Questions and Answers About LSI Products
   
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Choosing a chamber
How do I choose a chamber that is best suited to my applications since LSI offers many different models?
  1. Talk with us.
  2. Decide whether you may want to run experiments on one or two vessels. Dual vessel chambers enable one to:
    - Test a second, equilibrated vessel after the experiment on the first vessel has finished, or is equilibrating from exposure to an agent.
    - Connect the two vessels as in a bioassay.
    - Compare responses of two vessels that may be of different sizes, from different vascular beds, or superfused and/or perfused with different agents.
    For these features, choose one of the dual vessel chambers. Otherwise, consider choosing one of our single chambers, and see if its features are sufficient for your needs.
  3. Select a chamber which has the unique features in the table below required by your experiments.

Features

Chamber

A small volume for using peptides or expensive agents.

CH/1/SH, CH/1/AU/SH, CH/2/B, CH/2/B/SH

Ability to work with fluorescent markers (calcium, pH, etc.) where objectives have a short working distance

CH/1, CH/1/SH, CH/1/AU, CH/1/AU/SH, CH/1/R, CH/2/B, CH/2/B/SH

Enable a large diameter objective (e.g., confocal) to be placed very close to the vessel

CH/1, CH/1/SH, CH/1/AU, CH/1/AU/SH, CH/1/R, CH/2/B, CH/2/B/SH

Has an aluminum base for heating the solution

CH/1/SH, CH/1/AU/SH, CH/2/B, CH/2/B/SH

Capability to quick freeze or rapidly fix vessels

CH/1/QT

Release neurotransmitters from nerve varicosities using platinum stimulation electrodes.

All

Ability to rotate or invert a cannulated vessel and make confocal observations

CH/1/R

Be able to apply an extravascular pressure to a perfused, pressurized vessel, or perform long-term experiments in these sealed chambers

CH/1/AU, CH/1/AU/SH
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Non-superfused chambers
Do all chambers require superfusion of the vessel to maintain temperature and pH?
No, the CH/1/SH, CH/1/AU/SH, CH/2/B and CH/2/B/SH have an aluminum base heated by an electronic unit that sets and regulates the bath temperature when using buffers like HEPES or MOPS.
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Branched vessels
Can I make measurements on small vessels that have branches?
Some vascular beds from which small vessels are dissected have smaller side branches that lie between the cannulated vessel ties. These branches could cause leaks in a pressurized vessel. They must be located and carefully tied off while visualizing them under high power – perhaps requiring use of a compound microscope for higher magnification than a dissection microscope.
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Cannulas
Can I draw my own cannulas, or is it necessary to buy them from LSI?

yes, we can supply borosilicate tubing for those having access to a micropipette puller and grinder.
What size cannulas are best to use?
The size is not important when blind-sac experiments are to be done. However, for perfused vessel experiments we recommend roughly matching the outside tip diameter to the lumen diameter of the vessel.
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Coverslip Replacement
What cement is recommended for sealing coverslips in the chamber?  
A flowable windshield sealer, Dow Corning # 734 is best. It can be obtained from an automobile supply store, or from VWR.
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Measurement of Large Diameter Vessels

 
Can I visualize the diameter of large vessels – greater than around 350 µm, or those having thick walls?
Yes. Generally, these measurements are limited to only the outside diameter since the light is dispersed passing through the vessel making the inside diameter difficult to detect. Our instruction book details how this is easily done. Adapters can be purchased from LSI that will change the camera magnification so that larger vessels can be measured using the Video Dimension Analyzer. Contact us for specific details.
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Changing Optical Density Detection
What is the purpose of the internal switch in the LSI Video Dimension Analyzer, V94, which reverses the normal image detection circuitry of light to dark?
This allows measurements of diameter to be made on vessels imaged by incident light when using a dissection microscope, or a microscope having dark-field illumination.
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Flow Pulsations
Can the flow fluctuations from this peristaltic pump be minimized, and about how large are they?

When the pump has been calibrated and flow set either by the speed dials, or from an external voltage, the mean flow will be that desired. However, the pump will produce a pulsating flow. The amplitude and frequency of these pulsations depend on the pump speed, the tube set used in the pump, and possibly on the system perfused by the pump. For example, a 015 tube set delivering 100 µl/min may have flow fluctuations of about ± 3 µl/min, and half that at 50 µl/min.

A simple windkessel placed in the output line from the pump will reduce these fluctuations at the expense of slowing down the response to any flow changes that might be effected. The windkessel is a ‘tee’, connector with the branch at right angles to the flow path having a closed-off tube which traps some air. The larger the volume of the air trap, the more the pulsations are diminished.

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Recording Flow
Can a signal voltage be obtained from the pump so that the actual flow can be recorded?
Yes. There is an output jack on the FC unit that provides a voltage proportional to the flow. A simple calibration procedure is used to establish this factor (in volts/µl/min). Our Flow Indicator, FI-1 can be used with the FC and  PS/200 to obtain a similar voltage output.
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Flow Indicator
Is there a way to monitor the flow while running an experiment?
Yes, the Flow Indicator (Model FI-1) instrument has a digital meter that shows the flow directly in µl/min. It connects to the Model FC, or our PS/200 pump unit. An output jack for recording the flow is provided.

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Pressure > 200 mmHg
Can pressures greater than 200 mmHg be measured and used for control?
Yes, the analog voltage signal from the Pressure Out connector is linear to about 300 mmHg. Use a digital voltmeter to read this voltage (10 mV/mmHg). However, the digital panel meter of the Pressure Servo Control will read ‘1’, an overload condition.
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Computer Control
Can I use a computer to program pressure protocols?
Yes. Using a digital-to-analog (D/A) converter, program the amplitudes and time sequence of the voltages that will be outputted from the D/A into the External Input jack of the Pressure Servo Control unit.
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Pressure Pulsations
How can the peristaltic pump pressure fluctuations be minimized?
These pulsations are due to tube expansion when one of the rollers moves off the tube. First of all, use as large a tube set (e.g., the 093 tube set) in the pump as possible when the pump is providing a controlled pressure at the distal end of the cannulated vessel system. A simple windkessel may also be placed in series between the system’s distal end and the pump. The windkessel is a ‘tee’ connector with the branch at right angles to the flow path having a closed-off tube which traps some air. The larger the air trap volume, the more the pulsations are diminished, but at the expense of slowing the response time from 1 – 3 seconds to 15 seconds. It is possible in this way to reduce pulsations to as much as ± 1 mmHg. Alternatively, the pulsations may sometimes be minimized by raising or lowering the pump height to closely match the inlet and outlet pressures of the pump.
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Perfusion Pressure Control
Can I use the Pressure Servo to control the vessel pressure (PAV), or maintain the difference in pressure (dP) between two locations in an isolated vessel or vascular bed?
Yes, in conjunction with the Perfusion Pressure Monitor, use either the dP or PAV output and connect it to the PAV jack on the rear of the PS/200 (see instruction manuals).
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Necessity for Recalibration
How often do the ZERO and SCALE calibrations have to be made?
Normally, the drift of the ZERO calibration is less than 1.5 mmHg in 24 hours when the ambient temperature changes in the room are not significant. The SCALE calibration does not have to be readjusted from day to day.

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Use of upright microscope
Is it OK to use a non-inverted microscope to visualize the vessel in the chamber?
Yes, but there are a few disadvantages. Fluid surface movements caused by superfusion can cause some jumpiness in the image. Although, the Video Dimension Analyzer will follow these movements and measure correctly, it is a little disturbing to view on the TV monitor. Also, any pH or temperature probe located in the chamber will move when changes in focus are made if their associated holder does not move with the stage. Another problem is that moisture may accumulate on the objective to cloud the image. In sum, an inverted microscope is preferable.

 

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Large Coverslips useful for Confocal Microscopes
Which chambers have coverslips recessed from the base so that large diameter objectives such as found in confocal microscopes can be brought extremely close to the vessel?
The CH/1, CH/1/SH, CH/1/AU, CH/1/AU/SH, CH/2/B, CH/2/B/SH and CH/1/R are designed for this purpose.
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Sources for Tools & Supplies
Where can dissection tools, stopcocks, tubing, valves, etc. be obtained?
Sources for dissecting instruments such as Dumont # 5 or # 55 forceps and micro Vannas scissors are Fine Science Tools and Roboz. For other components see the Cole-Parmer or VWR catalogs; they contain a wide variety of components to choose from.