In vitro imaging: microscopy, histology and cytometry
What is imaging applied to biotechnology?
Imaging techniques make it possible to observe organisms functions at a molecular, cellular or tissue level by more or less invasive means. From the observation of living cells using fluorescent molecular markers to the labelling of tissues with specific antibodies, imaging techniques allow the biology of an organism to be visualized precisely.
The observation of the biology of organisms is done at several levels:
- At molecular level, observation is typically done by immunological labelling of molecules of interest, followed by observation by flow cytometry or microscopy.
- At cellular level, by labelling different organelles or by observation of the state of the cell, for example by flow cytometry.
- At tissue level, typically by histology, following different markings of cellular and molecular structures.
- At the level of the whole organism, using electron microscopy techniques after treatment of the organism, or imaging techniques on living organisms.
What are the advantages of using a service provider for imaging analyses?
Access to the latest technologies and expertise
Save time in the experimentation phase
Discover imaging services and connect with the best service providers
Histology, immunohistochemistry and anatomopathology
Histology refers to the microscopic study of tissues and their cellular composition. For this purpose, different labelling techniques are used, such as staining or the use of antibodies. Histology is a technique widely used in the diagnosis of abnormal cells, pathological anatomy or anapath, and the distribution of markers.
Confocal and electron microscopy
Microscopy is a set of imaging techniques at the microscopic scale. Different techniques can be used, such as confocal microscopy (fluorescence microscopy) or transmission and scanning electron (electron beam) microscopy.
Flow cytometry and cell sorting
Cytometry is a technique for studying cells suspended in a sample. It is used in immunology because it allows the labelling of numerous biomarkers simultaneously, up to several dozen with mass cytometry. Cell sorting, adapted from flow cytometry, allows cells to be recovered according to predefined criteria.
The different types of microscopes
There are multiple types of microscopes, here are the different types used specifically in biology.
The stereoscope, also known as a dissecting microscope and stereomicroscope, is a light-illuminated microscope that provides a three-dimensional view of a specimen. It uses two eyepieces at different angles.
Stereoscopes have lower power than compound microscopes. Images are magnified only up to about 100 times, and their application is limited to growth monitoring in cell culture, or basic imaging.
A compound microscope is an instrument for viewing enlarged images of small objects on a glass slide. It can achieve higher magnification levels than stereo microscopes (up to 2000x) or other low-power microscopes and reduce chromatic aberration. Applications remain relatively limited: direct observation without a coupled photographic system.
Confocal microscopes are also microscopes using light. Confocal microscopes allow to magnify specimens strongly with three-dimensional images. They also have higher resolutions, capable of differentiating details up to 120 nanometres from each other. The most common type of confocal microscope is the fluorescent microscope. This microscope uses intense light to excite the molecules in a specimen. These molecules emit light, or fluorescence, which is observed, allowing for higher magnification and resolution.
Transmission electron microscope
The first electron microscope was a transmission electron microscope (TEM) invented in Germany in 1931 by Max Knoll and Ernst Ruska. If light microscopes can magnify up to 1000x or 2000x at best, then the electron microscope magnifies objects up to 10,000x. A TEM works by focusing a single energy electron beam strong enough to pass through a very thin sample. The resulting images are then viewed by electron diffraction or direct electron imaging.
Scanning electron microscope
The scanning electron microscope (SEM) was invented in the early 1930s, and the development of the first commercial system in 1965 by the Cambridge Instrument Company. The SEM works by scanning the surface of a sample with an electron beam. This beam creates different signals, secondary electrons, X-rays, photons and others, all of which help to characterize the sample. The signals are displayed on a screen that maps the material properties of the sample.
Anatomopathology and Histology
Pathological anatomy, colloquially called "anapath", and officially called pathological anatomy and cytology (PAC) or Pathology, is based on the diagnosis of lesions based on their morphological appearance.
In concrete terms, this means making a diagnosis based on a sample of cells or tissues. For this, the specialist doctor will use histological or cytological methods, followed by microscopic observation.
Depending on the markers of interest to be studied, histology is based on different techniques: specific staining of cellular components, immunology to detect certain proteins, in situ hybridization to detect nucleic acids.
Commonly used in anatomopathology, particularly in oncology, histology is at the crossroads of many research projects: in research and "discovery" during the analysis of biomarkers on tissues, during preclinical studies on small animals, and throughout clinical studies.
Types of providers
Cytometry and microscopy require access to state-of-the-art instruments, and university facilities are able to provide such equipment and services.
Some specialized structures may offer microscopy studies, especially for high content screening (HCS) or other projects.
Freelance firms and experts
Small consulting firms offer histology services, and freelancers can carry out anapath studies.
Mass cytometry: what is it?
Unlike conventional or spectral flow cytometry, which is based on fluorescence, mass cytometry (MCM, CyToF), which emerged in the 1990s, is based on an analysis of the atomic mass of molecules.
Fluorophores that used to be coupled to fluorochromes are now coupled to non-radioactive metal isotopes.
Thus, 135 detection channels ranging from 75 to 209 daltons are available. Each channel can be assigned to an isotope, which will be coupled to an antibody or molecule targeting the molecules of interest.
The advantage of mass cytometry is therefore the wide availability of elements that can be used to mark cells: whereas classical cytometry is limited to a few markers used at the same time (largely due to the wavelength overlap of fluorophores and the background noise of the samples), MCM is free from these constraints. This also makes it possible to study rare events and to standardize analyses from quantifiable standards.
Finally, the MCM has a major advantage: sample preparation and study planning are done in a similar way (or sometimes even simplified!) compared to conventional cytometry. This facilitates method transfer and avoids having to start from a scratch.
Estimated fees for this type of service
€ 800 - € 2,500 per microscopic study
€ 100 - € 500 per immunohistochemistry slide
€ 250 - € 2,000 per electron microscopy observation
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