C.L.I.

P – Continuous Live
Imaging Platform for C. elegans
Dynamic Studies

Jan Krajniak and Hang Lu

Georgia Institute of Technology, Atlanta, GA
11/1/2012
 Understanding the Human Nervous System

Better understanding of nervous system crucial for

▪ Understanding neurodegenerative
diseases
▪ Discovering novel drugs and drug
targets

Hurdles are significant

▪ 100 billion+ neurons
▪ Many other interacting systems
▪ Direct in vivo observation difficult
▪ Genetic manipulation difficult

Model organisms very useful

fcweb.sd36.bc.ca/~taylor_paula/nervous

1
 C. elegans: The Great Model Organism

Simple Organism with a Fully

Functioning Nervous System
• 3-day generation time
• Hermaphrodite
• Transparent
• 302 neurons with mapped out 100 µm
anatomical connections
• Capable of behavior

Genes have mammalian or

human homologues
60 µm

Artufucial Brains: OpenWorm 3D project. www.artificalbrains.com/openworm

2
 Imaging C. elegans Nervous System

Specific promoters used in combination

with fluorescent markers
Confocal montage 40X objective (ruIs32(Histone-GFP); itIs44(m-Cherry:PH (PLC-d)) by: Ian Chin-Sang and
Tony Papanicolaou. Wormpics.com

Elucidate role of gene from

Sur-5-GFP expressed in the nuclei.
• Localization Confocal
3 D projection (with shadow) by:

• Change over time Tony

Papanicolaou. Wormpics.com

• Change as response
• Wild type vs. mutant Photo: H. Hutter, Max Planck Institut
, Heidelberg. Nobelprize.org

Many of these processes are dynamic

Animals have to be immobilized

3
Techniques Available for Dynamic Studies

Hallam SJ, Jin Y, Nature, Volume 395, Issue 6697, 1998cc

Klassen, M.P. et al, Neuron, Tsai et el., Analytical Biotechnology, Volume 20, Issue 1, 2009
Volume 66, Issue 5, 10 June 2010

What Is Needed:
• Continuous Observation
• Feeding During Experiment
• Benign immobilization
Krajniak et al. 2010

Chung et al. 2008 Hulme et al. 2010

Chronis et al. 2007 Rohde et al 2011

Chokshi et al. 2009

Seconds to Minutes Minutes to Hours Hours to Days

Time period of studied process
4
 C.L.I.P – Hybrid Platform for Intermediate
Length Studies
FLUSHING OUTLET NUTRIENT
DELIVERY
CHANNEL

LOADING CHAMBER
MAIN OUTLET

TRAPPING CHANNELS
• Immobilize animals with Pluronic WORM “MUZZLE”
F127 • Allows animal feeding

5
 Immobilization – Pluronic F127 and
Microbeads

Tri-Block Copolymer (PEO-PPO-PEO)

• Food additive, drug delivery system, etc.
• Thermo-reversible sol-gel transition

Controlled Transition Biocompatible

⚫ Temperature Controlled ⚫ No effect on C. elegans physiology

⚫ Concentration Dependent ⚫ No deformation

⚫ Reversible

Microbeads used to prevent tail movement

6
Simple 4-Step Loading Procedure

1. LOAD ANIMALS
• Open chamber Inlet

• Begin withdrawing through outlet

• Load solution with suspended animals

7
Simple 4-Step Loading Procedure

2. PRIME FOR IMMOBILIZATION

• Open flushing outlet

• Load 10% w/v PF127 solution

with 10um microbeads

7
Simple 4-Step Loading Procedure

3. IMMOBILIZE ANIMALS
• Close flushing outlet

• Load cooled (0 C) 25% w/v PF127 solution

Continue withdrawal until gel forms

7
Simple 4-Step Loading Procedure

4. BEGIN NUTRIENT DELIVERY

• Open delivery/stimulus inlet

• Continue withdrawal through duration

of experiment

7
 Loading Is Efficient

PROCEDURE OPTIMIZED FOR

• L4/ young adult animals
• < 2 minutes of loading time
• Single loading per channel

DEAL FLOW RATE (2mL/hr)

Average 4+ animals
Can be scaled to larger
array if needed
Higher rate: average <2

8
 Only Animals’ Bodies Get Immobilized

• Sol-gel transition occurs uniformly • Nutrient flow erodes gel in the

through the device undesired areas in <2 min

• Flow engineered to not erode

• Presence of gel is not desired in
gel inside trapping channels
media delivery and muzzle areas
9
Muzzle Design Facilitates Nutrient Delivery

Design allows for

• Perfusion of bacteria through muzzle
• Slow-down of flow compared to bulk
• Lower shear rate and higher residence time

Minimal bacterial buildup occurs over

the period of a standard experiment

10
 Immobilized Animals Feed Properly

Pharyngeal pumping is an
indicator animal feeding
behavior
• Pushes nutrients to gut
• Animals pump in presence of
food

Rate of pumping
• Not significantly different from
standard plate
• <5% lower

11
 Live Imaging with C.L.I.P

Animals’ bodies immobilized

Heads free to move and feed

Sub-cellular object imaging 70 µm

possible

100 µm 45 µm
 CONCLUSIONS

We have developed a hybrid microfluidic platform for short term to

intermediate length studies capable of

– Eficiently loading and immobilizing animals

– Feeding animals during experiments

– High resolution continuous imaging

Platform has broad range of applications

– Normal developmental and functional processes

– Response to stimuli

13
 ACKNOWLEDGEMENTS

Dr. Hang Lu & the Lu Microfluidics Group

ΜTAS 2012 Conference and Organizers

Funding Sources:
NIH NINDS
NSF CAREER
Sloan Foundation

14
 C. elegans MINIMALLY EXPOSED TO
TEMPERATURE OUTSIDE PHYSILOGICAL RANGE
PF127 is loaded cooled to 0 C to enable 0C
proper filling of channels

Physiological range of C. elegans is 15-25C

Temperature measured via fluorescence

• Ratio of Rhodamine B to Rhodamine 110
intensities in PF127
• Followed standard loading procedure

Average temperature at channel inlet 12C

17C transition temperature reached in <20 sec

16