FAQ

 

Frequently Asked Question

We have collected together some of the questions that we have been asked over the years. If you have a question related to our technology please contact us at info@prsbio.com.

 

  • Time-Gated Imaging FAQ

    What is the difference between fluorescence lifetime imaging and time-gated imaging?
    If a sample has both long-lived and short-lived fluorescence (or phosphorescence etc.) it is often desirable to distinguish between these. For example, a long-lived fluorescent label can be detected with high sensitivity against a highly fluorescent background if the latter has a short lifetime. One simple way to accomplish this is to use a 'gated' detector synchronised to a pulsed excitation source. If the detector is insensitive during and immediately after excitation but is turned on rapidly thereafter, then only long-lived emission is measured. If a similar measurement is made without 'gating', all emission is seen. The difference between the measurements with and without 'gating' gives primarily the short-lived component of emission. If the detector is a camera of some type, then a time-gated image can be measured.

    A fluorescence lifetime image is a calculated image where contrast is directly proportional to some function of fluorescence decay time or fluorescence ‘lifetime’. A fluorescence lifetime image can often look very different to the sample as seen by the naked eye and if the fluorescence is uniform then there the lifetime image will be similarly featureless. Lifetime images are often combined with normal intensity images to show how the lifetime variations relate to the overall sample.The component images from which lifetime images are calculated can be collected using a number of techniques including Time-Gated Imaging and homodyne modulation imaging where a the sensitivity of the detector and the brightness of the lightsource are modulated at high frequency.

    How can an intensified camera give fluorescence lifetime contrast?
    The intensifier most commonly used for fluorescence lifetime imaging is the so-called 'Gen-II' intensifier. In this design the photocathode is physically very close to an electron amplifier known as a microchannel plate. A potential of a few hundred volts or so accelerates emitted electrons from the photocathode and directs them into nearby regions of the microchannel plate amplifier. The microchannel plate is composed of an array of tiny channels in a glass ceramic matrix. The walls of the channels are doped so that energetic electrons striking them emit a number of secondary electrons in proportion to the energy of the incident electron. A potential of the order of 800 volts is applied across the microchannel plate. An electron entering the plate gives rise to a burst of secondary electrons from the rear of the plate as a result of a cascade process. These secondary electrons are accelerated by several thousand volts and strike a phosphor screen, giving amplified light output.

    Gen-II intensifiers are easily 'gated' by applying a voltage pulse to the photocathode. For fast gating a pulse of the order of 100 volts or so is adequate. Alternatively the voltage across the microchannel plate can be switched, though this requires a larger voltage change. The gain of a proximity-focused intensifier can also be modulated either by imposing a modulation voltage on the photocathode or by modulating the voltage across the microchannel plate (which is less common and can lead to thermal 'drift' of the intensifier's gain).

    If modulated operation at high frequency is required it is usual to operate with a reduced proximity-focusing voltage, so that a relatively small modulation voltage can be used. This helps to keep power dissipation in the intensifier to an acceptable level. If this is not done the photocathode becomes very noisy and can show time-dependent changes in properties. The disadvantage of this mode of operation is that the spatial resolution of the intensifier is degraded because the proximity focus is maintained by the accelerating voltage between the photocathode and microchannel plate. The lowering of the proximity-focusing voltage also lowers the overall gain of the intensifier.

    If fast gating or modulation is required the relatively high resisitivity of the photocathode can give rise to problems. One effect is so-called 'irising' where the response at the centre of the intensifier is delayed relative to that at the periphery and the modulation depth is reduced. This effect is most marked for intensifiers having large photocathode areas. Various attempts to minimise this problem have met varying degrees of success. A partially-transmissive metal undercoat on the photocathode can increase the speed of gated response and improve modulation bandwidth, but at the expense of reduced sensitivity. A fine metallic grid on the photocathode can be used to similar effect, but with potentially less loss of sensitivity.

    All intensifiers are easily damaged by exposure to bright light. Very bright light can adversely affect the photocathode properties if no voltage is applied. Even if no permanent damage results the 'noise' level is much higher for a period after such exposure. If protection circuitry is not built in the intensifier can be ruined if over-exposed while the voltage is applied. Photocathode composition can be tailored to give spectral sensitivity in selected regions. The most usual photocathode types have poor red sensitivity. Extended red sensitivity can be had at the expense of substantially increased 'noise' background.
     

    Which is best for Fluorescence Lifetime Imaging? Modulated CCD or Intensifier?
    The main advantage of the image intensifier for lifetime-resolved imaging is the ability to switch the sensitivity at very high speed. The modulation bandwidth of a typical 18mm diameter intensifier allows operation at frequencies in excess of 100-200MHz and a typical gating time of the order of 5-10nsecs, depending on the photocathode type and whether a conductive coating is used. Faster operation is possible for specially-constructed intensifiers, and Kentech Ltd quote sub-nanosecond operation for some devices. The main disadvantages of an intensifier from the viewpoint of gated or modulated imaging are the high 'noise' level of the intensified detector and the limited spatial resolution during modulated operation.

    The noise performance of the intensifier is much inferior to a typical CCD detector and very much worse than a cooled CCD. It is difficult to give a definitive value for the noise performance of an intensifier since this depends very much on how the device is operated and there is considerable variation between intensifiers within a given batch. To minimise noise the intensifier is best operated at the lowest gain that is within the design specification.

    The noise level is a crucial factor in fluorescence lifetime imaging using frequency-domain methods with modulated excitation. An intensified camera must typically be used with an integrating framestore to allow many images to be averaged. Alternatively, and preferably, the camera associated with the intensifier can be a slow-scan cooled CCD that allows an image to be integrated in the camera without interference from thermal noise. The latter approach is better in principle than integration of images in the computer because the readout noise is lower and is not repeated with each image that is accumulated.

    As a rough guide, images must be measured over integration times of several seconds to several minutes to give reasonable performance for time-resolved imaging with an intensified detector in our experience. In part this is because the operations of fluorescence lifetime imaging involve several image subtractions and ratiometric measurements which amplify noise in images. If fluorescence lifetimes of interest are long enough for the alternative PRS directly-modulated/gated CCD technology to be used, we believe this to be a very much better method than the use of intensified detectors. Our opinion is based on many years of experience with both techniques. For this reason PRS has expended much effort in developing time-gated CCD technology and we would only recommend the use of intensified detectors where they offer particular advantages of speed.
    How many 'bits' resolution are needed for fluorescence lifetime imaging?
    This is a deceptively simple question but the answer needs some careful thought. The first thing to remember is that the noise performance of the 'weakest link' in the imaging chain is the determining factor in the performance of a lifetime imaging system. If the 'front-end' of the imaging system is an image intensifier then this is almost certainly the noisiest component. Even if the camera viewing the image intensifier's screen only worked to 5-bits resolution (i.e. 32 grey levels) this would usually be better than the performance of the intensifier.

    At first sight this seems to contradict the information in published accounts (including our own papers), where cameras capable of 12-16 bits are used to collect data. In fact, there is no contradiction. The process of measuring a fluorescence lifetime image involves image manipulations such as subtractions and ratiometric measurements. If the resulting image is to be of high quality then the signal-to-noise ratio of the precursor images must be very high since the image processing operations inevitably degrade signal-to-noise. The best way to achieve the required signal-to-noise figure for an image to be used in such a calculation is to integrate the signal over time (i.e. to perform a time-average) until the data are adequate for the required purpose. This can be achieved by averaging many 'noisy' images acquired using a camera with a low-resolution digitiser or alternatively the signal can be accumulated for an extended time period in a camera with a wide dynamic range (such as a cooled CCD camera.)

    What is most important for a high quality lifetime image whichever technique you are using is that you have detected an adequate number of photons from your sample for good quality ratiometric images. The high gain of the intensifier does not overcome this basic requirement.
     

    My CCD camera is capable of 14-bit resolution. What does this really mean?
    The bit-resolution of a CCD camera relates to the operation of the A/D converter that digitises the charge packets in the camera. A 14-bit converter can resolve the full-scale output of the camera into 16,384 levels. However, this does not mean that the data recorded are valid to this precision. To illustrate this, consider a typical CCD camera with pixels each capable of storing up to about 200,000 charges (electrons or 'holes' depending on the camera design). Statistically the standard deviation of 200,000 randomly collected charges is the square root (about 447 charges). Thus, although we can measure the voltage level corresponding to the full well capacity of the pixel to very high precision with our A/D converter, we do not know the 'true' value of the photon count to anywhere near this precision

    It is reasonable to ask why high precision converters offer advantages in CCD cameras and similar devices. The answer is that the converter allows us to make measurements on high and low charge levels within a given image without having to change the 'gain' of the output amplifier to match the charge level to suit the converter for each pixel. Thus, for example, if an eight-bit A/D converter is used to digitise signals from a device with a full well capacity of 200,000 electrons the least number of electrons that can be sensed is 200,000/256 or about 781 charges. The standard deviation of 781 electrons is about 28 electrons. Another well having only 400 electrons with a standard deviation of 20 electrons would not be distinguished from the higher level using an eight bit converter, even though statistically the count levels in the two pixels are clearly distinct. A higher resolution converter avoids this problem.

    Another reason to use a high precision converter is that often signal levels in CCD cameras are increased at the expense of spatial resolution by combining the charge from adjacent pixels (pixel 'binning'). This can often be achieved within the CCD chip itself, leading to increased precision at the readout stage. The charge capacity of the readout stage is sometimes greater than that of an individual pixel to make this possible. A high-precision A/D converter allows the gain of the output amplifier to be set to suit the higher signal levels seen in the 'binned' pixel blocks while still allowing adequate resolution if the camera is used without pixel binning.

     

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