The electron microscopy unit EMil

Our EM lab routinely perform embedding, sectioning, immunolabelling, low temperature embedding and microscopy. Our aim is to help researchers to solve biological questions using various electron microscopy techniques. We have extensive experience in conventional preparation for TEM and SEM, preparation for immunohistochemistry, morphometric analysis, negative staining, low temperature techniques such as low temperature embedding and cryo-sectioning. The unit manages two transmission electron microscopes TEM (FEI Tecnai Spirit BioTwin and Hitachi HT7700) and one scanning electron microscope (Zeiss Ultra 55).

We have a fixed pricelist.

Services

Ultrathin sectioning - TEM

Ultrathin sectioning TEM, a preparation method for ultrastructural analysis of larger biological specimens such as various tissues, cells and bacteria. It involves chemical fixation, dehydration, plastic embedding and ultrathin sectioning. Typical questions are for example general morphology, cellular uptake of nanoparticles, morphological changes of intracellular organelles due to treatment by e.g. a chemical, siRNA etc. The method is routinely used in ultrastructural pathology for the diagnosis of renal, muscle and cilia related diseases.

1. TEM image of cellular uptake of silicon dioxide nanoparticles. 2. TEM image of Influenza virus budding from the plasma membrane. 3. TEM image of bacterium phagocytosed by a peripheral blood mononuclear cell, kindly provided by A. Sendel (MTC, KI).

Immunoelectron - TEM

Immunoelectron microscopy is a powerful tool used to detect and localize single proteins within the cell and different organelles. It allows for the study of various cellular processes and response to various changes in the microenvironment e.g. gene silencing, chemical treatment. Basically primary antibodies directed against the antigen (protein) of interest is used in a first step, the primary antibody can subsequently be detected using a secondary probe conjugated to colloidal gold markers for example species specific secondary antibodies or protein-A or G. The contrast of the colloidal gold markers are so strong that they can easily be detected in the TEM images.

1. Immunoelectron microscopy image of podocalyxin labelling in podocyte cells lining the urinary space of the glomeruli. 2. Immunoelectron microscopy image showing localization of LL-37 in E. coli.

Negative stain TEM

Negative stain TEM, a quick-and-dirty method to assess the morphology, purity, size, aggregation state etc. of suspensions containing various nanoparticles such as protein complexes, virus particles, exosomes, inorganic nanoparticles etc. The method can also be used to analyse suspensions of larger biological specimens such as bacteria and mycoplasma to assess the size and shape but also presence of cellular organelles such as cilia, fimbriae and flagellum. Various membranous materials such as liposomes, LNP’s and virosomes etc can also be analysed using negative stain TEM.

1. Negative stain TEM image of bacteria with protruding flagellum. 2. Negative stain TEM image of liposomes. 3. Negative stain TEM image of Semliki forest virus.

Scanning electron microscopy (SEM)

Scanning electron microscopy of biological specimens typically involves a topographical representation of the specimen which in many scientific questions can give unique and relevant morphological information regarding the overall specimen morphology. Similar to TEM, sample preparation involves chemical fixation and dehydration and prior to imaging the specimen is sputtered using e.g. platinum. The scattered electrons originating from this metal coating will then be detected when irradiated by a scanning beam of incident electrons.

1. SEM image of Influenza virus budding from the plasma membrane. 2. SEM image of Plasmodium falciparum infected erythrocytes (top middle) and a crenated erythrocyte morphology (middle) due to a hypertonic environment. Kindly provided by Madle Sirel (MTC, Karolinska institutet). 3. SEM image of Lactobacillus kunkeei. Kindly provided by Christian Seeger (ICM, Uppsala universitet). 4. SEM image of the parasite Spironucleus salmonicida penetrating ASK (Atlantic Salmon Kidney) cell layer. Kindly provided by Ásgeir Astvaldsson (ICM, Uppsala universitet). 5. SEM image of bacteriophages infecting E. coli.

Clinical electron microscopy

The EM facility also performs clinical services of primarily renal biopsies for the diagnostics of kidney related diseases but other diagnostics are performed such as cilia related diseases.

1. TEM image of tubuloreticular structures in the glomerular endothelial cells of a SLE patient. 2. TEM image of electron dense “humps” in the glomerular basement membrane in post-infectious glomerulonephritis. 3. TEM image of type II crystalline inclusions in mitochondria myopathy in muscle biopsies.

Equipment

TEM

TEM images are acquired using a FEI Tecnai Spirit BioTWIN or a Hitachi HT7700 120kV transmission electron microscope both microscopes equipped with 2kx2k Veleta OSiS CCD cameras.

SEM

SEM images are acquired using a Zeiss Gemini Ultra 55 equipped with on-axis SE detector (InLens SE), Everhart-Thornley SE detector (SE2), energy selective backscatter (ESB), angle selective backscatter (ASB) or STEM detector.

Sample preparation

For the TEM and SEM sample preparation we have access to Leica EM AFS, EM TP and UC6 and UC7 ultramicrotomes and cryo-ultramicrotome for the thin sectioning of tissue and cell specimens. SEM sample preparation is done using a Leica CPD030 critical point dryer and a Qourum Q150T ES sputter coater/carbon evaporation. The sputter coater also have glow discharging functionality for the hydrophilization of grids.

Contact

Lars Haag

Forskningssamordnare

Phone: 08-524 836 63

Email: lars.haag@ki.se

Department: H5 Department of Laboratory Medicine