The imaging of biological samples, or bioimaging, plays a key role in current life science research, enabling scientists to analyze molecules, cells and tissues from a range of living systems. Developments in microscopy techniques and associated tools now allow imaging across an extensive range of scales, from 1-2 nm to whole organism phenotyping.
Types of microscopes
Confocal microscopyBeams of focused light are scanned across the specimen, creating optical sections, eliminating out-of-focus glare.
Fluorescence microscopyFluorochromes enable the identification of cells and molecules with a high degree of specificity.
Transmission electron microscopyA beam of electrons passes through a very thin specimen to produce a highly detailed 2D black and white image.
Scanning electron microscopyA beam of focused electrons bounce off a sample (often coated in a thin layer of metal such as gold) to create a 3D black and white image of the sample surface.
Cryo-electron microscopyUsed in structural biology to study biomolecules, samples are studied at cryogenic temperatures without requirements of staining or fixing, enabling observation of close to a natural state.
Super-resolution microscopyAble to break the light diffraction barrier to image sub-cellular processes.
Benefits of bioimagingOne of the major draws of bioimaging is its ability to observe details and understand processes that are difficult to analyze in other ways. For example, in a recent study published in Frontiers in Cellular and Infection Microbiology, bioimaging approaches were used to help researchers from the University of Copenhagen uncover mechanisms of adhesion and invasion of the opportunistic pathogen Streptococcus zooepidemicus in epithelial cells. This organism, which is responsible for endometritis and infertility in mares, was thought to be able to hide for prolonged periods of time in the endometrium, but how it achieved this was unknown. The researchers aimed to determine whether S. zooepidemicus has an intracellular phase, enabling it to hide from the immune system. Field emission scanning electron microscopy (FESEM) allowed the researchers to observe three different invasion mechanisms of S. zooepidemicus, including internalization by the host cell membrane. Speaking about these observations, Bolette Skive, lead author of the paper tells us, “I think these different mechanisms of internalisation would be difficult to acknowledge without the use of bioimaging.” This is just one example of a discovery that may not have been possible without being able to look at the structure of the cells in such close detail, an ability that bioimaging techniques such as FESEM enable.
Bioimaging challengesDespite the advantages that bioimaging can offer, there are some drawbacks associated with the use of microscopes. In addition to the expense of purchasing, housing and maintaining equipment, there is often a need for considerable sample preparation. In the case of FESEM for instance, the sample must first be dehydrated and dispersed with gold particles, which can be a laborious and time-consuming process. Also, the super resolution of techniques such as FESEM can sometimes be a double-edged sword. For example, in the study outlined above, FESEM was extremely valuable for looking at the structure and interphase between the bacteria and the cell. However, when the researchers used less inoculant to study the cells over a 23 hour period to see if they were surviving, it was very difficult to look for small numbers of bacteria. In cases such as this, complimentary techniques like fluorescence microscopy are often needed to also gain an overview of the sample.
Overcoming quantitation challengesAnother potential challenge of bioimaging studies is quantitation, and finding some objective measures to substantiate findings. Often this will mean researchers must devise their own methods of quantitation, such as applying algorithms. In the study outlined above, researchers were required to develop a semi-automated algorithm to identify whether the S. zooepidemicus bacteria were dead or alive, as this could not be determined by the images alone. Bolette tells us that having to go and proofread everything for this “was really laborious,” and involved some degree of subjectivity due to difficulties characterizing cells. The issue of quantitation seems to be common across the bioimaging community, and even with the emergence of new methods, comparisons between studies will be difficult as a result of different techniques and analysis across laboratories.
The role of imaging centers and core facilitiesImaging equipment can also be bound up to software and require a significant amount of user training, presenting challenges for researchers unfamiliar with its operation. Some of these difficulties can be lessened by working with a core facility, where high quality equipment and knowledgeable staff are available to support researchers. Having a dedicated team to maintain the instruments and give instruction for use can give researchers access to, and the confidence to use a wider range of equipment. Facility staff are also usually able to assist with study design and optimisation, helping researchers to obtain the best images possible. Although the cost of these services must be factored in, most would agree that the value more than justifies the expense.
Looking to the future
Bioimaging technologies are rapidly developing, with continued improvements in resolution capabilities, image analysis and data management enabling new boundaries for scientific discovery. Improvements in quantitative methods will further increase the potential that imaging can offer researchers, and the ability to integrate image data with other data types is likely to be another key area of development.
Acknowledgement: With thanks to Bolette Skive, PhD student at the University of Copenhagen for her insights and time.