Light-sheet fluorescence microscopy (LSFM), also termed single plane illumination microscopy (SPIM), enables live cell fluorescence imaging with optical sectioning capabilities superior to confocal microscopy and without any out-of-focus exposure of the specimen. However, the need of two objective lenses, one for light-sheet illumination and one for imaging, imposes geometrical constraints that require LSFM setups to be adapted to the specific needs of different types of specimen in order to obtain optimal imaging conditions. Here we demonstrate the use of an oblique light-sheet configuration adapted to provide the highest possible Gaussian beam enabled resolution in LSFM. The oblique light-sheet configuration furthermore enables LSFM imaging at the surface of a cover slip, without the need of specific sample mounting. In addition, the system is compatible with simultaneous high NA wide-field epi- fluorescence imaging of the specimen contained in a glass-bottom cell culture dish. This prevents cumbersome sample mounting and enables rapid screening of large areas of the specimen followed by high-resolution LSFM imaging of selected cells. We demonstrate the application of this microscope for in toto imaging of endocytosis in yeast, showing for the first time imaging of all endocytic events of a given cell over a period of >5 minutes with sub-second resolution.

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sometimes another promoter that can be regulated like the GAL1 promoter comes in handy … here

Update – the paper is now accepted in YEAST

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fist paper on a systematic functional analysis of antisense RNAs in yeast  …

read here the open access pdf

and a commentary here …

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 we aim to create a new user interface for the software ImageJ in order to ease scientific image analysis. While keeping the core components of ImageJ and full compatibility with the current plugins, ImageJFX brings scientists closer to their goal by making the interface clearer for beginners and more practical for advanced users.

It’s still early days but Cyril’s project is gaining momentum … check it out… and if your are interested: register!

 

 

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PostDoc with a background in physics/engineering and a strong interest In microscopy, optical engineering and BioImaging (Feb. 2016)

Job description:

Our laboratory works at the interface of physics, informatics, cell- and molecular biology, we develop new methods and microscopy instrumentation and we employ these to answer important biological questions.

We are looking for a postdoc with strong background in physics or engineering with a passion to engage in collaborative and interdisciplinary projects. Experience in one or more of the following topics would be a big plus: optics, laser and microscopy instrumentation, electronics, computing – especially GPU programming, CCD camera technology or microfluidics, but we also offer the possibility to acquire complementary knowledge and skills with the help of our collaborators. Knowledge in biology is not required.

The candidate will be responsible for the development of a new and innovative microscope that will be central for the realization of a funded project.

Start of position: open (as soon as possible)

For application or further information, please contact:

Michael Knop: m.knop@zmbh.uni-heidelberg.de

Phone: 06221-544213

June 2016: the position has been filled, but still, if you like what we do and would like to be come part of it, enquire ….

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are you interested in systemic yeast research and you would like to have a new yeast library with a different feature, such as your favorite fluorescent protein, then please feel free to use the

SWAp-Tag strategy

go here!

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for science, Pizza and skiing ….

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We submitted a technical manuscript for publication and simultaneously uploaded it to BioRxiv … with excellent feedback! Several people contacted us in order to obtain reagents or to discuss data….

http://biorxiv.org/content/early/2015/07/23/023119

Update (Nov’15) : We actually were contacted by people who read the paper on BioRxiv, and we sent them reagents from the paper, so I consider the trial as a success.

Update (Jan’16): the paper is published: go check out our publications

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If you have no clue whether your microscope is working according to its ‘diffraction limit’ (=providing the resolution it is supposed to according of the specification of its optical part), you can now find out very easy by using a new and straight forwards software tool: PSFj along with some fluorescent beads. NO a priori knowledge required. NO manual PSF calculations etc.

Read a short INFO statement on the software here.

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Do you want to increase the gene dosage from one copy to two copies without the hassle of cloning the gene followed by chromosomal integration, then read our small PLoS One paper, and be surprised how easy this is….PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast.

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our evolved long-stokes shift fluorescent protein mKeima8.5 – based on mKeima – has made it into a paper … thanks to a great collaboration with Jagesh Shah and his group and the folks at Montana State University…and hopefully soon into the cytoplasm of many different cells in your lab! Mail to get it, or simply order your preferred codon-usage optimised version from your preferred full gene synthesis company. If it works for you – let us know — if not, please do so as well.

here is the link to the paper

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Do you want to use a particular promoter to drive your reporter gene, but you are too lazy to clone the promoter into your reporter plasmid, then read how you can save some time and hassle ……..PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast.

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Martin, Patrick and Florian together with Katrin and Bogdan made a sensational 2nd place in the annual Henkel run – congratulations!

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Cells are the smallest functional units of the most complex 3D objects that currently exist  ….

.. and we need to understand how they function!

My lab hopes to contribute to this goal by several ways.

  • First, we develop methods to study the spatio-temporal organisation of the proteins inside cells, how they are produced, distributed, regulated and degraded.
  • Second, we use these methods to study various cellular processes to demonstrate that we can learn something new by using these  methods.
    • meiosis and sporulation in yeast
    • yeast mating and MAPK signal transduction
    • protein degradation to study GPCR signaling in fish protein quality control at the inner nuclear membrane
  • Third, we are involved in various teaching activities and conference organisation.

Our reserach

Living cells are  highly dynamic objects – to understand them we play  around with fluorescent proteins – sometimes this yields useful new tools  observe various types of  protein behaviours right in the living creature.

Read our latest papers on fluorescent timers and how they allow direct visualisation of protein age: where are the old proteins and where are the new ones, and what do we learn form this?

 

  tFT timers to understand protein dynamics and  degradation.  Read the paper …

  tFT timers to visualise cellular communication in living embryos.  Read the paper …

Screen Shot 2015-02-25 at 21.47.48

  tFT timers to learn about the (terminal) fate of mislocalized proteins.  Read the paper …

We also love to play around with microscopes to make them better tools to visualise protein in their cellular environment. Read our papers on fluorescence correlation spectroscopy (FCS), and on light sheet microscopy, and how we (and you) can make sure that the a microscope’s performance does keep up with the manufacturers promises.


 PSFj – make sure your microscope is working well! Read the paper …Screen Shot 2015-02-27 at 08.56.59

 FCS and intracellular communication: how cells react to pheromones. Read the paper …

 FCS and nuclear organisation observed with a light sheet microscope (FCS-SPIM). Read the paper …

 FCS and Endocytosis: where does the music play in endocytosis? Read the paper …

We believe that we can learn the most from reductionist approaches: this is why we love to use yeast as our model organism: they grow fast, no ethical issues, yet, they are highly similar to human cells. Any new fundamental insight obtained with yeast does fuel directly research with mammalian cell.

 

Yeast as a model for evolutionary studies

For example yeast also allows us to understand the evolutionary dimension of molecular mechanisms. Some of our work in this direction is listed here. By comparing with the situation in related species we can obtain insight into the constraints that shape molecular processes. This is one of the reasons why we initiated an EMBO funded conference series so that people with similar interests can meet and discuss. See the meeting reports of previous meetings in various journals:  2008, 2010, 2012 and 2014 meetings, the next one will be in 2016.

For further reading, please also visit our specific pages on our previous research and our methods projects.

 

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but make sure you have a complete backup of the hard disc of your system installation. This lesson we had to learn the hard way, because our Nikon-TiE/FCCS System stopped working after Microsoft decided that it is time to update the system with some patches – unfortunately the software components that control the system (Micromanager, Easy-FCS automation software and the sort) turned out to be incompatible with this update —> full system crash!

Five days later and a heroic remote desktop session of Cyril from Martinique via TeamViewer, and the system is back in full operation again! Thanks everybody for his efforts!

PS: two backups of the system installation have now been made, even using different backup software….to leave Murphy no chance ….

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we recently decided that a 3D printer could solve several issues with custom made fabrication of microscopy parts, prototyping of metal pieces etc., …. now we bought such a device. Let’s see how useful it will become.

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Lots of cool microscopy  …..

link

 

 

 

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