ZEISS Lightsheet Z.1时序成像实验中的外部设备控制与天光照明集成技术指南说明书
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Control of External Devices During
Time Series Acquisition with ZEISS Lightsheet Z.1
Integration of Daylight Illumination into Time Lapse Experiments
Control of External Devices During
Time Series Acquisition with ZEISS Lightsheet Z.1
Integration of Daylight Illumination into Time Lapse Experiments
Author: Dr. Annette Bergter
Martin Beck
Carl Zeiss Microscopy GmbH, Germany
Date: May 2014
Introducing
The primary benefit of light sheet fluorescence microscopy (LSFM) is extremely low light exposure to the sample, in combination with optical sectioning [1,4,5]. This is achieved by illuminating the sample with a thin sheet of light from the side, exciting only the fluorophores within the focal plane of the objective lens (Fig. 1). The detection beam path is arranged at a perpendicular angle to the illumination, so all light emitted from the focal plane can be collected by a camera.
Due to this intrinsic efficiency, LSFM allows for high speed 3D fluorescence imaging on large, living samples, with virtually no photo-toxicity or bleaching.
For the first time it is feasible to image live specimens from multiple directions for a prolonged period of time covering hours or even days of development.
Figure 1
Furthermore, light sheet fluorescence microcopy allows for novel sample mounting approaches, mostly based on sus-pension of specimens in transparent aqueous gels. This allows for sample immobilization, while still permitting organism growth and development [2]. Maintaining appropriate environ- mental conditions are key to a successful long term experiment. In Lightsheet Z.1, the specimen is kept in needed aqueous medium within a sample chamber (Fig. 1). A cover on top of the sample chamber prevents eva-poration and therefore fluctuation of media concentration.
Additionally, temperature and CO
2
concentration are controlled by an integrated incubation solution, ensuring stable conditions during the experiment. To accommodate any additional en-vironmental or experimental needs, it is possible to integrate external devices (1) for activation during imaging pauses in a time lapse experiment. For example, trigger signals could be used to activate a light source for specimen stimulation and plant imaging, or perfusion pumps to exchange tissue culture media or modify its composition or temperature [3,6]. This Technology Note describes the setup of daylight illumination for plant imaging as one example.Trigger-Out Signal
Lightsheet Z.1 can be equipped with the option to send a trigger-out signal (2), delivered during the intervals between image acquisitions of a time series.
The interval of a time series is defined as the time between the start of two consecutive time points (Fig. 2). If acquisition of the data requires less time than the interval, an acquisition pause with no imaging is part of the time series. During the acquisition of a time point, the trigger signal is in its low level state. Once the acquisition is completed, the trigger signal switches to the high state during the pause, and will return to its low level just before acquisition of the next time point begins (Fig. 2). An external device can therefore be active during these pauses without interfering with LSFM imaging. The trigger-out signal is a level trigger with a high level of 3.3 V (nominal value of the high level: > 3.2 V < 4.0 V, and nominal value of the low level: 0 V ± 0.4 V). The minimal working resistance is 5 kΩ. The trigger signal is delivered via a BNC connector, which is found at the back side of the PC for system control of Lightsheet Z.1.
The BNC connector is coupled to the Lightsheet Z.1 PC for system control via PIN 9 and 10 of the 44 pole SUB-HD port of an ISG adapter, while PIN 18 is electrical grounding.
Up to 2 BNC connections can be made to external devices and one device can be chosen to be included in an experiment.
Figure 2
Notes
(1) T his external device is not provided or serviced by ZEISS.
(2) T his is a hardware option of Lightsheet Z.1 system configuration. Please talk to your ZEISS sales representative for further information.
It is found as Inter-Acquisition-Signal in the Time Series tool window in ZEN for Lightsheet Z.1.
Example Setup for Daylight Illumination
For imaging of plants over long periods of time, the trigger-out signal can be used to include daylight illumination. The example shows a setup in which a light bulb of the desired spectrum and power is installed outside the system (Fig. 3–A,C7), and a flexible light guide is used to illuminate the inside of the system cavity (Fig. 3–C3) above the sample chamber (Fig. 3–C 2,5). This installation allows to easily change the light source according to the experimental needs and op-tionally introduce filters for spectral specification of the light. The flexible light guide can be of different diameters.
Apart from the amount of light needed, it is important to consider the 8 mm width of the openings (Fig. 3–C4) leading into the system cavity. The flexible light guide used in this example (417063-9901-000 Flexible light guide 1500,
8/1000 mm, ZEISS Microscopy Online Shop) has a diameter of 8 mm but is actually thicker due to the outer coating. To use it, one of the partition walls in between the openings is removed with a small hand-held buzz-saw (e.g. Dremel®). This is done while this part of the cover is removed from the system to prevent dirt entering Lightsheet Z.1 and damage due to vibration. Removal and later reattachment of the system cover needs to be done by a ZEISS service technician.
For imaging with Lightsheet Z.1 the system cavity door must completely close. A motorized shutter (Fig. 3–A,C6) is posi-tioned in between the light bulb and the light guide, blocking the light while images are acquired with Lightsheet Z.1 (trigger at low level). This shutter opens during the acquisition pause (trigger at high level).
The motor is attached via a circuit board to the trigger out signal and a power source (Fig. 3–B,C8). The motor used for this example is specified to be operated at 24 V, but was used at 12 V to prevent unnecessary heat production.
References
[1] H uisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., and Stelzer, E.H.K. (2004).
Optical Sectioning Deep Inside Live Embryos by Selective Plane; Illumination Microscopy. Science 305, 1007–1009.[2] K aufmann, A., Mickoleit, M., Weber, M., Huisken, J. (2012).
Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope. Development. Sep;139(17):3242-7.[3] M aizel, A., von Wagenheim, D., Federici, F., Haseloff, J., Stelzer, H.K. (2011).
High resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy. The Plant Journal 68, 377-385.
[4] Reynaud, E.G., Kržič, U., Greger, K., Stelzer, H.K. (2008). Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage. HFSP J. Oct 2008; 2(5): 266–275.[5] Selchow, O. and Huisken, J. (2013). Light sheet fluorescence microscopy and revolutionary 3D analyses of live specimens. BioPhotonik 1/2013; 44-47.
[6] Vermeer, J.E., von Wagenheim, D., Barberon, M., Lee, Y., Stelzer, E.H., Maizel, A., Geldner, N. (2014).
A spatial accommodation by neighboring cells is required for organ initiation in Arabidopsis. Science 343, 178-83.A diode is positioned in a parallel circuit (Fig. 3–B) to absorb the stress-peaks induced by the motor coil. With this setup the plant can be illuminated during the experiments without interfering with image acquisition. A day-night rhythm (e.g. 16 h of light to 8 h of darkness) is easily added by using a plug-in timer for the daylight illumination lamp.
Conclusion
Light sheet fluorescence microscopy allows for imaging of live specimens for long periods of time with virtually no photo-damage. This opens new options for long term imaging, with even more demands on tending to environmental needs of the sample. The trigger-out signal, marking image acquisi-tion pauses, provides the option to add specific treatments for the specimen’s benefit, such as daylight illumination in the above example. Using this model circuit diagram, other devices can be added as needed for the experiment, such as a perfu-sion pump to regularly exchange media or even to introduce drugs or other environmental stimuli to the specimen.
Materials List
• Flexible Light Guide (e.g. 417063-9901-000 Flexible light guide 1500, 8/1000 mm)
• Shutter motor (e.g. 24 V/DC motor D23 LOL-F, Kuhnke; run at 12 V)
• Transistor (e.g. BDW 42G)• Resistors (as needed)• Circuit board
• Daylight illumination bulb (hardware store; spectrum and power as needed)• Timer (hardware store)
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