High Speed Acquisition of Spectra Triggering and Synchronization of the SamBa Camera to a Scanning Laser

High Speed Acquisition of Spectra Triggering and Synchronization of the SamBa Camera to a Scanning Laser Overview This note describes how to use the Sensovation SE-34 for high speed acquisition of Spectra. In essence one can perform hyper-spectral 2-D or 3-D imaging. The use of a CHARGE MULTIPLYING CCD (singleelectron detection) allows very high sensitivity. At the high rates needed for scanning spectroscopy, cooling of the CCD is not needed. Typical applications: Confocal Scanning Microscopy Multi-Photon Microscopy Scanning Laser Opthalmoscopy Live Cell Imaging Slide Based Cytometry Biochip Scanners, etc. One CCD Camera (SamBa SE-34) coupled with a spectrograph can replace a complex and expensive multi-photomultiplier or multiple-apd system, while giving full digitisation of the spectrum. Wavelength ranges to be measured can be software selected. Fig 1. The SamBa SE-34 Charge Multiplied CCD SINGLE ELECTRON DETECTION Fig. 2. Coupled to a Spectrograph (ISA CP-140) to measure spectra www.sensovation.com== 1= info@sensovation.com

Typical Laser Scanning Confocal Optical System REPLACE MULTIPLE PMTs WITH THE SamBa SE-34 + Spectrograph! Fig. 3: Typical Laser Scanning Confocal Optical System: Scanning laser systems typically use Photomultipliers for detection, since very fast acquisition times and high sensitivity is required. The dwell time of the laser spot together with its scan speed determine the resolution of the imaging system. For fluorescence imaging, other factors come into play such as the tradeoff between laser power and photobleaching of dyes. As shown in the above example, multiple photomultipliers (PMT) or avalanche photodiodes (APD) are used in order to detect many wavelengths simultaneously. The disadvantages of such systems are: Only few wavelength bands can be measured. As the number of colors go up, optical efficiency & sensitivity is lost. Optical filters fix the wavelengths, and mechanical filter wheels are big, unreliable. Optical filters throw away light that is not in the band to be measured. Expensive and complex. Now there is a better solution www.sensovation.com== 2= info@sensovation.com

Sensovation s SamBa SE-34, Charge Multiplying CCD The SamBa SE-34 was designed for high-speed spectral scanning applications to replace multiple PMTs. In order to do this, the following are absolutely mandatory features: Readout Sequence of the CCD is user programmable: You decide where you want to position the spectra on the CCD, how it is read, which wavelength bands. Triggering is precise for synchronization with a fast scanning laser. You can trigger anywhere in the readout sequence! It is necessary here to trigger on SPECTRA, which may relate to a particular sub-area of the CCD! Since this is not an imaging situation, user control of the interface to the host (PC) and the timing is necessary. For example, how do frame grabbers acquire spectra? The SamBa SE-34 allows you to control the data transmission timing. The Charge Multiplying CCD with the capability to detect single electrons is the key to achieving the sensitivity at speeds high enough for these applications. Line binning, unlimited and user programmable This increases sensitivity, and allows ultra-high speed capture of a burst of spectra on the CCD (described later). With the following advantages: Higher performance for multi-wavelength detection Confocal and multi-photon strategies can be preserved (in comparison to normal imaging), Background scatter and crosstalk minimized. Software programmable wavelength bands. Digitization of the wavelength axis better discrimination of dyes Solid State reliability, calibrated and repeatable measurements Compact and less complex Less expensive www.sensovation.com== 3= info@sensovation.com

Architecture of the Charge Multiplying CCD ACTIVE AREA Spectrum Rows are summed here! STORAGE AREA On-chip Charge Multiplier Detection of SINGLE ELECTRONS! Fig. 4 Architecture of the Charge Multiplying CCD The architecture of the Texas Instruments TC253SPD Charge Multiplying CCD is shown above. First of all we notice that it is a frame transfer type of CCD, which has an active area (where the pixels are exposed), and a shaded storage area. This frame transfer feature is useful in capturing spectra fast, and guaranteeing that no further light will affect the captured spectra. Secondly, the serial or readout register at the bottom of the storage area is extended such that it has a region 400 multiplication pixels. It is in this region that the electrons are multiplied by a gain of up to x100 as a line is read. The idea is that since the read noise of the output of the CCD is about 20 electrons rms, we need to multiply a single electron by a gain of at least 60 (3 times the read noise) to get a minimum SNR of 3. Then we can say that we can measure single electrons. The high clocking speeds of the TI CCD (12MHz readout, 3MHz line shift) allow us to use it in laser scanning spectroscopy. www.sensovation.com== 4= info@sensovation.com

Schemes for the Acquisition of Spectra Case 1: Projection of a single spectrum onto the CCD, with continuous or burstmode acquisition in time. The most likely scheme for acquiring a single spectrum is to position the image of the spectrum in the active area, just above the storage area as shown in the figure above. This situation is shown in Fig. 4 above. The SamBa AS-34 is programmed such that upon receiving an external hardware trigger, one spectra is captured i.e., shifted fast down into the storage area. In this process, the lines containing the spectra are summed ( binned ) into one line. This process can continue continuously as the laser scans, thereby building up a hyper-spectral image of the focal plane. Continuous reading of spectra: Burst rate of capturing up to 500 spectra: 17,000 spectra/s. 150,000 spectra/s. Background Subtraction, Fixed-pattern Correction: Unwanted offset signal comprising of dark response (fixed-pattern variances, dark current) is eliminated by acquiring spectra in the dark, and then subtracting this fixed component from each subsequently acquired spectrum. To do this, the exposure of the CCD is pulsed off, and a dark spectrum stored. Such calibration spectra can be updated at any time, and at the limit each spectra can be acquired paired with a dark spectrum. Such in-line correction of data (data processing) can be implemented in the SamBa on a custom basis. Relative Measurement: In some applications, a change in intensity and/or spectral properties with time is measured. An examples is the detection of kinetic molecular binding via fluorescence intensity or wavelength shift. In these cases, the capture of spectra is timed and synchronized to match the rate of the kinetic event. For example, pairs of spectra with a specific time interval can be captured. Or a burst of spectra can be captured, synchronized to the start of an event. Rejection of background scatter and fluorescence: In other applications, the unwanted varying background caused by the illumination or excitation source must be eliminated. This cannot be done using a fixed calibration. A common technique uses pulsed sources, together with the gating of spectral measurement during the lifetime decay of fluorescence dyes. This can be implemented using the SamBa SE-34 by synchronizing the capture of the spectrum such that the integration occurs during a precise time period after the excitation pulse. The decay of the dye itself can be measured via capture of multiple spectra after the light pulse. In effect, relative measurements can be done on-the-fly, at high speeds. www.sensovation.com== 5= info@sensovation.com

Rejection of Ambient Light: This can be done by a simple pulsing (strobing or chopping) the light source such that a higher intensity is concentrated in the pulses, and synchronizing the capture of spectra to the pulse frequency the same concept as a lock-in amplifier. The captured Spectra correspond only to the intervals when the source in on. The longer dead (dark) periods in between are discarded. Since ambient light is at DC or have other specific frequencies such as 60Hz, it can be strongly attenuated. Case 2: Projection of a many spectra onto the CCD simultaneously In some applications, a number of spectra must be measured simultaneously often in order to increase throughput. An example of this is the spectral measurement of samples in an array, where one row at a time is projected onto the CCD. In the case of a 96-well micro-plate, either 8 spectra or 12 spectra (a row of wells) are measured simultaneously. An effective use of the 2-dimensional area of the CCD is shown below in this typical projection of the spectra on the CCD: Regions of Interest to be binned and measured Spectral axis Discrete spectra Wavelength Bands are binned here STORAGE AREA On-chip Charge Multiplier Detection of SINGLE ELECTRONS! Fig. 5 Typical projection of multiple spectra on the CCD In this acquisition scheme, all of the discrete objects (samples) in a row are excited/illuminated simultaneously. The resulting spectra are projected onto the active area of the CCD. www.sensovation.com== 6= info@sensovation.com

The exposure and readout of the CCD is synchronized to the indexing of the rows of objects perhaps mechanical motion in one direction. The advantage of the frame transfer CCD is that the image in the active area is stored fast, and can be read simultaneously while the mechanical motion and exposure of the following row is occurring. This means that speed and light collection efficiency is increased. Furthermore, the user can programmatically select the wavelength bands to be measured, as well as the precise location of the samples. These settings define Regions of Interests to be measured. Through onchip binning of these areas, sensitivity can be increased while greatly reducing the amount of data and increasing readout speed. Also by varying binning levels, the dynamic range of measurement can be adjusted to the signal intensity. The SamBa allows this flexibility. Case 3: Projection of the spectrum of a continuous line onto the CCD (hyper-spectral line scanning) If one wanted to hyper-spectrally scan an area with a high resolution in the direction of the scan width (in the spatial axis), then a continuous line on the object would be excited/illuminated. The spectra of the emission from this line would be projected onto the CCD as shown below: Regions of interest Spectral Axis Wavelength bands of interest are binned here STORAGE AREA On-chip Charge Multiplier Detection of SINGLE ELECTRONS! Fig. 6: Typical projection of the spectrum of a line onto the CCD The acquisition scheme is similar to case 2 above, where the surface to be measured is scanned in one direction. The lines are read at a high pixel resolution. Regions of interest can be defined, together with on-chip binning of the wavelength bands to be measured. www.sensovation.com== 7= info@sensovation.com

Programming the SamBa SE-34 to acquire spectra The key is the Readout Sequence, which can be programmed by the user. This Readout Sequence is a text list comprised of steps, where each step performs shifting of pixels, shifting of lines, etc. Therefore the user can define how the pixels are read, down to individual pixels. There is no limitation to binning, clearing (skipping) pixels or lines, etc., up to the limits of what the CCD architecture will allow. Other steps in the sequence may perform I/O the most useful here are the generation of framegrabber timing signals such as Frame- and Line Enable Signals. The other important step is the wait for trigger. After you decide which Readout Sequence suits your detection scheme, you then send the sequence to the SamBa. With a START command, the sequence is executed in blazing hardware speed and precision, and the spectral data is yours. The SamBa SE-34 can send data to your host computer either via a standard digital framegrabber, or over Ethernet. Scheme 1. Capture a "burst" of spectra and then transfer them to a framegrabber board: In this case, the image of a single spectrum is positioned on the top N rows of the CCD chip, to maximize the number of spectra that can be captured. It is assumed that only the top N rows are exposed (perhaps the rest of the CCD is mechanically shaded, or optically not exposed). For every new sample location (laser scanner position), we "acquire" a spectrum on the CCD by vertically shifting the image say N rows in the direction of the storage area. Upon each trigger, this is repeated until the CCD is "full of spectra". Since the frame transfer CCD has a total vertical resolution of 1000 lines (counting both the storage plus the active areas), you can collect a burst of 1000/N spectra before having to read them. Naturally the better you can focus the spectrum into less lines, the more of them you can collect in one burst. The burst of spectra are then read form the CCD chip, and transferred to the host as one "image". www.sensovation.com== 8= info@sensovation.com

The SamBa SE-34 readout sequence would look like this: READOUT SEQUENCE, BURST OF 50 SPECTRA, NO BINNING SEQ:BEG SEQ:STEP CLRPULSE Clear entire CCD. SEQ:LOOP BEG 1000/N Loop to capture 1000/N spectra, one per trigger. SEQ:STEP TRIG Wait for rising edge trigger. SEQ:STEP INTTIMER Integrate. SEQ:STEP VSTORE,20 Capture a spectrum by shifting the image N rows toward the storage area. SEQ:LOOP BEG 3 Loop to send 3 dummy lines to framegrabber SEQ:STEP SETLEN,1 This is simply a framegrabber timing requirement. SEQ:STEP HREAD,SIZE_X SEQ:STEP SETLEN,0 SEQ:STEP SETFEN Readout the CCD. SEQ:LOOP BEG 1000 Loop to read 1000 lines SEQ:STEP VSERIAL,1 SEQ:STEP HCLR,710 + START_X Charge Multiply & skip to start of line SEQ:STEP SETLEN,1 Read a line SEQ:STEP HREAD,SIZE_X A programmable line length is read. SEQ:STEP SETLEN,0 SEQ:STEP SETFEN,0 SEQ:STEP REPEAT,1 SEQ:END Assuming that N=20 lines, since the vertical shift speed is 3MHz, with this sequence you can capture 150,000 spectra/s max. burst rate (not including additional desired integration time). Each frame contains 1000 rows, where there are 20 rows/spectrum. An improvement of this is if you perform vertical binning, in which the spectrum is summed into one line on the CCD chip. This allows you to store more spectra on the chip, as well as increase sensitivity at the same time. This line-binning occurs in the first row of the storage area, located between the active and storage areas. Note however that if the intensity is too bright, this summed line will be the first to bloom. it is better to position the image of the spectrum at the bottom N rows of the CCD chip, near the storage area. www.sensovation.com== 9= info@sensovation.com

The SamBa SE-34 Readout Sequence would look like this: READOUT SEQUENCE, BURST OF 500 SPECTRA, BINNING SEQ:BEG SEQ:STEP CLRPULSE Clear entire CCD. SEQ:LOOP BEG 500 Loop to capture 500 spectra, one per trigger. SEQ:STEP TRIG Wait for rising edge trigger. SEQ:STEP INTTIMER Integrate. SEQ:STEP VSUM,N Capture a spectrum by binning N rows into one row of the storage area. SEQ:STEP VSERIAL,1 Shift the binned row once toward the serial register. SEQ:LOOP BEG 3 Loop to send 3 dummy lines to framegrabber SEQ:STEP SETLEN,1 This is simply a framegrabber timing requirement. SEQ:STEP HREAD,SIZE_X SEQ:STEP SETLEN,0 SEQ:STEP SETFEN Readout the CCD, all 500 rows of the storage area. SEQ:LOOP BEG 500 SEQ:STEP VSERIAL,1 SEQ:STEP HCLR,710 + START_X Charge Multiply & skip to start of line SEQ:STEP SETLEN,1 Read line SEQ:STEP HREAD,SIZE_X Desired line length is read SEQ:STEP SETLEN,0 SEQ:STEP SETFEN,0 SEQ:STEP REPEAT,1 SEQ:END Scheme 2: Continuous acquisition of spectra. In this case, the image of the spectrum is positioned at the bottom N rows of the CCD chip i.e., closest to the storage area. Only these N rows are exposed. For every new sample location (laser scanner position), we "acquire" a spectrum on the CCD by vertically shifting the image N rows in the direction of the storage area. Here again we will bin the N rows into one such that the time to read one spectrum is minimized, and sensitivity is maximized. Also, if we only read a portion of the line (the first n pixels), speed will be increased. Upon each trigger, one spectrum is read. www.sensovation.com== 10= info@sensovation.com

Here the focusing of the spectrum into a smaller number of lines is not as critical to speed, since this is limited by the time to read a line. Since you can control the Frame Enable and Line Enable in the Readout Sequence, you can either transmit the spectra to the IMAQ board as a continuous stream of lines (like a line-scan camera) or formatted as "images", where an image comprises of many spectra. Remembering that in imaging mode many framegrabbers (including the IMAQ-1422) require a number of dummy lines before the frame, in order to synchronize. This will of course cause periodic loss of spectra, which may not be acceptable. It is therefore better if the framegrabber can operate in a streaming line scanning mode, in which there is no concept of frames. Such a sequence is shown below, where the Frame Enable signal is not used at all. It is to be verified that the framegrabber can receive this. The SamBa SE-34 readout sequence would look like this: READOUT SEQUENCE, CONTINUOUS SPECTRA, BINNING SEQ:BEG SEQ:STEP TRIG Wait for rising edge trigger. SEQ:STEP INTTIMER Integrate. SEQ:STEP VSUM,N Capture a spectrum by binning N rows into one first row of the storage area. SEQ:STEP VSERIAL,1 Shift the binned row once toward the serial register. SEQ:STEP HCLR,24 + START_X Charge Multiply & skip to start of line SEQ:STEP SETLEN,1 SEQ:STEP HREAD,SIZE_X Read the binned spectrum (one line) SEQ:STEP SETLEN,0 SEQ:STEP REPEAT,1 SEQ:END With this Sequence you can sample spectra continuously (656 pixels/spectrum) at 17,000 spectra/s max. (with no additional desired integration time). Note that since this is a continuous mode, the CCD is not initially cleared in the readout sequence. You may want to use a "clearing sequence" prior to loading this sequence. Nevertheless, the first few spectra will likely not be valid, until all of the CCD lines have been read once. www.sensovation.com== 11= info@sensovation.com

Triggering When using asynchronous external triggering, there are two ways that the integration time can be defined: a) Programmed integration time. As shown in the above sequences, a desired integration time is set via the SCPI command: ACQ:TINT <time in ms, with range of 0.001 to 65535> Then within the Readout Sequence, this step is used immediately after the trigger: SEQ:STEP TRIG SEQ:STEP This sequence would typically continue after the integration step to store the spectrum (capture it on the CCD) is, and eventually read it. b) One can use two trigger pulses to define an integration time. Note that while waiting for trigger, the CCD is integrating: SEQ:STEP TRIG SEQ:STEP TRIG Or: SEQ:STEP TRIG SEQ:STEP CLRPULSE (clears the ENTIRE CCD in 5) SEQ:STEP TRIG Trigger Timing: Note that the external trigger input is rising edge triggered, and requires a 1 us high pulse width minimum. This input is the LED of an opto-coupler, and the external driver must be able to drive 10mA. There is a 10us max delay from rising edge of the trigger signal to clocking of the CCD. This is mainly due to the opto-coupler and debouncing. www.sensovation.com== 12= info@sensovation.com