Background of Electron Microscopy-based Lysosome Monitoring
An important step in understanding cellular regulation is to explore the importance of the interconnections between different types of organelles for function. This requires new microscopic approaches to study the spatial and temporal regulation of intracellular processes at the nanoscale. The advent of electron microscopy (EM) has greatly contributed to the nanoscale understanding of the intra-lysosomal structures that underlie many cellular functions. Despite considerable heterogeneity in size and morphology, lysosomes can be identified by transmission electron microscopy (TEM) as electron-dense organelles surrounded by a single membrane. In the past, EM has been used to help diagnose lysosomal storage disorders. Since lysosomal morphology may be heterogeneous, varying between cell types or conditions or even within individual cell populations. Therefore, EM can be popular for providing details of lysosomal ultrastructure.
Fig. 1. High accuracy 3D correlation workflow from live-cell-to-volume EM with iCLSM. (Loginov S V, et al., 2022)
Our Electron Microscopy-based Lysosome Monitoring Services
Deciphering the biogenesis and function of lysosomes and lysosome-associated organelles (LROs) and their dysfunction requires their visualization and detailed characterization by electron microscopy at high resolution. Here, CD BioSciences provides a detailed protocol for studying lysosomal morphology by transmission electron microscopy. We take the following approaches to facilitate lysosome identification and provide additional information. Importantly we will combine the advantages and disadvantages of the following methods to develop the most effective and cost effective solution for you.
- Internalization Probes
We use pulse/tracking experiments with gold particles or horseradish peroxidase (HRP) labeled probes to aid in lysosome visualization.
- Labeling endocytic organelles without the need for immuno-EM.
- Allowing study of the kinetics of lysosomal delivery.
- Can use a large number of antibodies or ligands.
- Can only be used to trace the endocytic pathway to lysosomes.
- Immunoelectron Microscopy
We perform specific antibody staining of lysosomal proteins to facilitate lysosome identification, where the secondary antibody is bound to nanogold rather than fluorescent labeling.
- An easy-to-use immuno-EM technique.
- Any lab can do the labeling.
- Only applicable to labeling the cytoplasmic structural domain of lysosomal proteins.
- Due to the variable size of the enhanced nanogold, only one protein can be labeled at a time.
- 3D Electron Microscopy
Based on a 3D tomographic reconstruction platform containing Electron Tomography (ET), Serial Block Scanning EM, Focused Ion Beam Scanning EM (FIB-SEM), Array Tomography/Automated Tape Collection Ultrathin Slicer SEM (ATUM-SEM). We can achieve exploration of lysosomal physiology with high resolution ultrastructural detail throughout the lysosomal volume.
- Allowing imaging of relatively small sample depths.
- Requiring expensive specialized equipment.
- Can be imaged through large volumes.
- Non-destructive method, sections are preserved on slides for future use.
- Correlation Photoelectron Microscopy
We use fluorescent probes or live-cell light microscopy of ectopically expressed/gene edited fluorescent lysosomal proteins to study lysosomal dynamics.
- More than 108 fluorescent proteins can be visualized in a near-native state in a nanoscale cellular landscape.
- Highly specialized equipment is required.
- Technically challenging and time consuming.
CD BioSciences can meet any reasonable requirements of our clients, taking time and budget into consideration for you. Our aim is to be customer-centric and to provide the highest quality services to customers. Our customer service representatives are enthusiastic and trustworthy 24 hours a day, 7 days a week. If you are interested in our services, please feel free to contact us for more information or a detailed discussion.
Reference
- Loginov S V, Fermie J, Fokkema J, et al. (2022) Correlative Organelle Microscopy: fluorescence guided volume electron microscopy of intracellular processes[J]. Frontiers in cell and developmental biology. 10.