MiCAM Ultima -An ultra fast low noise imaging system for real time neural activity imaging

IntroductionFor voltage-sensitive dye (VSD) optical imaging, we need a fast and low noise imaging

system. We had already developed a practical imaging system that employed a CCD chip until1997, however this system was not optimal in terms of both well depth and speed. An imagingsystem we developed afterwards is a nearly ideal one for VSD recording, if we can providesufficiently intense illumination. The system can detect less than 0.1% fractional change from VSDwithout averaging or filtering.

(1) Image SensorImage sensor is the most important component of the system. Our basic design parameters are:

· Large well depth about 10M electrons · Fast imaging more than 10,000 frames per second · High fill-factor and more than 50% quantum efficiency · 10mm X 10mm and 100 x 100 resolution · Optimized dark noise

The sensor has 16 output lines to increase readout speed and also exhibits high resolution inintensity. The image sensor is consisted by active pixel array, sample amplifiers and readout circuitsas typical CMOS APS. The light exposure on pixel photo diode (PD) is converted to electron andstored in its parasitic capacitance. A pixel of CMOS APS has voltage follower transistor (T2) for

current buffer, reset transistor (T1) and select transistor (T3) for each pixel. Accumulation time isdetermined by reset pulse duration. Normally, signal is sampled just before reset. Y-select and X-select are driven by scanning logic circuit for sequential read out through whole pixels. The imagesensor has no snapshot shutter feature. It means actual timing of a frame has line delay timeaccording to scanning. Essential difference between typical sensors and our image sponsor is thatthe latter has 16 outputs for fast readout and very large pixel photo diodes for large well depth. The image sensor detects wide spectrum in visible light; wavelength 400-700nm. The peaksensitivity wavelength is about 500-600nm and its practical quantum efficiency exceeds 60%. Thisconversion practical efficiency is almost the highest compared with other image sensor except a fewlow speed cooled CCD image sensor adapted to the back-illumination technology.

On the other hand, large demerit of CMOS image sensor compared with CCD is fixed pattern noiseand kTC noise from pixel charge reset. In order to cancel both of them, the sensor can be derived byCDS (correlated double sampling) readout sequence by using digital processing. In CDS sequence,we need to scan twice for horizontal multiplexer both before and after reset. By the second scan,reset level for the next frame is acquired. An acquisition system should hold this signal in itsmemory and retrieve it at the next frame readout for reset level subtraction. Figure below (left)shows simplified pulse sequence, readout signal of normal mode and CDS mode with capturedpicture.

(2) Acquisition SystemFigure above (right) shows simplified block diagram of whole acquisition system. The

system is designed by very direct parallel processing way. Total data acquiring rate is 320Mbytes/sec and memory capacity is 1G bytes. Data resolution is 14bits in minimum, 20bits foraveraged signal. Signals from camera unit are sent to processor unit via complex cable with

independent 16 coax cables for each output. To transfer the data, we designed a bus master PCIinterface card on computer. The sensor is mounted in camera unit with 250g weight and 60mm x50mm x 130mm dimension. (3) Signal to Noise ratio

The figure below shows noise waveform of dark noise (A) and with light (B), both samplingspeed is 0.1msec and light source is green LED. Waveforms from a single pixel are indicated. Asfor this noise waveform, dominant noise source in bright measurement condition is photon-shotnoise. But the dark noise might be problem when illumination is not enough, like 3M electrons.Fortunately, many recordings require the sampling rate slower than 0.5msec in the practicalexperiments. The imager can scan whole image more than 6 times within 0.5msec on the average.Due to this technique, noise is reduced to half in 0.5msec sampling and also to a third in 1msecsampling as shown in right-below figure.

Thw next figure shows conclusion of noise properties of the system. A curve of total noiseindicates RMS noise (Qn) versus illumination (Qi) for a pixel. In range of practical illumination,like 1Me- to 5Me-, dominant noise component is photon shot noise at 1msec sampling.

(4)Experimental results

Single cell recording by using Di2-ANEPEQA CA1 pyramidal cell was stained by Di2-ANEPEQ via internal perfusion application. The cell wasvoltage clamped at –70mV, resting potential, and commanded hyper polarization and subsequentlydepolarization. High NA 20x objective lens (Olympus) was used. Due to light source (150Whalogen lamp) weakness, fluorescent light intensity was about 200K electrons for each pixel, whichis less than 2% of the saturation level of image sensor. We have performed 100 times averaging toimprove signal to noise ratio.

Each image shows fluorescent image (B/W) and its differential component (color) from time zeroimage. This experiment is done by Takashi Tominaga, Riken BSI

Intrinsic light scattering measurement for rat brain slices

We confirmed intrinsic (without dyeing) optical signal of rat hippocampal slices. The next figureshows the optical setup to measure light scattering by transmitted light. We provide polarizer andanalyzer in optical pass of transmitting microscope. By this setup, we can characterize lightscattering properties in detail.

In single stimulus, large artifact is observed at the point of stimulation electrode (blue trace infigure) as previously reported by Grinvald A. In addition, small but clear fast signals (red lines) are observed from near stimulus portion. Very

small fast component travels along axons and slow component appears more than 200msec afterstimulation (orange line). This is the first recording of intrinsic signal induced by single stimulationpulse in rat brain slice.

Tetanic stimulation causes large slow component in st. rad. , which signals are regarded asreducing light scattering according to cell volume change (Andrew R.D., Macvicar B.A., SomjenG.G.).

These results suggest that intrinsic signal can be used to activate the map work even in brain slices.In other hand it can be balanced out by using porlarizer and analyzer, when it become biologicalnoise for extrinsic optical probe. This experiment is done by Takashi Tominaga and MichinoriIchikawa, Riken BSI

(5)Another applicationThe imaging system has both of high speed and high dynamic range will able to uniqueapplication which can not be expected from conventional fast video systems. For instance ofindustrial application, it succeed to take images at moment of melt brake filament in halogenlamp and its sparking. It may possible to see flare of electrospark machining system and itmay play some role for astronomical physics of the sun.

(6)Modification in the futureOur system is fully programable because all digital electronics are composed by FPGA. And,we have plan to built 40,000FPS system by using same sensor and the latest analog and digitaltechnology.

The system was developed by Riken BSI and Stanley electric Co.Ltd. Its patents and licenses are provide forBrainvision Inc. by Riken. Sensor was developed by FillFactory NV.

History: Document originally written in Japanese 2003.10 and translated to English 2004.6

BrainVision Inc. 3-46-8 Narimasu, Itabashi, Tokyo 175-0094 Japan

info@brainvision.co.jp