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Over the past several centuries, we have learned to manipulate light to peer into previously invisible worlds—those too small or too far away to be seen by the naked eye. Through a microscope, we can examine microbial cells and colonies, using various techniques to manipulate color, size, and contrast in ways that help us identify species and diagnose disease. This chapter explores how various types of microscopes manipulate light in order to provide a window into the world of microorganisms. By understanding how various kinds of microscopes work, we can produce highly detailed images of microbes that can be useful for both research and clinical applications.
- 3.1: How Microscopes Work
- Visible light consists of electromagnetic waves that behave like other waves. Hence, many of the properties of light that are relevant to microscopy can be understood in terms of light’s behavior as a wave. An important property of light waves is thewavelength, or the distance between one peak of a wave and the next peak. The height of each peak (or depth of each trough) is called the amplitude.
- 3.2: Staining Microscopic Specimens and Descriptions
- In their natural state, most of the cells and microorganisms that we observe under the microscope lack color and contrast. This makes it difficult, if not impossible, to detect important cellular structures and their distinguishing characteristics without artificially treating specimens. Here, we will focus on the most clinically relevant techniques developed to identify specific microbes, cellular structures, DNA sequences, or indicators of infection in tissue samples, under the microscope.
- 3.3: Cells as Living Things
- The theory of spontaneous generation states that life arose from nonliving matter. It was a long-held belief dating back to Aristotle and the ancient Greeks. Experimentation by Francesco Redi in the 17th century presented the first significant evidence refuting spontaneous generation by showing that flies must have access to meat for maggots to develop on the meat. Louis Pasteur is credited with conclusively disproving the theory and proposed that “life only comes from life.”
Thumbnail: A compound microscope in a Biology lab. (CC -BY-SA 4.0; Acagastya).
Many organisms (bacteria) and parts of organisms (cells) that biologists study are too small to be seen with the human eye. We use microscopes to enlarge specimens for our investigation. The word microscope means “to see small” and the first primitive microscope was created in 1595.
There are several types of microscopes but you will be mostly using a compound light microscope. This type of microscope uses visible light focused through two lenses, the ocular and the objective, to view a small specimen. Only cells that are thin enough for light to pass through will be visible with a light microscope in a two dimensional image.
Another microscope that you will use in lab is a stereoscopic or a dissecting microscope. This type of microscope uses visible light view thicker, larger specimens, such as an insect, in 3D. Since you are viewing larger samples, the magnification range of the dissecting microscope is lower than the compound light microscope.
Your instructor will review the parts and functions of the compound light microscopes that we will be using throughout the semester. Fill in the table on the next page to help you remember this important information. You will likely refer back to this page frequently. Here is a picture of a light microscope for you to label and take notes on.
|Part of Microscope||Function|
|Stage control knob|
|Substage lamp (Illuminator)|
3 - Microscopy and Cellular Diversity
This exercise introduced the different types of microscopes used in our laboratory to study cells and cellular structure. You should know the parts of these microscopes and their functions, and the procedures for preparing wet mounts, focusing and viewing specimens, and proper care and storage of microscopes.
You examined a variety of cell types, ranging from prokaryotic cells (cyanobacteria and bacteria) to eukaryotic cells (plant cells and animal cells). You should be able to recognize each of these cell types as well as any subcellular structures visible with the light microscopes. In addition you should be able to name bacterial types and identify Oscillatoria , Anabaena , and Gloeocapsa . You will be expected to be able to estimate the size of a cell or organism as seen through the microscope.
Review the images on this page to test your knowledge. Concentrate on those structures in bold face type and those that you were asked to label on your diagrams.
To access these images in PowerPoint format, click here.
Novel microscopy method at UT Southwestern provides look into future of cell biology
DALLAS &ndash June 28, 2021 &ndash What if a microscope allowed us to explore the 3D microcosm of blood vessels, nerves, and cancer cells instantaneously in virtual reality? What if it could provide views from multiple directions in real time without physically moving the specimen and worked up to 100 times faster than current technology?
UT Southwestern scientists collaborated with colleagues in England and Australia to build and test a novel optical device that converts commonly used microscopes into multiangle projection imaging systems. The invention, described in an article in today&rsquos Nature Methods, could open new avenues in advanced microscopy, the researchers say.
&ldquoIt is a completely new technology, although the theoretical foundations for it can be found in old computer science literature,&rdquo says corresponding author Reto Fiolka, Ph.D. Both he and co-author Kevin Dean, Ph.D., are assistant professors of cell biology and in the Lyda Hill Department of Bioinformatics at UT Southwestern.
&ldquoIt is as if you are holding the biological specimen with your hand, rotating it, and inspecting it, which is an incredibly intuitive way to interact with a sample. By rapidly imaging the sample from two different perspectives, we can interactively visualize the sample in virtual reality on the fly,&rdquo says Dean, director of the UTSW Microscopy Innovation Laboratory, which collaborates with researchers across campus to develop custom instruments that leverage advances in light microscopy.
Currently, acquiring 3D-image information from a microscope requires a data-intensive process, in which hundreds of 2D images of the specimen are assembled into a so-called image stack. To visualize the data, the image stack is then loaded into a graphics software program that performs computations to form two-dimensional projections from different viewing perspectives on a computer screen, the researchers explain.
&ldquoThose two steps require a lot of time and may need a very powerful and expensive computer to interact with the data,&rdquo Fiolka says.
The team realized it could form projections from multiple angles by optical means, bypassing the need to acquire image stacks and rendering them with a computer. This is achieved by a simple and cost-effective unit consisting of two rotating mirrors that is inserted in front of the camera of the microscope system.
&ldquoAs a result, we can do all this in real time, without any noticeable delay. Surprisingly, we can look from different angles &lsquolive&rsquo at our samples without rotating the samples or the microscope,&rdquo Fiolka says. &ldquoWe believe this invention may represent a new paradigm for acquiring 3D information via a fluorescence microscope.&rdquo
It also promises incredibly fast imaging. While an entire 3D-image stack may require hundreds of camera frames, the new method requires only one camera exposure.
Initially, the researchers developed the system with two common light-sheet microscopes that require a post-processing step to make sense of the data. That step is called de-skewing and essentially means rearranging the individual images to remove some distortions of the 3D-image stack. The scientists originally sought to perform this de-skewing optically.
While experimenting with the optical de-skewing method, they realized that when they used an incorrect amount of &ldquode-skew,&rdquo the projected image seemed to rotate.
&ldquoThis was the aha! moment. We realized that this could be bigger than just an optical de-skewing method that the system could work for other kinds of microscopes as well,&rdquo Fiolka said.
&ldquoThis study confirms the concept is more general,&rdquo Dean says. &ldquoWe have now applied it to various microscopes, including light-sheet and spinning disk confocal microscopy.&rdquo
Using the new microscope method, they imaged calcium ions carrying signals between nerve cells in a culture dish and looked at the vasculature of a zebrafish embryo. They also rapidly imaged cancer cells in motion and a beating zebrafish heart.
UTSW co-authors include Bo-Jui Chang, Etai Sapoznik, Theresa Pohlkamp, Tamara S. Terrones, Erik S. Welf, Vasanth S. Murali, and Philippe Roudot.
Researchers from the MRC Laboratory of Molecular Biology, Cambridge, United Kingdom Calico Life Sciences LLC, South San Francisco, California and the Walter and Eliza Hall Institute of Medical Research and the University of Melbourne, both in Australia, also participated.
The research received support from the Cancer Prevention Research Institute of Texas (RR160057), and the National Institutes of Health (T32CA080621 F32GM117793 K25CA204526, R33CA235254, and R35GM133522). Fiolka has filed a patent for the scan unit and its applications to microscopy. Additional disclosures are in the paper. The researchers made both the data used in the study and the software code available online. Access details are in the study.
Fiolka is a member of the Harold C. Simmons Comprehensive Cancer Center. Visit the Fiolka lab here.
About UT Southwestern Medical Center
UT Southwestern, one of the premier academic medical centers in the nation, integrates pioneering biomedical research with exceptional clinical care and education. The institution&rsquos faculty has received six Nobel Prizes, and includes 24 members of the National Academy of Sciences, 16 members of the National Academy of Medicine, and 13 Howard Hughes Medical Institute Investigators. The full-time faculty of more than 2,800 is responsible for groundbreaking medical advances and is committed to translating science-driven research quickly to new clinical treatments. UT Southwestern physicians provide care in about 80 specialties to more than 117,000 hospitalized patients, more than 360,000 emergency room cases, and oversee nearly 3 million outpatient visits a year.
These advances have been the result of contributions from scientists in many different fields. Physicists have provided much of the technology, such as the advanced electron detectors that increased the speed and sensitivity of modern cryo-EM devices. Chemists have developed brighter florescent probes that illuminate targets for longer. Statisticians and computer scientists have improved image processing and analysis techniques. “The acceleration in imaging has come about through this incredible synergy,” says Jennifer Lippincott-Schwartz, a cell biologist at the Howard Hughes Medical Institute’s Janelia Research Campus in Ashburn, Virginia, who helped to lay the foundations for the development of super-resolution microscopy with work during the 1990s on the use of green fluorescent proteins to visualize cellular trafficking pathways in living cells 2 .
Many advances have been made using these microscopy tools. Lippincott-Schwartz and her colleagues, for example, used a form of light-sheet fluorescence microscopy with confocal microscopy to capture 3D colour footage of the interactions between different types of organelle. “We were able to map out the relationships between six types of organelles, how fast they were moving and the contacts they made with each other,” says Lippincott-Schwartz, whose paper 3 was published in Nature in 2017. “That’s important if you want to understand the cross-communication between organelles, which is one of the big interests among cell biologists right now.”
Jennifer Lippincott-Schwartz demonstrates a microscope capable of super-resolution imaging. Credit: Matt Staley
The growing availability of these advanced techniques presents opportunities for early-career cell biologists. Most obviously, it increases the number of processes cell biologists can probe. “These techniques open up tremendous vistas for the types of questions we can answer,” says Lippincott-Schwartz. Structural biologist David Barford at the MRC Laboratory of Molecular Biology in Cambridge, UK, has used cryo-EM to advance understanding of some of the cellular mechanisms involved in mitosis 4 , a type of cell division that results in the formation of two daughter cells with the same chromosomes as the parent cell. “For academic scientists, the ability to determine structures at atomic resolution with electron cryo-microscopy can be very important in the design of new experiments and testing of biological hypotheses,” he says.
Barford adds that the potential benefits to early-career researchers of acquiring an in-depth understanding of the latest imaging techniques could extend beyond the immediate research questions they are seeking to answer. “Drug companies are becoming very keen on electron cryo-microscopy as a means to determine the structures of proteins and drug targets, so moving into it could be a very good career choice,” he says. Barford also thinks these techniques will grow more important and overtake older techniques used by biologists. “It will probably supersede crystallography in the job market.”
It is impossible to become proficient in the use of all or even many of the latest imaging tools. Early-career cell biologists seeking to use them need to decide whether to specialize in a particular technique, or to identify collaborators who can do it for them (see ‘Meetings of minds’ for some of the conferences popular in cell biology). Ridley, who studies the role of cell migration in cancer progression, advises those doing PhDs to take up any opportunities available to them to get a flavour of the different techniques. “I would recommend that anyone doing a PhD programme with the option to do rotations in different labs and gain experience of different imaging areas to do so,” she says. “Even if you don’t become an expert in electron microscopy, for example, working in that area for a couple of months will give you an understanding of what it can and can’t do.” Barford adds that researchers who leave it to collaborators to do their imaging for them risk falling behind in other ways. “If you become just a user rather than a developer, it limits your future potential to contribute to the field through developing and advancing the technology.”
Meetings of minds
Delegates at the joint meeting of the American Society for Cell Biology and the European Molecular Biology Organization in 2018. Credit: Paul Sakuma Photography
Symposia and conferences are good for getting updates and overviews of a field.
Researchers often have to choose between going to broad or specialized meetings. For those seeking an overview of the state of the field, the joint meeting of the American Society for Cell Biology and the European Molecular Biology Organization is by far the largest annual gathering of cell biologists in the world. Around 6,000 people are expected to attend this year’s, in Washington DC on 7–11 December. Subjects to be covered will be wide-ranging, including emerging topics such as non-conventional model organisms, computational modelling and synthetic biology.
Bruce Stillman, president and chief executive of Cold Spring Harbor Laboratory in New York, will give the keynote lecture on his work on chromosome duplication in cells. There will be a variety of symposia, workshops, poster sessions and special-interest sessions. On the day before the main meeting, there will be a full day of session on careers and professional development for academics, and a one-day mini biotech course, at which attendees can learn how scientific discoveries are turned into bioscience ventures. Other sessions will cover careers in non-profit science advocacy, science policy, outreach, scientific infrastructure management and bench-based research in industry.
There are many other options for researchers wanting to dig deeper into a particular branch of the discipline. A symposium called Seeing is Believing, for example, brings together the developers of cutting-edge imaging techniques with those applying them in the lab. This meeting attracted some 400 participants when it was last held, at the European Molecular Biology Lab in Heidelberg, Germany, in October this year. It featured sessions on the latest tools and methods transforming researchers’ abilities to visualize proteins, protein complexes, organelles, cells, tissues, organs and whole organisms.
One of the draws of imaging for Lippincott-Schwartz is its purity as an empirical method for acquiring knowledge. “When you are imaging, you are first observing, then generating hypotheses and then designing approaches for testing your hypotheses. It’s the perfect avenue for fulfilling the scientific method.” She adds that the proliferation of advanced tools has made microscopy all the more attractive as a focus for cell biologists. “It can make imaging a very creative direction to take,” she says.
Nature 575, S91-S94 (2019)
This article is part of Nature Career Guide: Cell biology, an editorially independent supplement. Advertisers have no influence over the content.