Fluorescence Time-Lapse Imaging of the Streptomyces venezuelae Life Cycle

Abstract

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 automation and control software. Use a high numerical aperture (NA) oil immersion objective for optimal signal collection and spatial resolution, such as a 100X, 1.46 NA oil DIC objective. Select appropriate filters and dichromatic mirrors to acquire differential interference contrast (DIC) images and images of yellow-fluorescent and red-fluorescent protein fusions.
3. Place a drop of immersion oil onto the objective and add immersion fluid to the bottom of the imaging window on the microfluidic plate to improve cell-focus during image acquisition. Carefully mount the sealed microfluidic device onto the stage of the inverted microscope. Ensure that the plate is securely placed in the stage holder and does not shift over the course of the experiment.
4. Bring the imaging window of the microfluidic culture chamber into focus using the embedded position markers for orientation. Focus on the leftmost part of the first flow chamber (labeled "A") with the trap size 5, corresponding to a trap height of 0.7 µm.
5. In the microfluidics software, load cells from inlet well 8 at 4 psi for 15 sec. Check the cell density in the culture chamber by moving the stage across the imaging window. If no spores were trapped, repeat the cell loading step or alternatively increase the loading pressure and/or time until the desired cell density is achieved (1-10 spores per imaging window with 2,048 x 2,048 pixels). Avoid overloading the culture chamber.Note: We normally use trap size 5 for imaging Streptomyces, but we have also obtained good results with trap sizes 4 and 3.
6. Start the flow program in the control software and allow the microfluidic plate to heat-equilibrate for 1 hr in the microscope stage before starting image acquisition.
7. In the microscope control software, set up a multi-dimensional acquisition to take multiple images at multiple stage positions over time:
   1. For Autosave: specify a directory for automatic saving of the image files.
   2. For Illumination settings, determine optimal illumination settings for each specific construct in advance. For the outlined experiment, use the following exposure times: DIC 150 msec, YFP 250 msec, RFP 100 msec.
   3. For Time-series: Set up a time series to acquire images at the desired time points in sequence. For imaging the life cycle of S. venezuelae, select a 40-minute time interval over a 24 hr period.
   4. For Stage positions and autofocus: scan the culture chamber by moving the stage and store stage positions for each imaging position of interest. Ensure that the single-stage positions are located sufficiently apart to minimize photobleaching and phototoxicity. Typically, use up to 12 positions. Run autofocus routine for each time point to correct for slow focal drift. If available in the microscope control software, set the autofocus strategy to "local surface update by hardware autofocus". Once the Z-coordinates of the selected stage positions are verified, activate the hardware autofocus.
8. Start the time-lapse experiment in the microscope control software.
9. Check that all stage positions are still in focus at later points. For time-lapse experiments running over several hours, we occasionally observe a stage drift even when using autofocus. If stage positions need to be refocused, stop the experiment at an appropriate time point, adjust the focus and restart the experiment within the defined imaging time interval.
10. Stop image acquisition after 24-30 hr or when the hyphae in the region of interest have differentiated into spores. Then, stop the flow program in the software and disassemble the microfluidic device.
11. Prepare used microfluidic plate for short-term storage. Remove remaining media from well 1 to 6, empty waste well 7 and cell loading well 8. Under sterile conditions, re-fill used wells of lane “A” and wells of unused lanes (“B” to “D”) on the plate with sterile phosphate-buffered saline (PBS). Seal the plate with parafilm to prevent it from drying out and store at 4 °C.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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