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Many of our experiments make use of a home-built 2-photon laser scanning microscope. This system is based on a pair of high-speed galvo mirrors from Cambridge Technology (6210). The light source is a Mira femtosecond oscillator laser pumped by a 10 watt Verdi laser (both from Coherent). The intensity of the laser illumination is controlled by a pockels cell attenuator (ConOptics) and an electronic shutter from Vincent Associates. Standard silver-coated mirrors (New Focus) are used to direct the beam through a telescope (to adjust the beam width) and then to the XY mirror set. The position of the mirror galvos are governed by two 67021 controllers that are connected to the DAC output of the NI ADC interface card (PCI 6111) through a 5X voltage attenuator. The scanned beam is directed into a video port of the microscope (Olympus BX51WI) through a 50 mm diameter doublet scan lens (#47317, f=100 mm; Edmund Optics). We typically use the Olympus 60x water immersion objective for both IR-DIC visualization and 2-photon imaging
Our current system uses a single cooled PMT detector (H7422P-40; Hamamatsu) that that is mounted directly on the objective turret holder (i.e., it goes up and down with the objective when you focus the microscope.) The very high sensitivity of this detector to green light makes it well-suited for detecting most common anatomical and functional probes used in neuroscience. In the past several years, Hamamatsu began offering to modify H7422Ps to increase the current threshold limit (to 50 uA) which makes these PMTs much easier to use for brain slice imaging. We use the M9012 controller to regulate the cooling and gain of the PMT; the PMT voltage setting is driven by one of the DAC outputs on a LabJack USB ADC interfaces. We use the other digital and analog lines on the LabJack to control the pockels cell attenuator and to determine the system status. Emitted light is directed toward the PMT using a 2” square long-pass dichroic mirror (700DCLPXR; Chroma Technology). The mirror is mounted on a linear slide with magnetic stops that allow us to easily position the mirror in the correct position. We use a set of aspheric and doublet lenses to relay the light emerging from the back aperture of the objective onto the entrance face of the PMT. In most of our experiments we use a single BG39 emission filter although we have the capability of adding additional bandpass filters into the detection path using a Spinder/Hoyer manual filter wheel. The current output of the PMT is converted into an analog voltage using a high-bandwidth current preamplifier (SR570; Stanford Research Systems). We use the same NI ADC board to acquire the detector signal and to generate the X and Y galvo mirror position commands. This system can run relatively easily with 1 us pixels (the ADC and DAC modules of the PCI 6111 updating at 1 MHz) with a moderately fast PC.
We wrote custom software in Visual Basic 6 (Microsoft) to control the XY galvo scanner and acquire raster 2-photon images. Unfortunately Microsoft has ended support for VB6, which will eventually limit the lifespan of our software. Though much maligned, we have found VB6 to be an ideal language for creating the “glue” that binds together different software components (e.g., the low-level driver routines that control the NI ADC card, interprocess command libraries, Matlab-based image analysis routines.) Visual Basic also excels at creating workable user interfaces very efficiently. We currently operate a suite of five VB6 programs that interact using Windows Registered Messages (encapsulated through the very handy MBInterProcesss ActiveX control.) Three VB6 programs control different hardware devices (the 2-photon system, our Photometrics Quantix 57 cameras, and the Instrutech ITC-18 data acquisition system). The other two programs display images and electrophysiology data. Both display programs can be used to view both live data and to browse through previously-recorded data sets. Both display programs also run a background instance of Matlab for filtering, import/export functionality and image processing. All five programs run under either the Win2000 and WinXP operating systems.