Research interests

Microwell Cell Arrays

Reverse transfection on cell arrays is a high throughput method for parallel transfection of mammalian cells. GFP tagged cDNAs or small interfering siRNAs are printed together with a transfection solution at defined locations (spots) on glass slides resulting in “ready to transfect” substrates (solid phase transfection). The dried and “ready to transfect” replicates can be stored for more than 15 months.
Disadvantages of the standard “array” reverse transfection technique are the risk for cross contamination and the lack of reference markers for fully automated screening pipelines. A big drawback of the well based transfection is the limited number of samples (96, 384 or 1536).
For this, we develop microwell cell arrays overcoming the problem of cross contamination by using physically separated cavities on glass slides by a titanium coating hostin 9216 samples.
Reymann J. et al. (2009), Next generation 9216 microwell cell arrays for high content screening microscopy, BioTechniques

Automated correlative microscopy for high content screening

In fluorescence microscopy three major techniques are commonly used for different biological approaches:

Wide field microscopy

  • Pros: robust, rapid, HIGH throughput capable, probe requirements
  • Cons: LOW information content

Confocal microscopy

  • Pros: high information content (3D structural information), medium throughput capable
  • Cons: Bleaching of dyes

Super high resolution microscopy

  • Pros: single molecule resolution
  • Cons: NO screening procedures, probe requirements, no automation (so far)

 

Thus, the "ideal" microscope would combine all the advantages of these techniques and get rid of limitations given by the optical setup.

Within the ViroQuant-CellNetworks RNAi Screening Facility we are developing an advanced screening platform for correlative microscopy. This platform will collect data ranging from high throughput procedures to a single molecular level thus collecting information from phenotypic penetrance to interactions on a molecular level.


Developments and grants

1. nc RNAs gain more and more attention as regulators of gene expression. To get a complete systematic view of biology we need to consider the regulatory potential of ncRNAs to a higher degree. The RNAi screening facility coordinates the BMBF funded “SysTec” project “Functional analysis of non-coding RNAs in living cells, resources and technologies for the systematic high-throughput and high-resolution functional analysis of non-coding RNAs as potential regulators of secretory membrane trafficking”. Partners in the project are 6 scientific groups and two industrial partners. Within this project we will develop strategies and produce molecules for an efficient knock-down of ncRNAs.

2. miRNAs belonging to nc RNAs play a highly recognized role in gene regulation. At the moment bioinformatics mirna target predictions vary enormously. The RNAi screening facility coordinates as well the Baden-Württemberg Stiftung funded project “An integrated high-throughput and super-high resolution platform for fluorescence microscopic analysis of miRNA-targets in living cells”. Partners in the project are 6 scientific groups. We want to establish within this project molecules for labelling miRNAs and mRNAs to detect “real” miRNA-mRNA interactions.

In the course of both projects we will further develop the cell array technology and strengthen correlative light microscopy.

Improvements of the cell array technology

Our goal is to improve the cell-array technology towards high sample numbers per cell array (20.000 spots or more) and reduced cost per screen. Proof of principle results from collaboration with Graffinity Pharmaceuticals GmbH in Heidelberg showed that we could increase by usage of microtiter plate size substrates the number of spots from 384 to 9216 per substrate with reduced risk for cross-contamination. These spots are in addition clearly detectable enhancing the automation capability of those arrays (Reymann et. al, BioTechniques, 2009).

Developments on correlative light microscopy

Wide-field high-throughput microscopy detects cellular changes in a relatively coarse resolution level. Informative details are so far hidden by a lack of resolution at the screening step. For increasing the information content we are planning to build a bridge between low-resolution wide-field microscopy and high resolution STED and STORM microscopy (Johann Engelhard (DKFZ, group Prof. Stefan Hell) and Dr. M. Heilemann) by advanced image processing (F. Hamprecht and K.Rohr).