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Schematic of Speckle Imaging Instrument  Example of Speckle Imaging Instrument  Camera Our software is currently designed to work with Basler camers with an IEEE1394 interface. A new version of the software that should work with most fire wire cameras is currently being tested. A monochrome camera with 8 bit resolution is sufficient for most in-vivo experiments. Suggestions: Zoom Lens The purpose of the zoom lens is primarily to provide magnification, but it also helps in focusing the sample on the camera sensor. Some camera manufacturers provide a focusing lens that fits with their camera. While this would function in theory, the quality of the image obtained is much better with some magnification. The Zoom lens can also be replaced by just two lenses or a lens with an objective to obtain high resolution images. Suggestions: Camera Mounting (not shown in figure) It is convenient to mount the camera on a vertical translation stage. This helps adjusting the focus of the camera, and to counter variations in sample heights. The mounting components are merely to provide course adjustment for focusing and are not crucial to the setup. The Zoom lens can be used to achieve fine focusing. Note that if you are using a microscope like setup, then the mounting hardware can help greatly. Suggestions: Laser It is best to use an infrared laser since it gives good penetration of light through tissue. Also the speckle signal would be less affected by absorption because the wavelength is away from the absorption peaks of hemoglobin. An advantage of speckle is that high laser powers are not required so diode lasers are usually sufficient. An important point to be remembered in getting a laser is that it has to be single mode to generate reliable speckle measurements. Suggestions: General Comments on Setup The Collimation kit is used to limit the divergence of the diode laser. The collimation package can be used to adjust the laser divergence such that the entire field of view is evenly illuminated. It is easier to use white light (from a flash lamp) when trying to focus the camera on the sample. Use a camera exposure time (in software) of 1ms to 5ms for physiological measurements for good sensitivity. When performing the experiment, ensure that ambient light is OFF or is dimmed down. If the illumination is only from the laser, effects due to presence of incoherent light can be minimized. Dimmed ambient light with low exposure time (5ms) should be good. Make sure that there is no saturation. Saturation in the image would artificially reduce the contrast. The histogram of the image when the laser is the only illumination source should be centered about 100-140 on a 0 to 255 scale. It is better to have the laser operating at its operating current (well above the lasing threshold) and controlling the intensity at the sample by deliberately letting the laser diverge to prevent saturation. It is also better to have the aperture stop on the zoom lens open all the way, and not use it to control the recorded intensity. When directing the laser onto the sample, set the laser up as close to the vertical normal as possible. |
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