MDC_Juli_2017_5394

Advanced Light Microscopy & Image Analysis

Anje Sporbert

Profil

Aim of the Advanced Light Microscopy & Image Analysis (ALM) technology platform is to give researchers from the MDC, the BIMSB and the ECRC on the campus Berlin-Buch access to a broad range of high-end, state-of-the-art imaging techniques and data analysis workflows. We provide scientific and methodological support to enable scientists to perform advanced imaging experiments and to acquire high quality microscopic images from different specimens. This includes all samples in the meso scale, for example from fixed cells to tissue sections and modell organisms. Further information:  internal ALM webpages.

Available microscopy techniques include          

  • confocal laser scanning microscopy
  • two-photon microscopy
  • light-sheet microscopy
  • wide-field fluorescence microscopy
  • TIRF microscopy
  • stimulated emission depletion (STED) superresolution microscopy
  • fluorescence lifetime microscopy (FLIM)
  • laser-assisted microdissection and catapulting

As part of collaborative projects ALM offers expertise to find optimal image acquisition and data analysis tools for advanced imaging experiments, i.e. by consultation about experimental design and specimen preparation, the optimisation of image acquisition conditions and customised image processing and analysis workflows.

We also contribute to the establishment of new imaging methods, i.e., light-sheet microscopy, functional imaging (FLIM-FRET), live/intravital microscopy or image analysis.

Membership and funding

ALM of the MDC is member of the steering committee and actively contributes to the GermanBioImaging, a network of german microscopists and imaging specialists. http://www.germanbioimaging.org/wiki/index.php/Main_Page

ALM is co-funded by DFG, BIH and DZHK.

Deutsche ForschungsgemeinschaftBerlin Institute of HealthDZHK

Microscopy techniques

Advanced Light Microscopy & Image Analysis  - Technology Platform offers several high end state-of-art microscope setups for a wide range of imaging techniques and applications.

Many of the setups are equipped for live imaging.

 Confocal and Two-Photon Microscopy  

 

Our confocal microscopes are equipped with multiple laser lines, various objectives and spectral detectors (PMTs or high sensitivity detectors). Two-photon excitation is possible for thick samples. Observation of subcellular structures can be done with the high-resolution Airy scan detector. A dedicated FLIM microscope can be used to study intracellular protein-protein interactions.

More information is available for internal users here.

Instruments

  • Leica Stellaris 8
  • Leica TCS SP8
  • Leica TCS SP8 DLS
  • Zeiss LSM 700
  • Zeiss LSM 880 NLO
  • Zeiss LSM 980 Airyscan
  • Nikon/Andor CSU-W1
  • Becker & Hickl FLIM

Some Applications

  • Sequential multi-color fluorescence imaging (UV/green/red/far-red dyes) of adherent fixed cells and tissue sections up to 100 µm thickness
  • z-stacks for 3D reconstructions
  • Tile scans for large area samples
  • Multiposition imaging
  • Spectral unmixing of overlapping emission spectra
  • Live cell imaging with very fast time series (e.g. vesicle movement, Ca2+responses)
  • Long-term live imaging of adherent cells
  • FRET (Förster resonant energy transfer) for detecting protein-protein interactions in situ measured via sensitized emission (fluorescence intensity ratio) or fluorescence lifetime imaging (FLIM)
  • Laser photomanipulations such as FRAP (fluorescence recovery after photobleaching) or photoswitching/ photoactivation
  • Confocal imaging with Airyscan technology (about two-fold improved resolution, speed and sensitivity)
  • 2-Photon excitation for imaging in thick samples
  • (Intravital) imaging of small organs/organisms with long working distance objective and/or 2-Photon excitation and non-descanned detection
  • DIC microscopy
  • Combination of confocal and selective plane imaging on the same system with the Digital LightSheet technology

Two-Photon Microscopy for in vivo deep tissue imaging

 

Deep tissue and organ imaging is possible due to the high penetration depth of the infrared radiation in Two-Photon microscopy. Our dedicated setup is completed with equipment for maintaining conditions for long-term live imaging. Large area imaging is possible for cleared or un-cleared tissue samples.

More information is available for internal users here.

Instrument

LaVision Biotec Trimscope II

Some Applications

  • Two-photon flourescence imaging for non-transparent tissues labelled with flourescent proteins, selected flourophores or quantum dots using up to three different excitation wavelengths and six detection channels simultaneously and possibilities for spectral unmixing
  • in-vivo flourescence imaging of cells expressing flourescent proteins and/or of blood vessels labelled with flourescent dextranes or quantum dots
  • SHG (second harmonic generation) of collagen fibres
  • Functional imaging of intracellular signals such as Ca2+ activation
  • FRET imaging of endogenously expressed biosensors via sensitized emission (flourescence intensity ratio) or FLIM (flourescence liftetime imaging)
  • Label free imaging of cellular autoflourescence (NADH, FAD)
  • Photoactivation of bacteriorhodpsins for optogenetic studies
  • Tile scans and multipostition imaging

     

Light sheet microscopy

 

Our 2 light sheet microscopes are applied for fast imaging of large volume tissue and organ samples after clearing. We also offer conditions for long-term imaging of live small samples.

More information is available for internal users here.

Instruments

  • LaVision Biotec Ultramicroscope I
  • Zeiss Lightsheet 7
  • (LifeCanvas Tissue Clearing and Labeling Device)

Some Applications

  • selective plane illumination for 3 dimensional imaging of large samples
  • multiview acquisition with different rotation angles
  • multicolor fluorescence imaging (blue/green/red/far-red dyes) of cleared samples like spheroids, tissue and organs, or transparent specimen like Zebrafish embryos or Drosophila larvae
  • long-term and short-term live imaging

Superresolution & TIRF Microscopy

 

Breaking the resolution limit is possible using several microscopy techniques. We offer wide field TIRF microscopy for studying dynamic membrane processes in live cells and possible applications to localisation microscopy (e.g. PALM). Our STED microscope can be used for both fixed and live samples as well as for the RESOLFT technique.

More information is available for internal users here.

Instruments

  • 3i Vector TIRF
  • Olympus TIRF
  • Abberior STED

Some TIRF applications

  • observation of membrane-associated cellular processes
  • FRET microscopy by sensitized emission
  • fast, sensitive single molecule imaging and tracking

Some STED applications

  • subdiffraction-resolution imaging of subcellular structures and organelles in membranes, cytoplasm and nucleus of flat adherend cells and thin tissue sections
  • multicolor superresolution imaging

Wide field microscopy & laser microdissection

 

Laser microdissection is applied for specific isolation of individual cells or larger areas of a tissue sample, which will undergo further molecular analysis, i.e. contamination-free molecular analysis of DNA, RNA and proteins.

More information is available for internal users here.

Instrument

Zeiss PALM MicroBeam (Axio Observer Z1)

Some applications

  • multicolor wide-field fluorescence imaging (software: ZEN Blue) of adherent fixed cells and tissue sections up to 100 µm thickness, esp. tile scan and multiposition imaging for large area samples
  • laser-guided precise microdissection (software: PALM Robo) of histological sections on glass or membranes slides and of living cells in membrane dishes

Image Analysis Tools

Advanced Light Microscopy & Image Analysis Platform also provides access to several high-end computer workstations and server with different image processing and analysis software packages and support with image analysis and data processing for, i.e.:

Stitching

 

Reconstruction of multiple imaged tiles with overlapping fields of view into a single big image. Depending on the software reconstruction can be done in two and three dimensions and time laps recordings. For a robust computation, tiles need to overlap with each other by 5 – 15 % of their width and/or height.

Deconvolution

 

A mathematical transformation by which out-of-focus light or blur in the image is reduced. Images acquired on optical systems suffer from a systematic error caused by the Point Spread Functions (PSF). Out-of-focus light arises from the sum of the PSF of fluorescent molecules in the sample. Deconvolution aims to deblur images and remove out-of-focus light. It thereby restores image contrast and resolution.

3D Reconstruction

 

Large structures in organisms or cells can be imaged as a series of two-dimensional slices or a z-stack throughout their entire depth. From these stack of two-dimensional images, structures can be assembled and reconstructed as a three-dimensional surface render to visualize and analyse the entire structure independent of the image series.

Filament tracing

 

An image analysis method to reconstruct filamentous structures from 2D or 3D images. The operation helps to determine pathways of structures such as axons, dendrites and others and allows further visualization and analysis of the reconstructed structures.

Deep Learning and Machine Learning

 

Deep Learning (DL) and Machine Learning (ML) are specialized subsets of artificial intelligence (AI). With DL approaches one can use multilayer networks to perform segmentation or automatic pattern recognition. Through ML approaches, neural networks can be trained and optimized to perform segmentation, denoising or object classification.

Quantitative colocalization

 

Fluorescence signals of molecules in two different channels of a fluorescence image may colocalize when the distance between them is below the resolution of the microscope. Colocalization analysis aims to determine quantitative aspects of this special proximity. Quantitative analysis thereby helps to identify possible interactions between molecules and to determine between colocalization and co-occurrence. 

For creation of robust and reliable analysis workflows, including custom-designed software and macro-programming for users, please contact us.

Available Software packages (always up to date)

 

  • ZEN: for analysis and converting of Zeiss data files

  • LAS X: for analysis and converting of Leica data files, advanced adaptive deconvolution (Stellaris)

  • Imaris: 3D reconstructions, analysis, build-in Machine Learning Pixel Classification and LabKit integration

  • Arivis Vision4D: 3D reconstruction, analysis, Cellpose integration for segmentation and build-in Machine Learning tools, stitching of large image files

  • Huygens (SVI) for deconvolution and advanced image reconstruction

  • MathLab

  • Fiji / (ImageJ)

 

Publications

Projects

 

Our projects will be shown here. This page will be updated.

MDC_Juli_2017_5394

 

Publications by ALM users 

 

Publications by ALM Team members

 

 

TEAM

 

Dr. Anje Sporbert

asporb@mdc-berlin.de

Phone: 030 9406 2734

Building 31.1, Room 1001

 

Dr. Anca Margineanu

Anca.Margineanu@mdc-berlin.de

Phone: 030 9406 2662

Building 31.1, Room 1008

 

Dr. Sandra Cristina Carneiro Raimundo

Sandra.Raimundo@mdc-berlin.de

Phone: 030 9406 2662

Building 31.1, Room 1008

 

Matthias Richter

m.richter@mdc-berlin.de

Phone: 030 9406 3781

Building 31.1, Room 1007

 

 

 

ADMINISTRATIVE ASSISTANCE

 

Elena Gubanova

elena.gubanova@mdc-berlin.de

Phone: 030 9406 2884

Building 87, Room 1.13