumin
labeled cells. 10x

Category:EMBO2014