Microscope
In general, large numerical apertures should be used to maximize light intensity. However, when the signals of interest come from multiple focal planes, a smaller numerical aperture may be required. The interface between the microscope and all three cameras is a C-mount connector.

Microscope Light Source
In most cases the brighter the light source the better. Both cameras are excellent at detecting small changes on a large resting background. For absorption and population fluorescence signals (where the light levels are high) a tungsten-halogen source is often used. The microscope tungsten-halogen lamp housing will often be satisfactory. However, the DC power supply supplied by the microscope manufacturer may not be optimal and a well-stabilized DC supply may be essential. We recommend the VS-20ML power supply by Astron (9 Autry, Irvine, CA 92618, (949) 458-7277 l (949) 458-0826). For single cell fluorescence (where the light levels are low) we recommend an arc lamp. Cairn Research Ltd (Graveney Road, Faversham, Faversham, Kent, ME13 8UP, U.K. tel: +44(0)1795 590140) is a highly recommended supplier of Xenon arc lamps and power supplies. Used with their monochromator the 75W Xenon is brighter than the 150W. The opposite will be true without the monochromator. Their light source has a built in shutter (see below).

Shutter
A shutter which can be driven by a 5-volt pulse should be available; shutter opens at 5 volts, closes at 0 volts.
In addition to reducing bleaching and photo toxicity, using a shutter will reduce or eliminate a DC drift one sometimes sees at the beginning of the recording. This DC drift is usually caused by the fact that in order to synchronize the electrical (BNC) recording with the camera, NeuroPlex switches the camera from the free running mode to trigger mode shortly before the acquisition. The trigger mode allows the camera to be triggered frame-by-frame by the clock signals that control the BNC recording. Once the camera is switched to the trigger mode, it will not read out or empty the photons in the CCD until the trigger pulses arrive. If the shutter is not implemented and controlled by NeuroPlex and is open before the acquisition starts, photons will accumulate in the CCD and it will take many read-outs to completely empty the charges and reach a stable level. This is why a DC drift can be seen. Using a shutter controlled by NeuroPlex will avoid such photon/charge accumulation and thus this initial DC-drift.
We use shutter drivers and shutters from Vincent Associates. Choose a shutter opening that is larger than the diameter of the incident light beam. We use a VS35S1ZM0R1-with heat sink shutter (designed to withstand high intensity light and high temperatures) and a VCMD1 Shutter driver. We mount the shutter independently of the microscope so that its vibration does not affect the recording.



Stimulator
The software generates a signal to the stimulator with a rising edge to 5 volts.

Splitters
For recording emission from 2 wavelengths simultaneously we have used and are supporting the Optical Insights, LLC Dual-View splitter. A similar solution for doing emission ratio imaging, the OptoSplit-II, is offered by Cairn Research. With these optical devices, each of the two images created by the splitter is recorded simultaneously on half of the camera chip. When using a low spacial resolution camera (e.g. NeuroCCD-SMQ) for recording very fast transients, a better solution is to use a dichroic mirror as a beam splitter and record simultaneously with two cameras.

Filters
The specific filters needed for fluorescence measurements - excitation, emission and dichroic mirrors - are dependent on the dyes used for the measurements. We therefore include filters only with Macroscope II. Excitation filters could be obtained from Chroma Technology Corp . For emission, we find Schott Glass filters, very good.

Camera Stands
Users who are not using a microscope, need a stabilized optical arrangement to align the camera, via a Macroscope or other lens, to look at the preparation. We have helped several laboratories to put together such a stand, using components from Linos . A list of parts is available from us.

Vibration-Isolation
Optical recordings are very sensitive to vibration. Most disruption is caused by vibrations in the horizontal plane. A vibration isolation system, air table or table supported by inner tubes is required. In situations where the vibrational noise is not severe, a platform made from an optical table­top mounted on air­filled rubber tubes (obtained from Newport: isolator, air cushion, #5274) is adequate. For more severe vibration problems, Minus K Technology sells vibration isolation tables with very low resonant frequencies.

Secondary Camera (for cardioPDA)
Because of the relatively low spatial resolution of the CardioPDA system, it is useful to relate the optical signals to the preparation by obtaining a higher resolution image. A high resolution camera (placed either in the usual position of the array or a second view port) can be used to produce a TIFF or a BMP file of the image of the preparation. The CardioPlex software can import these files and overlay the images with the pseudo-colored optical data. A Dage-MTI CCD-300-RC (CCD Camera with RC300 Control) (Michigan City, IN) with a 3045 Integral Technologies FlashBus MV Pro Frame Grabber from I-Cube (Crofton, MD) has been successfully used for this function.

Computer Monitor
We strongly recommend purchasing a high resolution monitor (1600x1280 or greater) because it is helpful to be able to visualize as much data as possible, and the software display is designed for a very high resolution monitor. For those who would like a NeuroPlex display larger than that provided by a 1600x1280 21 inch monitor, two monitors can be used simultaneously A video card that supports two monitors is the Matrox, G200MULTI-MONITOR (DUAL); part number G2/DUALP-PL (for PCI slot).(contact Brian Salzberg and AnaLia Obaid).

Room Lighting
The outputs of ordinary room lights often introduce 60 or 120 cycles into the optical recordings. DC powered lights should be available for experiments.