Control Circuit Overview: Confocal and 2-Photon Microscopy:
Before attempting to build the control circuit, it is recommended that the function of each part the circuit is understood. The schematic below shows the basic functions only.
Refer to the circuit diagram for specific details.
Note: The details given below incorporate revisions made though November 2006.
Click on figure for pdf
CRS Controller board: The key synchronization signal or Scan Line Clock (SLC) for the scanning microscope is generated automatically by the CRS Controller board. The rising edge of the square pulse of the SLC indicates the start of each sinusoidal oscillation of the CRS mirror. This is available on pin 4 of connector J2. The exact position of the CRS mirror relative to the timing of the SLC can be adjusted with a potentiometer on the CRS board (R48). R48 should be set to 0 to align the SLC with the 0 position of the mirror. The factory setting is for a 5 µsec delay.
The angle of the CRS mirror oscillation (smaller angle = shorter scan distance = larger magnification) is controlled by an input voltage at pin 2 of connector J3 on the CRS board. This voltage may be manual regulated by a front panel potentiometer or by computer-controlled digital potentiometer (AD5204) for automatic zoom control.
Horizontal Sync Signal: The SLC from the CRS board serves to form the horizontal sync signal to coordinate the line-acquisition by the frame grabber (Raven). Before reaching the Raven frame grabber the H sync signal is processed via a CD4050 to provide sufficient current to drive the Raven board. In addition, the signal is processed via a monostable multi-vibrator with Shmitt-trigger (74LS221) to generate a fast pulse signal. The Raven temporarily stores the data captured from a forward and reverse scan as a single line in image memory before processing.
Frequency Doubler (CD4070): The strategy for using an oscillating mirror is to avoid the requirement for a fast fly-back of the scanning mirror and to utilize the symmetrical reverse scan to collect image data. The result of this approach is that 2 horizontal lines of data can be collected for each full mirror oscillation. Consequently, the synchronization signal needs to be doubled to track the vertical progress. The CD4070 IC creates a pulse at the positive and negative edge of the scan clock to double the clock frequency.
12 Stage Counter (CD74HCT4040E): Each sync pulse entering the counter serves to increment a line-count and this is digitally represented as a binary scale on the output lines (using Q0 - Q9). Counts of 128, 256, 512 and 1024 are indicated by HIGH signals at Q6,7,8 and 9, respectively. Depending on the count position selected with the rotary switch, a HIGH signal representing the vertical sync signal is sent to the frame grabber to advance the image.
Digital to Analog Converter (AD7393 AN): The binary output of the counter is instantly feed into a 10 bit digital-to-analog converter. This results in an output voltage at pin 19 with a saw-tooth waveform that progressively increases with each input pulse. The saw-tooth ranges from 0 volts with a full scale referenced to 5 volts (count of 1024). This saw-tooth will drive the the motion of the vertical scan mirror (M3H/S). The required line count is selected by the rotary switch which functions by connecting addition binary lines to the converter. A reset is not required for values of 128, 256, or 512 because these values divide exactly into 1024 and reset automatically during sequential counting.
Offset Control (Op Amp 1, LT1012): To center the saw-tooth around zero volts (and to center the mirror scan at all image acquisition rates) the saw-tooth is processed by an op amp configured as a difference amplifier. The appropriate fixed difference voltage for each line-count (2.5 v x line-count/1024) is supplied via the rotary switch.
Saw-tooth Amp (Op Amp 2 LT1012): The magnitude of the saw-tooth signal controls the height of the vertical scan (smaller scan = higher magnification). The magnitude is controlled by 2 processes: a manual or digital potentiometer (AD5204) varies the saw-tooth voltage input applied to a fixed-gain, non-inverting op amp. The capacitance (also selected by the rotary switch) applied to this op amp serves to smooth the saw-tooth waveform to dampen the mirror movement. The final waveform is applied to the differential input of the M3H/S controller board.
A manual position control is applied to the second differential input. With the zero-centered circuitry, this control is not critical and is used for the initial mirror alignment. However, it is useful if a line-scan mode for the microscope is required; the position of the single line scan be adjusted.
Raven Frame Grabber: According to the H sync and V sync pulses, the frame grabber digitizes the resulting luminance signal arising from each of four PMTs (if required). Each image is processed by Video Savant software and stored in computer memory.
Rotary Switch: A double rotary switch which has (5 poles, 8 ways) allows for the simultaneously selection of the digital-to-analog lines, vertical sync, offset voltage, damping capacitance and LED indicators. This allows the selection of frame rates of 15, 30, 60 and 120 frames per second. Each frame may consist of 1 - 4 images from the PMTs. The image height is reduced by half the number of lines for each increase in speed. For exact wiring see full schematic.
Digital potentiometer (AD5204): The height and width of the mirror scans determines the image size. In order to gain a square aspect ratio, the height and width of the scans needs to be calibrated and manual set with potentiometers. The ability to change the image height and width provides the microscope with an electronic zoom. Manual control requires that each potentiometer is set individual. But, by routing the control signals via a digital potentiometer under computer control with a predetermined look up table for H and V sizes, an automatic zoom control can be implemented from Video Savant.