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High speed or high resolution imaging

Frequently Asked Questions

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Choosing Your Equipment

Is a laser absolutely required to perform localized point photolysis (uncaging)?

If short exposure times and highly localized spatial control are of interest, then a laser is the best solution.

Uncaging is a function of the power vs. time required to deliver the energy needed to uncage a molecule. A laser can deliver concentrated amounts of energy of a specific wavelength in the shortest time frame with the fastest on/off switching. If a spot size of 5 microns or less is desired, a laser is required as it is not possible to focus the image of a mercury (or xenon) lamp down to this size while preserving its energy.

Can I use a laser to uncage the entire field of view or a large area simultaneously?

No. Since the power delivered to the specimen decreases as the radiusĀ² of the illumination spot, there is simply not enough energy from any of the commonly available lasers once their beams have been expanded to fill a large area.

What is the difference between the Ultima and the SGS Mini-Scanner?

Our Ultima system is designed to be extensible, with multiple accessory ports to adapt the system to changing research needs and emerging technologies.

The Single-Galvo System (SGS) is a budget-conscious model built for static applications. It can be upgraded to an imaging-and-uncaging system, but unlike the Ultima, requires a complete scanhead replacement to do so.

What is the difference between the uncaging/photoactivation optics and the Photoactivation Module?

The uncaging optics (UO) is used to point a laser beam (visible or 2P) to points of interest on a sample for uncaging/ablation purposes.

The Photoactivation Module (PA) is used to couple the visible laser light into the scan head such that it is collimated and aligned for point uncaging. The PA also provides the ability to change the spot size.

When should I use an Acousto-Optic Tunable Fiber (AOTF)?

An AOTF is ideal if you want to photoactivate several points quickly. AOTFs have a very high extinction ratio and are very effective high-speed shutters as they have no moving parts. They are also useful if there is a need to modulate the laser power from one point to another. Prairie's Aurora laser launch comes standard with AOTF.

How does the piezo objective holder integrate with my current objectives/Prairie's other systems?

The Z-piezo Module is designed to work with both smaller and larger objectives, and is integrated into Prairie View software in the same way as the Z-motor. The Z-piezo Module can also be controlled externally through third party software by using an analog output (0 to 10v).

Can I add a photolysis rig to my existing microscope?

Our moving spot Single Path Photolysis head is compatible with most upright light microscopes that allow placement of the unit below the epi-fluorescence illuminator and above the objective lens.

The Laser Point Module is compatible with virtually all current microscopes equipped with standard epi-fluorescence.

Can I use a single high speed Ti: Sapphire laser for multiple two-photon scanners?

Yes. In fact, it is more and more common for researchers to place two Ultima systems on a single table with either one or two lasers shared between them. This is a good way to maximize the investment in lasers by allowing two independent experiments to be conducted simultaneously.

Instrument Specifications

How big can my in vivo specimens be?

Prairie's in vivo systems include taller pillars to raise the scanhead if needed; the objective can be 13 inches or more above the table allowing ample space for both the specimen and a stage.

What is the beam size at the back aperture with 6mm scan mirrors?

Approximately 15mm.

I need larger scan mirrors and high movement speed. Will the size of the mirrors affect the performance of the galvos controlling them?

No, you shouldn't notice any speed reduction with the larger optics.

Do Prairie microscopes offer flexibility in the placement of the detectors?

Yes. The initial configuration is custom-built to your specifications; however, as your needs change, you may wish to change the layout.

Our multialkali PMTs are attached with four screws each and can be swapped with each other or with GaAsP detectors.

How does Prairie's detection pathway compare to those of other microscopes?

Our engineers designed the detection pathway to accommodate multiple configurable dichroic filters in the epi turret. With these and larger exit cone angles, our design combines flexibility with efficiency.

I'm using both multialkali and GaAsP detectors. Can I combine the signals?

Because they have different current outputs, you cannot combine signals from multialkali and GaAsP detectors. However, you can combine the signal from two of the same type of detectors and view it as data on one channel.

Can I use a Prairie system to simultaneously image and uncage?

Yes. Our systems use two independent sets of galvos to control the imaging and uncaging beams, thus providing simultaneous imaging and uncaging capability.

Getting the Most Out of Your Prairie System

Which method of Z-series acquisition is right for me?

Z-motor is the most flexible option, allowing the user to adjust Z position, Pockels cell, PMT high voltage, and other control signals. As a stepper motor, it requires about 300msec to make adjustments to the control signals and move to the next Z position.

For faster acquisition, Prairie also offers a Z-piezo Module. The piezo option has very little time overhead; hence it does not allow adjustments for laser power and PMT high voltages in between slices when used in the "fastest acquisition" mode. The Z-piezo module also provides a higher degree of positioning accuracy than the Z-motors. Travel range is restricted when compared to Z-motor, but is sufficient for most people.

What are the settings for the fastest possible Time series scan?

If you are acquiring a Time series at the same Z position, set your output trigger to "EOF" (end of frame). Then you can select "Max Speed" for the fastest possible scan.

How do I improve the time estimate for acquiring a Time series?

Due to the highly customizable nature of the T-series, it is difficult to provide an accurate estimate for how long acquisition will take. Prairie View gives a best estimate, and it also learns from the most recent acquisition. One way to get a better estimate is to run a T-series and then re-run it with the exact same settings.

How do I use Prairie View to render a linescan in 3D?

To do a freehand 3D linescan:

  1. Collect a Z-stack
  2. Export to a MATLAB program
  3. Mark the points across which you would like to do a linescan in 3D. This creates a text file of points.
  4. Import the text file into Prairie View.

Troubleshooting

My in vivo stage axis seems to be stuck in one position. What should I do?

First, try turning the stepper motor knob in the opposite direction. Make sure the screw(s) that secure the knob on the stepper motor are tight. Often, this is enough to fix a frozen stage.

If the stage is still stuck, the coupling (bellows) between the stepper motor shaft and stage lead-screw may need to be tightened. Follow the photos below to tighten the screws. (Note that your stage motors/mounts may differ slightly.)

After tightening the screws, rotate the stepper motor knob clockwise to translate the x-axis to the right, or counter-clockwise to translate the x-axis to the left.

If this does not resolve your problem, contact our service department and they will be happy to help.

General

In addition to my Prairie system, I need some other items that Prairie doesn't manufacture. Can I order everything through Prairie?

Yes! Prairie offers the convenience of bundling all your microscopy requirements together in one quote. We do the ordering and we make sure everything ships out on time. If you are restricted to a single purchase by grant or other requirements, or if you just don't want to deal with multiple invoices, this service is for you.

Does Prairie guarantee their products? How long does the warranty/extended warranty last?

Yes, Prairie products are covered by a limited warranty for one year from the date of shipment. An extended warranty may also be purchased by the year. During the warranty period, we will repair or replace any defective items purchased through Prairie except consumables such as batteries and computer disk drives. Other restrictions may apply; contact Prairie for more information.

DATA SPOTLIGHT

C. elegans embryo expressing B-tubulin GFP
Image courtesy of Koen Verbrugghe and Chris Malone, Laboratory of Molecular Biology, University of Wisconsin-Madison, Madison, WI

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Time-lapse recordings of tubulin GFP C. elegans embryos during mitosis.
One image was acquired every second with a 100x Super Fluor lens using the SFC.
Image courtesy of Kevin Eliceiri and Koen Verbrugghe, LOCI, University of Wisconsin-Madison, Madison, WI.

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SFC

Download SFC Brochure (PDF)

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