Wednesday, July 15, 2020

A Dedicated Solution for Microbial Growth Analysis


The global microbiome is composed of the microorganisms such as bacteria, algae, and fungus that surround us and are foundational to the world we live in. While microbiologists have been studying microorganisms for centuries, we are barely starting to understand the complexity and diversity, of microbes as a tool and as an agent in the pathophysiology of disease. The growth characteristics of microbes are some of their most commonly studied phenotypic traits and are used for a wide variety of applications, including screening for new antibiotics, characterization of new microbial isolates, and interrogating clones for the bioremediation of toxic waste or wastewater treatment.

Microbial growth is traditionally measured using optical density measurements—most often taken at 600 nm-- as a proxy for turbidity. Originally, these were conducted growing the microbes in an Erlenmeyer flask with shaking incubators, and then measuring the optical density of the suspension in a 1 cm cuvette with a spectrophotometers over time to get a kinetic analysis of the microbial growth. These studies provide robust data on the lag time, log phase, and the time to reach the stationary phase of growth, but are typically laborious, requiring multiple samples from each culture during a 6-, 12-, 24- or even 72-hour time course.

These 3 phenotypic characteristics are used to understand microbial growth and the difference between microbial samples.


The use of multi-well microtiter plates in incubated microplate readers has greatly increased throughput and automated the data acquisition steps. However, microplate readers have some constraints when it comes to microbial growth analysis. In normal aerobic growth conditions, accurate growth measurements require efficient mixing of the samples and controlled incubation temperatures. Also, as the plates require constant shaking and incubation, growth analyses are limited to one plate per instrument.

Mixing is critical to delivering accurate results in two ways. One is to keep the samples properly aerated so dissolved oxygen is not a limiting factor to the growth conditions. The second is to maintain cell suspension. Inconsistently suspended cells can lead to artifacts in the turbidity measurements. Surface tension in the small diameter wells makes it difficult to get proper mixing of microbial samples in microtiter plates. As efficient mixing to provide aeration and to maintain cell suspension are critical to proper microbial cell growth, this limitation have restricted researchers ability to fully realize the throughput and automation aspects of microplate readers. The rigorous shaking required to break the surface tension in microwells can also take a toll on the mechanics of many plate readers.


The other critical element to microbial growth is temperature. Microorganisms are very temperature sensitive. Fluctuations and gradations in incubation temperatures will result in inconsistent growth and have a negative effect on growth analyses in microplate readers, causing erroneous results, and edge or plate effects. Without proper temperature control in microplate readers, the results are variable and unreliable.

The LogPhase™ 600 is the first microplate reader dedicated to microbial growth analysis, addressing the constraints of microplate readers for these assays. The shaking profile of the LogPhase 600 has been tuned, in both the shaking speed and amplitude, to rigorously shake samples assuring proper aeration and suspension of the samples. The shaking mechanism has been designed from the ground up with limited mechanical connections and a robust motor, followed by painstaking testing to make sure this instrument delivers a lifetime of robust performance for microbial growth analyses. The incubation chamber has also been carefully designed with 11 independent thermistors to measure and adjust temperature of the incubation chamber to ascertain consistent temperature conditions across all four plates the reader can measure in a single run. With only one application in mind, LogPhase 600 delivers the highest data quality, reliably, four microplates at a time.



Tell us about your microbial growth studies and learn more about BioTek’s LogPhase 600 here.

By: BioTek Instruments, JD Herlihy, Product Manager

Tuesday, January 21, 2020

Whole Slide Imaging (WSI) and Region of Interest (ROI)


Most folks have used a conventional microscope at some point in their life to view specimens on a glass slide, prepared either in their native state or stained using a variety of methods to help identify different structures within the sample. Over the past several decades, there has been a shift in the way microscopy samples are viewed. Physical slides are now being transformed into digital images using whole slide imaging (WSI). The digitized format provides a resource that is easily shared for a wide range of uses, such as educational aids for scientists and physicians, research tools for academia and industry, and for diagnostic purposes in pathology and clinical medicine.

The specimens come from a wide spectrum of sources that are just as varied as the methods in which they are prepared on the slides. Physical slides can contain a single or several tissue sections, cell types, organisms or structures from both related and unrelated sources. Whole slide imaging is meant to capture the entirety of the slide so as not to miss any important samples that may be present. However, there is a cost to imaging such large areas, especially when higher magnification objectives are used for increased resolution. Of primary concern is the large amount of data that is generated, given that many samples require several images per field of view to capture all of the colors that may represent the true visual aspects of a sample.
Recent advances in imaging instrumentation can help to alleviate the need to image an entire slide at high magnification to capture the detail needed for analysis. The recent launch of the Cytation™ 7 by BioTek Instruments provides a method to rapidly image a whole slide or part of a slide to determine the region(s) of interest (ROIs).


Once the ROI selection(s) is made (as shown outlined in green above), subsequent imaging of those particular regions at a higher resolution and/or with more complex imaging methods, such as fluorescence imaging, high contrast brightfield, phase contrast or color brightfield, can be performed using automated methods as seen below.


The image below shows a ROI of cardiac tissue (tissue section on the left in the above image) imaged at 10x with one individual tile shown in an exploded view.


The combination of an upright microscope and inverted fluorescence microscope in a single instrument provides a unique opportunity for users to determine the most important areas to image, using more sophisticated, automated imaging techniques while saving valuable time and data storage resources.

By: BioTek Instruments, Peter J. Brescia Jr., MSc, MBA



Thursday, October 24, 2019

Monarch butterfly: A Beautiful Creature That Feasts on Poison

A recent Facebook post by a work colleague regarding his son being a budding scientist caught my attention. The two of them had found a Monarch butterfly wing in their yard and were looking at it under a microscope.
Monarch
Figure 1.  Female Monarch butterfly.
The Monarch butterfly (Danaus plexippus) is certainly one of the most beautiful of all butterflies and as its name suggests, is considered the king of the butterflies by many. As with all insects, Monarch butterflies go through four stages during one life cycle. The four stages of the Monarch butterfly life cycle are the egg, the larvae (caterpillar), the pupa (chrysalis), and the adult butterfly.
Life cycle stages of the Monarch butterfly
Figure 2.  Life cycle stages of the Monarch butterfly. (Composite of images provided by Wikipedia.)
At the start, the eggs are laid on milkweed plants. They hatch into baby caterpillars, also called the larvae. It takes about four days for the eggs to hatch. Then the baby caterpillar doesn’t do much more than eat the milkweed in order to grow. After about two weeks, the caterpillar will be fully-grown and find a place to attach itself so that it can start the process of metamorphosis. It will attach itself to a stem or a leaf using silk and transform into a chrysalis. Over the 10 days of the chrysalis phase, the body parts of the caterpillar undergo a remarkable transformation, called metamorphosis, to become the beautiful parts of the butterfly. The Monarch butterfly will emerge from the chrysalis and fly away, feeding on flowers for its remaining short life of about two to six weeks.

When I was child, it was fascinating to capture a few Monarch caterpillars from milkweed plants and watch them form a chrysalis and eventually turn into a butterfly. The observations took place at home or in the school classroom.
Figure 3. Milkweed (Asclepias syriaca) showing flowers and latex.
Figure 3. Milkweed (Asclepias syriaca) showing flowers and latex. (Courtesy of Wikipedia)
The interesting point of this is that milkweed (Asclepias), which the larva (caterpillar) feeds on exclusively is quite toxic. Asclepias is a genus of perennial flowering plants known as milkweeds, named for their latex, a milky substance containing cardiac glycosides termed cardenolides, exuded where cells are damaged. Cardiac glycosides affect the sodium (Na+/K+-ATPase) pump of cells. Sodium ion pumps create ion imbalances in cells critical for cardiac and nerve cell function. These are the type of compounds that Agatha Christie featured in a number of her murder mysteries such as Herb of Death, Postern of Fate, and Appointment with Death. Milkweed should kill the caterpillar, but it doesn’t. In fact, the caterpillars store the toxins in their bodies as a defense mechanism against birds that would like to eat them. The bright orange coloring of their wings is actually an “Unsafe to eat” message to animals. So what should kill them actually makes them stronger; the question is how? In a recent article in Nature, researchers used genome editing to retrace the evolution of toxin resistance in the Monarch butterfly. It turns out that only three gene mutations are necessary. These involve amino acid changes at position 111, 119, and 122 of the ATPĪ± subunit of the Na+/K+-ATPase pump. These gene mutations did not occur at once, but rather developed sequentially. Using CRISPR/Cas-9 to edit genes, the group was actually able to make Drosophila fruit flies resistant to milkweed. Studies showed that the mutant flies were 1000 times more resistant to milkweed than the wild type. Using fruit flies, biologists found that these adaptive mutations are not without a cost. It turns out that these mutations had to occur in a specific order. The first mutation, while altering the structure of the pump and conferring some resistance to milkweed also causes neurological problems. The second mutation amended the pump slightly and fixed the neurological problem. This allowed the last mutation, which confers most of the milkweed resistance. The third mutation alone resulted in intolerable neurological seizure issues. Only with the second mutation, would the neurological issues with the third mutation be alleviated.
Figure 4.  The budding scientist at work in his lab.
Figure 4.  The budding scientist at work in his lab.

Oh, back to the budding scientist. His Dad works with me at BioTek, where we manufacture a wide variety of research instrumentation including automated microscopes and imagers, and the software necessary to capture and process image files. He is also a camera buff who is obviously sharing his passion with his son. They managed to take a number of microscopic images of the Monarch butterfly wing, and using BioTek’s Gen5™ software, stitched the individual images into an amazing composite. You can see their work in Figure 5. I think he has a future in science!

Figure 5.   Composite stitched image of wing cells from a Monarch butterfly wing using Gen5.
Figure 5.   Composite stitched image of wing cells from a Monarch butterfly wing using Gen5. 


By: BioTek Instruments, Paul Held PhD, Laboratory Manager

Wednesday, July 31, 2019

Laser Focused and Then Some


When most people hear the word “laser”,  they think of sci-fi thrillers such as Star Wars,  with its hand- held laser blasters, but Albert Einstein proposed the idea of a laser over 100 years ago. Einstein based his theory of stimulated light emission on fundamental physics, more specifically quantum theory. Having recently shown that light was derived of packets of energy (photons), Einstein postulated that if the atoms making up the material are provided with excess energy, individual excited atoms emitting photons could stimulate other excited atoms nearby to do the same. As a result, all photons will have equal energy and move off in the same direction. While the theory was sound, it would take decades before suitable technology was available that would allow the idea to be put into practice. Ultimately, it was shown that when a material is pumped with energy in a mirrored cavity, photons bounce back and forth amplifying the emission of photons. The photons were then allowed to escape through a transparent section in the mirrored surface as a laser beam. Charles Townes at Columbia University produced the first device proving the theory in 1953. The device was capable of amplifying microwaves and was coined the maser.
Tatoute. 2006. Wikipedia.
In 1960, Theodore Maiman, at the Hughes Research Labs in California, produced the first visible-light laser with ruby as the laser medium. However, at this time the laser had few applications,  as did many discoveries stemming from basic research. This was to change toward the  end of the 20th century,  as laser research saw a large expansion and development of high-powered gas, chemical and semiconductor-based lasers. It was not until the development of a laser that worked at room temperature with little or no cooling, that the first widespread use outside of research was realized, in the form of  the compact disk (CD). Today most lasers are of the semiconductor diode type and found throughout industry as well as consumer products of all types. Many lasers have been adapted for biological and biomedical applications ranging from basic research to medical procedures.
Demonstration of a Helium-Neon laser at the Kastler-Brossel Laboratory in Paris. Monniaux, D. 2004. Wikipedia.
Over the past several years, BioTek has implemented lasers in several instruments. The first  was a 680 nm semiconductor laser incorporated into the Synergy Neo for use with PerkinElmer AlphaScreen® technology. BioTek then incorporated a red semiconductor laser as a rapid focusing method during image acquisition for our Cytation™ and Lionheart™ product lines. Most recently, a nitrogen laser operating a 337 nm is an option for the Synergy™ Neo2 Hybrid Multi-Mode Reader
for peak TR-FRET performance. The laser produces approximately 6x more energy than a xenon flash lamp at that wavelength and can flash approximately 2x faster. This results in ideal performance for high throughput screening where both sensitivity and high sample throughput are required. Typical assays include GPCR, kinase, biomarker and cytokine assays using technologies such as Cisbio HTRF®, LanthaScreen™, DELFIA® and LANCE®.
BioTek Instruments, Inc. Synergy Neo2 w/ laser module.



Learn more about BioTek’s patented Laser Autofocus utilized in the Lionheart Automated Microscope and Cytation Cell Imaging Readers.



By: BioTek Instruments, Peter J. Brescia Jr., MSc, MBA

Friday, June 28, 2019

Scratch Assay Starter Kit: Simplifying and Streamlining the Wound Healing Assay

Cell migration is a fundamental biological process important for normal tissue development and pathological processes such as wound healing, tissue repair, and cancer metastasis. Migrating cells undergo coordinated changes in shape and size via carefully orchestrated polymerization and depolymerization of cytoskeletal components. This dramatic rearrangement of cellular components generates motile force and establishes directionality to move the cell along toward its destination. With so many moving parts and biochemical pathways involved, keeping track of the details quickly becomes overwhelming. To model cell migration in vitro, scientists use the well-known scratch assay to measure, probe, and characterize this phenomenon in pursuit of medical research and drug discovery. At first glance the scratch assay is simple; a confluent cell monolayer is physically scraped to leave a gap or wound that the remaining cells can migrate into and “heal”. Look closer though and it is easy to quickly get lost in the details. Whether it is generating uniform and consistent scratches, acquiring high-quality time lapse images, or processing and analyzing images in a robust and objective way, there are many opportunities to introduce variability and prevent accurate quantification.

That’s where BioTek’s Scratch Assay Starter Kit comes in. Consisting of the AutoScratch Wound Making Tool, the Scratch Assay App software, and the necessary cleaning reagents, this kit provides a simple workflow to automate and analyze your scratch assay experiments. At the push of a button, the AutoScratch generates consistent, 800 micron wide scratches in 24-well or 96-well microplates. After scratching, move your plate to a BioTek imager and use the Scratch Assay App to automatically acquire time lapse images of each of your scratches and apply an optimized image analysis. Wound closure rates are tracked and graphed automatically; the healing process is easily visualized with time lapse movies. The Scratch Assay Starter Kit streamlines and automates your assays to increase throughput and ensure reproducibility and accuracy. This lets you focus on the complex biology of cell migration without getting bogged down by data collection and analysis.


Watch BioTek's Webinar on Demand:
A fully automated solution for conducting cell migration assays using the AutoScratch Wound Making Tool
Presenter: Joe Clayton, PhD., Principal Scientist

By: BioTek Instruments, Michael Sfregola, Product Specialist, Imaging

Thursday, April 25, 2019

Black Holes: From the Abstract to Reality

I am a cell biologist at BioTek Instruments, where we manufacture a variety of research instrumentation, some of which are digital microscopes. Like many scientists, I’m also a geek that is interested in fields of science other than my area of research, astronomy for example. One thing about astronomy that has fascinated me is that images of stars in space and cultured cells on a slide look very similar. With astronomy, very large objects (stars) are imaged from very far away. As such, they appear really small and need to be magnified in order to see them. With microscopy, things are very close; the objects are very small and need to be magnified to be seen. Both project as bright objects on a dark background.
Figure 1. NIH3T3 Mouse fibroblasts expressing GFP.
Figure 1. NIH3T3 Mouse fibroblasts expressing GFP.
Then just a few days ago the first images of a black hole was released. Black holes are one of those things you always read about in science fiction novels or see in SciFi movies or shows, not on the front page news. I’m not going to lie; my first thought was it looked like the eye of Sauron in the movie adaptation of The Lord of the Rings.

Image from the Event Horizon Telescope showing the supermassive black hole
Figure 2. Image from the Event Horizon Telescope showing the supermassive black hole in the elliptical galaxy M87, surrounded by superheated material. (EHT Collaboration)
Certainly this is a watershed moment for physics, but it took years of work and the collaboration of hundreds of scientists to make it happen. It also required about half a ton of hard drives. Yes, 1000 pounds of computer hard drives. This is another example of the similarity between microscopy and astronomy - data storage requirements. Before I started working with digital microscopes, my experiments required very little data storage. With digital microscopy, I quickly needed a 1Tb hard drive for my PC, then a 4Tb drive, then a 10Tb drive. Currently, I use a 110 Tb RAID array, but soon that won’t be large enough!

Data collection for the historic black hole image began in 2017 with a coordinated effort called the Event Horizon Telescope (EHT), which is a collection of seven radio telescopes from around the world that are linked to combine the capacity of all those telescopes, creating a “virtual” telescope the size of the Earth.

The now-famous image of a black hole comes from data collected over a period of seven days. At the end of that observation, the EHT didn’t have an image — it had a mountain of data. Scientists at MIT had to develop algorithms to take 5 Petabytes of data and make sense of it. That’s 5000 Terabytes!

While not on the same scale, BioTek Instruments provides Gen5™ as a software tool to combine, process and analyze microscopy images for biomedical research. In the end we hope to provide the tools that our customers need to solve their scientific questions in the microscopic world, but we still salute the cosmic success of MIT and the EHT team.


By: BioTek Instruments, Paul Held PhD, Laboratory Manager

Friday, April 5, 2019

Win Cash, or Better Yet, Bragging Rights! Enter BioTek’s 2019 Imaging Competition


If you've been following us for a while, you probably remember that last year we kicked off our 1st annual Imaging Competition called Imaging Perspectives. Researchers from around the world submitted images captured with their Cytation or Lionheart for a chance to win cash prizes and have their image featured in BioTek's annual wall calendar. Well, the competition proved a success! We loved seeing all of the images and learning about the various applications customers are running with their instruments. So much so that we decided to do it again!

Here’s your chance to show the world what you are working on, and to share the art and beauty that’s often found in science. If you have a favorite image (or three!) that you’ve captured using a BioTek Cytation or Lionheart imager, visit our contest page and submit your entry today! You could win one of three cash prizes (1st place = $1,000, 2nd place = $500, 3rd place = $250) as well as the chance for your image to be featured in our 2020 wall calendar.

To get your creative juices flowing, here are the top three photos from last year:

Imaging Perspectives 2018 Winners

You can click here to see all of our 2018 winners.

Entries for our 2019 competition will be accepted now through the end of July. We can’t wait to see what this year’s submissions will bring!!

Enter now at www.biotek.com/perspectives.

By: BioTek Instruments, Tara Vanderploeg, Marketing Specialist