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Seeing the Living Brain

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The Question

How does the brain work?

How does a collection of individual neurons coordinate to make a muscle move and understand what a scent means all in the same few milliseconds of time?
Scientists have been searching for answers to this question for decades, building up a complete picture from isolated bits and pieces of in vitro and tissue culture systems, stained tissue, and functional imaging technologies such as MRI.

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What’s been missing is an actual image of
the complete picture—the ability to directly view neurons firing in a whole, living brain in real time.

This view is exactly what Misha Ahrens and Philipp Keller deliver1 as they push the limits of light-sheet microscopy to image the larval zebrafish brain in action, using wide fields-of-view at single cell resolution, and imaging every 1.3 seconds.

Above: Dorsal and lateral projections of whole-brain, neuron-level functional activity, reported by the genetically encoded calcium indicator GCaMP5G in an elavl3:GCaMP5G fish via changes in fluorescence intensity (ΔF/F), superimposed on the reference anatomy.
Video courtesy of Philipp Keller, Lab Head, Janelia Farm Research Campus.

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Did you know
The oldest PubMed entry about the brain dates from 1809:John Yelloly. A case of Tumour in the Brain, with remarks on the Propagation of Nervous Influence.
Medico-Chirurgical Transactions (now published as Journal of the Royal Society of Medicine). (1809); 1: 183-223. PMCID: PMC2128793.
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Read the Paper (login may be required)
Ahrens, M. B., Orger, M. B., Robson, D. N., Li, J. M. & Keller, P. J. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat. Methods (2013) 10, 413–420. PMID: 23524393.
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Whole Brain GCAMP5G Recordings

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Camera:
(2) Two ORCA-Flash4.0 cameras were used
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Filter:
Brightline BP525/50 band pass (Semrock)
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Objectives:
PlanApochromat 20x/1.0 W (Carl Zeiss) or CFI75 LWD 16x/0.80 W (Nikon)
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SNR:
I/σ = 180 + 11
(mean + s.e.m., n=31)
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Light sheet thickness:
4.25 + 0.80 μm
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Laser power:
0.3 mW at 488 nm and 1.5 μj laser light exposure, slice, volumetric scan
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Photobleaching:
Negligible
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Avgerage data set size:
1 TB specimen 1 h continuous volumetric high-speed imaging per specimen
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Acquisition rate:
0.8 Hz (for a volume of the size 800 × 600 × 200 μm3)
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Lateral resolution:
0.65 μm
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In Nature News
For additional commentary on Ahrens, et al., with no login required, read the Nature News article.
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The Authors Interviewed
See the Reuters interview with Misha Ahrens and Philipp Keller.
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In the Popular Press
Read the article at Neurophilosophy, a science blog hosted by The Guardian.
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THE BARRIERS

To acquire these groundbreaking images of the brain, Ahrens and Keller had to extend the capabilities of existing light-sheet technology to speed up volumetric acquisition time.

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Fast frame rates are important for imaging moving, living systems, whether you are looking at an entire brain or watching microtubules dynamics. See how the chip architecture of the second generation scientific CMOS (sCMOS) cameras help scientists capture biology in motion. Download the quick guide to camera chip architecture.

Above: Sea urchin spermatozoa imaged in rolling shutter mode using 100x objective and 6.5 pixel ORCA-Flash 4.0 camera. Each frame is ~10% of the camera’s field-of-view. Points on the flagellum typically move at 15 microns/ms. Images courtesy of Shalin Mehta, HFSP Postdoctoral Fellow, Marine Biological Laboratory

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Which camera technology is right for me?
Read our guide on choosing between CCD, EM-CCD, and CMOS technology
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Biology in Action
Download our guide to imaging time-sensitive experiments.

Recording images every 1.3 seconds.

The light-sheet microscopy method developed by Ahrens and Keller required continuous acquisition of the whole brain volume, recording images every 1.3 seconds. How many frames per second do your imaging experiments need?
Find out using our quick guide to imaging dynamic bioloigcal systems.

Above: Another example of fast image acquisition—high-speed calcium imaging of an iPS cardiomyocyte. Below: Ahrens and Keller's experimental setup. Image courtesy of Philipp Keller, Lab Head, Janelia Farm Research Campus.

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In light-sheet microscopy, a thin section of the sample is illuminated with a laser light entering from the side.

The fluorescence emitted by reporter molecules in this thin volume is then collected with an objective lens oriented at a right angle to the light sheet. As a direct result of this optical sectioning strategy, light-sheet fluorescence microscopy provides substantially improved imaging speed and signal-to-noise ratio, while minimizing the light exposure of the specimen. Light-sheet microscopy is thus particularly well-suited for biological live imaging applications and has an outstanding potential in the systematic, quantitative study of development and function of complex biological systems. — Misha Ahrens and Philipp Keller

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THE SOLUTION

Advancing light-sheet microscopy technology required a redesign of the existing imaging strategy. The team optimized the hardware components and configuration to streamline communications throughout the microscope control system.

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"In the current implementation of the acquisition workflow, camera performance is approximately on par with the performance of the microscope control framework.”

Ahrens, et al. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat Methods. 2013 May; 10(5):413-20.
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Changing the Game
What’s so special about sCMOS technology? How does it compare to EM-CCD and what experiments is it good for? Learn more.
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Rolling Shutter
Will rolling shutter exposure cause problems for my experiments? Read what Shalin Mehta, PhD., Marine Biological Laboratory, has to say.
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THE POSSIBILITIES

Advanced light-sheet microscopy technology.

With advanced light-sheet microscopy technology, made possible, in
part, by the fast frame-rates and wide fields-of-view of the ORCA-Flash 4.0, neurobiologists can start exploring how collections of neurons act in concert to perform a multitude of sensory, motor, and homeostatic functions. And these advances go beyond neurobiology to study not only the function but also the development of complex biological systems.

Above: Functional imaging of the entire, isolated central nervous system of a Drosophila larva. Image data were acquired using Philipp Keller’s newest lightsheet microscope. The hs-SiMView (high-speed multi-view light-sheet microscope) captures images at a sustained speed of 370 frames per second, offering 25-fold faster multi-view imaging than other state-of-the-art imaging techniques. Courtesy of William Lemon and Philipp Keller (HHMI, Janelia Research Campus).
Read the paper: Whole-central nervous system functional imaging in larval Drosophila

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Next generation light-sheet microscopy

The promise of light-sheet is real-time imaging of neuronal ensembles, allowing scientists to probe functional patterns associated with sensory, motor and homeostatic behaviors. First demonstrated in 2013, the Keller lab radically advances the possibilities in 2015 with a multi-view hs-SiMView. In both systems, the speed of the ORCA-Flash4.0 V2 is essential. Above: The multi-view hs-SiM view set-up.

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By taking a multi-directional approach and revising the optical, mechanical and computational imaging strategy, the new hs-SiMView achieves a 25x speed improvement over the 2013 version. Because of these enhancements, it’s now possible to perform functional imaging of non-transparent biological samples such as the Drosophila larva above. Find details of this 2015 work by Lemon et al. at
DOI:10.1038/ncomms8924

“Philipp Keller ’s work on light sheet imaging of developing animals has been breathtaking, and his latest live imaging of calcium signaling in zebrafish brains is stunning. When I saw these movies for the first time, it left me in awe realizing that we had here a live view of a thinking brain.”

NICO STUURMAN, PHD., UCSF

“To explore neuronal activity in wide areas of the brain by calcium imaging, it is necessary to achieve single-cell resolution deep in the brain with high acquisition rates...The use of light sheet microscopy may solve both penetration and temporal resolution problems, and has been applied to the entire brain imaging”

AKIRA MUTO AND KOICHI KAWAKAMI,2 CITING AHRENS, ET AL.1

“This is a remarkable advance, one that could revolutionize our understanding of the brain circuits generate behaviors and encode learned experiences.”

ADAM C. ROBERTS, BRENT R. BILL, AND DAVID L. GLANZMAN,3 CITING AHRENS, ET AL.1

“Imaging whole-brain activity isn't really new. Neither is measuring the activity of single neurons. Doing both simultaneously, however, is really impressive, and hugely valuable from an experimental standpoint – akin to being able to see the individual dots of a pointillist masterpiece and the painting as a whole all at once.”

ROBERT T. GONZALEZ, SCIENCE WRITER, IO9, IN "THIS IMAGE COULD BE THE FIST STEP TOWARD MAPPING HUMAN THOUGHTS"

REFERENCES

  1. 01. Ahrens, M. B., Orger, M. B., Robson, D. N., Li, J. M. & Keller, P. J. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat. Methods 10, 413–420 (2013).
  2. 02. Muto, A. & Kawakami, K. Prey capture in zebrafish larvae serves as a model to study cognitive functions. Front. Neural Circuits 7, (2013).
  3. 03. Roberts, A. C., Bill, B. R. & Glanzman, D. L. Learning and memory in zebrafish larvae. Front. Neural Circuits 7, (2013).
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