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Team SPIMApes |
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EMBO 2014 Team SPIMApes report |
{% include image src="Pernisova.jpg" width="50%" caption="Markéta Pernisová" %} {% include image src="Wang.jpg" width="50%" caption="Yong Wang" %} {% include image src="Burri.jpg" width="50%" caption="Olivier Burri" %} {% include image src="WP_20140823_003.jpg" width="50%" caption="First official group photo" %}
{% include image src="Schmied.jpg" width="50%" caption="Christopher Schmied" %} {% include image src="Schindelin.jpg" width="50%" caption="Johannes Schindelin" %}
T configuration dual excitation (488, 561) microscope
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.
| {% include image src="Zebrafish_PTF1A_GFP_Large_FOV_YongWang.jpg" width="70%" caption="Large FOV" %} | {% include image src="Zebrafish_PTF1A_GFP_Small_FOV_YongWang.jpg" width="70%" caption="Small FOV" %} |
{% include video id="63MTaybr680" width="400" height="243" caption="Video showing heart-beating of live zebrafish sample" %}
The major locations of cells expressing Ptf1a-GFP are at the regions of hindbrain, retina, and pancreas.
{% include video id="LDbxF_6glJY" width="400" height="243" caption="Reconstructed whole fish (stitching)" %}
{% include video id="uS834ediTVY" width="400" height="243" caption="Zoom-in at the hindbrain (20X)" %}
{% include video id="TNA8R_N3g8o" width="400" height="243" caption="Zoom-in at one side of the hindbrain (50X)" %}
{% include video id="VnTQ1b3cPos" width="400" height="243" caption="Hindbrain (20X)" %}
{% include video id="N3yFSJt6jiY" width="400" height="243" caption="Hindbrain (50X)" %}
{% include video id="bSRMEokMcSU" width="400" height="243" caption="Multi-view" %}
{% include video id="_8imBuffGyU" width="400" height="243" caption="Intensity distribution" %}
{% include video id="UlCv3L56-hs" width="400" height="243" caption="View A" %} {% include video id="6-yrOQK00u8" width="400" height="243" caption="View B" %} {% include video id="qyq60uccVY4" width="400" height="243" caption="View C" %}
Imaging of a zebrafish expressing HuC-GFP by Zeiss Lightsheet Z.1.
{% include video id="nncFcm6m8_c" width="400" height="243" caption="Reconstructed whole fish" %} {% include video id="wtFXwl-jgS4" width="400" height="243" caption="Head" %} {% include video id="U-j_AikE8qQ" width="400" height="243" caption="Expression level" %}
Task: In vivo realtime imaging of primordium development.
Objective: 40x/0.75W.
{% include image src="Primordium4cells.jpg" width="70%" caption="4 cells primordium. Color coding shows the depth." %}
Movie:
{% include video id="GtCEFEaUhqs" width="400" height="243" caption="" %}
{% include image src="Primordium2days.jpg" width="70%" caption="2 days old primordium. Color coding shows the depth." %}
Movie:
{% include video id="DgKXA2t05xY" width="400" height="243" caption="Expression level" %}
Z projection of older primordium.
For clearing the brains I used the DBE technique. It worked very well on the primate cortical tissue. I tried only this method.
{% include image src="Picture1.jpg" width="70%" caption="Uncleared and cleared (DBE) cortical tissue" %}
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.
{% include image src="Picture1.jpg" width="70%" caption="Uncleared and cleared (DBE) cortical tissue" %}
{% include video id="53VcgpFr0Ho" width="400" height="243" caption="Somatostatin labeled interneurons in the primate cortex. Single sided illumination. 20x" %}
{% include video id="4jIXsT0XRvE" width="400" height="243" caption="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 interneurons in the cortex.
{% include image src="Pv_depth.jpg" width="70%" caption="Parvalbumin labeled interneurons in the primate cortex. Color coding shows the depth. 20x" %}
{% include image src="3intn.jpg" width="70%" caption="Parvalbumin (green), calretinin (purple) and somatostatin (blue) labeled interneurons in the primate cortex. 20x" %}
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.
{% include image src="Um1.jpg" width="70%" caption="One overview plane of the cortical tissue. 10x" %} {% include image src="Um2.jpg" width="70%" caption="2 parvalbumin labeled cells. 10x" %}