A New Tool Comes to BSCEB

By: Steven Hart

This news may take your breath away…especially because Dr. Torres’ new microscope is the only anaerobic set-up on campus, and because it has so many possible applications for your environmental biotechnology research!  Confocal BRIGHTFIELD, DIC, and EPIFLUORESCENCE modes are all possible, within an anaerobic glove box (+ or – O2), and with the guidance of Dr. Steven Hart, who presented an overview during the Torres/Krajmalnik-Brown Lab Meeting, today (5/20/20).  Steven is developing a detailed SOP for the instrument.  However, he can assist you, now, with training and optimization for your unique experimental needs.  This system is equipped with most excitation and emission filters that work with the most common dies.  Using ThermoFisher Spectraviewer, Steven can help you select viable dyes that don’t damage your cells, that don’t spectrally interfere with each other, and that allow you to evaluate multiple parameters simultaneously.  In addition to a mercury lamp for standard microscopy procedures, four rapid, scanning LED laser channels allow you to pinpoint discreet excitation bands for enhanced intensity to create 3D reconstructions and movies of your biofilms, flocks, and even bulk systems.  You can even image from an open reactor because this microscope can be in an anaerobic environment and we have two extremely good water dipping objectives that can be directly in your media with little interference.  We might even consider building some reactors to accommodate dipping objectives.  By the way, sludge responds very well to FISH.  Even though it’s so opaque, you can image it well by combining confocal and fluorescence techniques.  Almost all of the controls can be managed through the software making them accessible from home, which opens opportunities for long time courses or growth experiments.  Steven can even show you how to make spectacular PowerPoint displays of tiled images. 

Here Steven ascertains sizes of an amoeba and cellulose crystals.
DIC shows distinct biomass edges. You can look at unstained samples to count or assess morphology
Here, Steven used dye-labeled probes to look at the surface of an opaque sample. 
Gray line is a mercury bulb emission. The dotted lines are excitation and emission filters. DAPI is a common DNA stain. How we pair our excitation and emission filter sets increases our flexibility.
If you’re not careful, you would think a lot of this is geobacter but much is crosstalk from DAPI – this is an example of interference
The difference between confocal and epifluorescence microscopy is rastering, or exciting one small region at a time using an aperture to focus small areas of time and increase resolution.
Rastering reduces interference and allows you to stain substructures using multiple colors to generate images with complex information
You’ll get in-focus images of all parts of whatever you’re looking at because layers are scanned individually and then compiled
…but you cannot see around an opaque round or jagged object, since optical sections are dependent on where you collect the image.
Steven has a simple protocol that utilizes counting chambers, with specified volumes and areas, that will help you determine cell counts at very low concentrations, in cells/mL, while they are still alive, using DIC.  You could even do multiple counts over time. 
Here’s a biofilm grown in a MEC, blue are geobacter.  It’s a good example of simple stains (blue = DNA, red = lipids).
We had contamination issues.  We used microscopy to determine what was infecting our system, and where.   Here you see larger structures (fungal hyphae). And we found them on the cathode.  Once you’re trained, if something is going on with your reactor, you can quickly assess what might be going on.
Here’s the same system on a felt cathode but with different stain…you can see fungal structures; the gray image is using a reflection technique.  Carbon felt gave a strong signal. 
FISH allows you to track the spatial distribution of community members, in this case 16S ribosomal DNA and then overlay several layers. You can use genomic DNA or RNA (more complicated); all require specifically designed probes and optimization.
Growing on an activate carbon granule, we found geobacter on the inner layer of a biofilm and others were outermost. Here, we created a 3D reconstructions and movie and we were able to quantitate cell numbers at various depths.
You can combine reflection techniques with stains. 
You can see where Diana’s hollow fiber membrane got nicked as we grabbed it.  We can see very fine biofilm processes you otherwise couldn’t see without a dipping lenses because they would normally get squished during mounting. 
OMG Steven, the way you used PowerPoint, here, was so cool!  (It scrolls the length of the sample.)  Here’s a cross section of a felt anode. We tiled the image to get a continuous picture across the whole depth to see where the fungus was propagating.
This system is really useful for dynamics. If you use the right probes, you can see gradients in a living system over time, including pH and ion concentrations.