The aim of the Advanced Light Microscopy & Image Analysis (ALM) technology platform is to give researchers from the MDC/ BIMSB and the ECRC on the campus Berlin-Buch access to a very broad range of high-end, state-of-the-art imaging techniques and image analysis tools.
We provide scientific and methodological support to enable scientists to perform advanced imaging experiments and acquire high-quality microscopic images from different specimens. This includes all samples in the meso scale, from fixed cells to tissue sections, organoids and different model organisms.
In addition to individual training at up-to-date, well-maintained imaging setups, the ALM offers hands-on expertise for the optimisation of image acquisition conditions and customised image processing and analysis workflows as well as consultation about experimental design and specimen preparation.
Supported microscopy techniques include
confocal laser scanning microscopy
multi-photon microscopy
light-sheet microscopy, incl. sample clearing and active labelling
wide-field fluorescence microscopy, incl. medium throughput imaging
In close interaction with the research groups, the ALM actively contribute to the establishment of new imaging methods, i.e. light-sheet microscopy, functional imaging (FLIM-FRET), live/ intravital microscopy or the update and customization of existing imaging setups.
Membership and funding
The MDC and the ALM are members of the GermanBioImaging - a network of German microscopists and bioimage analysts and actively contribute to several of its working groups.
Part of the ALM resources is co-funded by successful applications to DFG, BIH, DZHK and MDC Tandem grants.
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 detector units (using photomultiplier tubes – 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.
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 (also using white light lasers)
Confocal imaging with Airyscan technology (about two-fold improved resolution, speed and sensitivity)
Long-term live imaging of adherent cells
Live cell imaging with very fast time series (e.g. vesicle movement, Ca2+responses)
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
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 fluorescence imaging for non-transparent tissues labelled with fluorescent proteins, selected fluorophores or quantum dots using up to three different excitation wavelengths and six detection channels simultaneously and possibilities for spectral unmixing
z-stacks, tile scans and multipostition imaging
in-vivo tracking of cells expressing fluorescent proteins
Imaging of blood vessels labelled with fluorescent 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 (fluorescence intensity ratio) or FLIM (fluorescence lifetime imaging)
Label free imaging of cellular autofluorescence (NADH, FAD)
Photoactivation of bacteriorhodpsins for optogenetic studies
Light sheet microscopy
Our light sheet microscopes are applied for fast imaging of large volume tissue and organ samples after clearing. We provide solutions for active clearing and labelling. We also offer conditions for long-term imaging of live small transparent samples.
More information is available for internal users here.
Instruments
Zeiss Lightsheet 7
Miltenyi UltraMicroscope BlazeTM
LaVision Biotec Ultramicroscope I
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
Olympus TIRF
Nikon TIRF (with 4 EMCCD cameras)
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
Olympus CellSens
Leica wide-field system
Zeiss PALM MicroBeam (Axio Observer Z1)
Some applications
Multicolor wide-field fluorescence imaging of adherent cells and thin tissue sections (up to 10 µm thickness)
Tile scan and multiposition imaging for large area samples on 1-4 microscope slides
Multi-well plates imaging
Long-term imaging of live cells
Imaging of fast intracellular processes (e.g. Ca2+ imaging)
Imaging of biosensors and of protein-protein interactions using FRET (Förster resonant energy transfer) via sensitized emission (fluorescence intensity ratio)
Prefinding of cells and regions using multicolor wide-field fluorescence imaging (software: ZEN Blue), followed by laser-guided precise microdissection (software: PALM Robo) of histological sections (up to 100 µm thickness) on glass or membranes slides and of living cells in membrane dishes
Ultra-high content microscopy
The ultra-high content microscopy system on our platform is based on cyclic immunofluorescence imaging technology, which allows for sequential imaging of tens to hundreds of different cellular markers. A fully automated workflow is set up to perform labeling, imaging in 4 spectral channels, and removal of the fluorescent signal (via photobleaching) or the labeled antibodies (via enzymatic reaction). The omics datasets are analyzed with dedicated software to generate complex interpretations of the spatial biology data.
Instrument:
Miltenyi MACSima platform
Some applications:
Ultra-high-plex spatial protein profiling with panels featuring over 200 validated protein markers
High-plex imaging of 27 RNAs
Combined protein and RNA detection within the same tissue section
Applicable for both mouse and human tissues and cell lines (with PFA or FFPE fixation methods)
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
