Fluorescence Time-Lapse Imaging of the Streptomyces venezuelae Life Cycle

Samenvatting

Source: Schlimpert, S. et al. Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device. J. Vis. Exp. (2016)

This video demonstrates fluorescence time-lapse imaging of the complete life cycle of the bacteria Streptomyces venezuelae using a microfluidic device to visualize germination, hyphal growth, and sporulation in real time.

Protocol

1. Set Up of the Microscope and the Time-lapse Protocol

Note: This method was implemented on a fully motorized and automated inverted widefield microscope equipped with a sCMOS camera, a metal-halide lamp, a hardware autofocus, a stage holder for 96-well plates and an environmental chamber.

1. Pre-warm the environmental chamber to 30 °C in advance in order to prevent problems with the autofocus after starting the experiment. The time required depends on the environmental chamber used, the microscope and the heating system. Start to heat up the system the night before the experiment.
2. Turn on the microscope and the microscopy auto....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
B04A CellAsic ONIX plate for bacteria cells	Merck-Millipore	B04A-03-5PK	Microfluidic culture plates
CellAsic ONIX Microfluidic Perfusion System and ONIX FG (version 5.0.2)	Merck-Millipore	EV-262	The latest ONIX vesion (July 2015) and instructions on how to use the programme can be found here: http://www.merckmillipore.com
Axio Observer.Z1 Microscope	Zeiss	431007-9902-000	Fully automated and motorized inverted widefield microscope
Incubator XL multi S1 with Temperature Module S1 and Heating unit XL S2	Zeiss	411857-9061-000	Environmental chamber surrounding the microscope
Plan-Apochromat 100x/1.46 Oil DIC objective	Zeiss	420792-9800-000
Ocra FLASH 4 V2	Hamamatsu Photonics K.K.	C11440-22CU
Illuminator HXP 120V	Zeiss	423013-9010-000
FL Filter Set 46 HE YFP shift free	Zeiss	489046-9901-000	Fluorescent filter set, excitation 500/25 nm, emission 535/30 nm
FL Filter Set 63 HE RFP shift free	Zeiss	489063-0000-000	Fluorescent filter set, excitation 572/25 nm, emission 629/30 nm
Mounting frame K-M for multiwell plates	Zeiss	000000-1272-644	Stage holder for microfluidic plate
ZEN pro 2012	Zeiss	410135-1002-120	Microscope control software
ZEN Module Time Lapse	Zeiss	410136-1031-110	Software module to set up time-lapse microscopy experiments
ZEN Module Tiles/Positions	Zeiss	410136-1025-110	Software module to save specific stage positions (xzy)
Fiji	open-source software package	http://fiji.sc/Fiji	Generation of time-lapse movies
PBS (phosphate buffered saline)	Sigma	P4417-100TAB	Used to refill inlet wells of unused lanes in B04A plates in order to prepare plate for short-term storage.
SV60	John Innes Centre strain collection		S. venezuelae strain expressing divIVA-mcherry and ftsZ-ypet