# Single-Image Fluorescence Lifetime Imaging Microscopy

Toggel features a unique image sensor that was designed and optimized specifically for fluorescence lifetime imaging applications. It enables lifetime imaging at unprecedented frame rates with the single-image fluorescence lifetime imaging microscopy (siFLIM) method [1].

"We used a prototype of the Toggel camera to develop a method for acquiring quantitative lifetime images from a single exposure." says professor Kees Jalink of the Netherlands Cancer Institute. "siFLIM takes advantage of the technical capabilities of the Toggel camera and simultaneously records two 180°-phase-shifted images. This allows for video-rate lifetime imaging with minimal phototoxicity and bleaching."

During a single exposure, the Toggel camera records two images. The electrons in each pixel of the sensor are toggled between two storage areas at the same frequency as the modulation frequency of the light source. This results in two images that are shifted 180° in phase with respect to each other.

Because both images are recorded simultaneously, there are no artifacts of cellular movements and there is a significant improvement in the photon efficiency of the image acquisition.

These two images of convallaria were recorded simultaneously, and because of the 180° phase shift between the two images, the recorded light intensity differs. The difference in light intensity between these two images depends on the phase shift and demodulation of the fluorescence light and can be used to calculate changes in the fluorescence lifetime of a sample [1].

[1] siFLIM: single-image frequency-domain FLIM provides fast and photon-efficient lifetime data, M. Raspe et al, Nature Methods 13, 501–504 (2016)

Schematic representation of one pixel in the image sensor of the Toggel. The electric field (rotating black line) in the pixel directs the photo-electrons (green) to one of the storage areas (gray).

# Intensifier Gating for Ultra-Short Exposure Times

The photocathode of an image intensifier can be used as an ultra-fast shutter. By varying the voltage on the photocathode, the image intensifier gate can be switched between open and closed. When the gate is open, incoming photons can enter the image intensifier and the light intensity is boosted. When the gate is closed, incoming photons can't enter the image intensifier.

Switching the gate between open and closed states can be done very quickly, thus allowing the gate to be opened for a very brief moment. This enables effective exposure times in the order of nanoseconds.

By opening the image intensifier gate only once during each exposure of the camera sensor, you can eliminate motion blur even when imaging fast-moving objects. The video below illustrates the effects of image intensifier gating.

The first part of the video shows a bullet recorded at 15000 fps. Despite the high frame rate, the bullet moves so fast that it is blurry. The second part of the video shows the benefits of image intensifier gating. By opening the image intensifier for just 2 microseconds during each frame of the camera, the bullet is no longer blurry. It is perfectly sharp in each frame of the video.

# The Difference Between Rolling Shutter and Global Shutter Sensors

Image sensors are available in many shapes and sizes, and with different capabilities. But in this post, we will focus on one very important thing: the electronic shutter methods that are available.

### Rolling Shutter

Most consumer cameras use a rolling shutter method. With this method, the pixels on the sensor are read sequentially. When you press the shutter button, the camera scans through all the pixels and stores the information digitally. This means that the first pixel will be read out at a different time than the last pixel. And everything that happens after the first pixel is read out will still be captured by the last pixel, and the pixels in between.

### Global Shutter

Global-shutter sensors read out all pixels of the sensor simultaneously, so the entire frame represents image data that was captured at the same moment in time. This method is not subject to the same motion artifacts as the rolling-shutter method.

### Consequences

In everyday use, you won't notice if your camera uses the rolling shutter method. Only when you're capturing an image of a fast-moving object (like a fan), you may notice some motion artifacts like deformed fan blades.

In situations that require high-performance imaging, rolling shutter can severely affect your data. In such cases, it is better to use a global-shutter sensor, to ensure that your image represents the same instant in time and to prevent rolling shutter artifacts.

# Enhanced Intensifier Techniques

Apart from the obvious advantages of gain and intensification, the intensifier offers additional possibilities. It can serve as a fast shutter, by use of gating. At a negative cathode voltage, the intensifier is open. It closes at a positive voltage. Switching can be done very quickly and at high repetition rates, resulting in very short exposures (down to nanoseconds), synchronized with a camera that can operate at very high frame rates. Ultrashort exposure will reduce any motion smear to a minimum. The figure below shows a recording sequence of a combustion cycle of a fuel injection engine at 22000 fps, made with a gated intensified high-speed camera.

An image intensifier can also serve as a radiation converter. Images in the part of the spectrum that are invisible to the human eye (for example UV or NIR) can be converted to a different part of the spectrum that can be detected by an image sensor. The spectral sensitivity of the image intensifier is determined by the type of photocathode that is chosen.

# CoaXPress

CoaXPress (CXP) is a communication standard for imaging data. It transfers data over one or multiple coaxial cables. The main strengths of this standard are its high transfer speeds and the long cable lengths. CXP can also power cameras with Power-over-CXP, removing the need for a dedicated power supply for the camera.

### Transfer speeds

Because of its high transfer speeds, CXP is ideal for streaming high-speed imaging. Each CXP cable can transfer up to 6.25 Gbps. Our cameras have 4 CXP ports for a total transfer speed of up to 25 Gbps.

### Computer interface

You need a frame grabber to capture the data that is transferred over CXP. A frame grabber is an expansion card for a computer that captures the incoming data and displays it on the screen or stores it on the computer. Most frame grabbers offer a software development kit (SDK) to develop your own specialized image acquisition software.

# GigE Vision

GigE Vision is a framework for transmitting images over an Ethernet connection. It consists of protocols that define how to configure a camera and to transfer the image data. Every computer with a fast Ethernet card is compatible with the GigE Vision framework. So GigE Vision requires only an ethernet card, whereas CoaXPress and Camera Link require a framegrabber.

The maximum transfer speed of a GigE Vision camera (assuming a gigabit Ethernet card in the computer) is 1000 Mb/s.

# GenICam

The Generic Interface for Cameras (GenICam) standard aims to provide a generic programming interface for cameras and other camera-related devices. Every step in the imaging process -from configuring the camera to getting the recorded images off the camera- can be configured using GenICam.

No matter what type of camera or data transfer interface you are using, if all your devices are GenICam compatible then it will be much easier for them to communicate.

# Fiber-Optic Coupling

It is important that image quality is maintained as much as possible when using intensifiers. At the same time, light efficiency should be maximized. This can be achieved by using a fiber-optic window as the output of the first stage and as the input of the second stage.

A fiber-optic window is a solid piece of glass consisting of millions of parallel glass fibers sealed together. Each fiber acts as an independent light conductor. The shape of the window can either be flat (parallel input and output faces), or concave. Fibers with a concave surface are used for distortion correction in electrostatic image inverters.

Often the second stage will also have a fiber-optic output to allow coupling to a third stage, or to the image sensor of the camera. In the latter case the image sensor of the camera should be equipped with a fiber optic input window. In addition, take the following into consideration when you need to make a choice for either fiber-optic coupling or lens coupling:

• Fiber-optic coupling is a permanent connection; the connection is made during the manufacture of the integrated intensified camera.
• A fiber-optic window transfers an image from one face to the other. If the fiber optic has a tapered form, the image is reduced or enlarged. This characteristic can be used to match it to the format of a coupled imaging component.
• While fiber-optic coupling between intensifiers is the standard technique, coupling to the camera can also be done by lens optics. Disadvantages of lens coupling are the greater loss in efficiency (compared to fiber optics) and the lenses are more bulky.
• Lens coupling offers the flexibility of easy decoupling, allowing you a choice to make camera recordings with or without the use of an intensifier.

Camera Link is a serial communication standard. It has three main configurations: Base, medium and full.

### Base configuration

This configuration requires one cable and has a data throughput of 2.04 GBit/s.

### Medium configuration

This configuration requires two cables and it can transfer twice as much data as the base configuration. The maximum data throughput of this configuration is 4.08 GBit/s.

### Full configuration

This configuration also requires two cables and it has a maximum data throughput of 5.44 GBit/s.

# TIRF FLIM

Total Internal Reflection Fluorescence (TIRF) microscopy is a super-resolution technique with high sensitivity of fluorescence near the cover glass. TIRF does not disturb cellular activity, and enables tracking of biomolecules, and the study of their dynamic activity and interactions at the molecular level. TIRF enables the selective visualisation of processes and structures of the cell membrane and pre-membrane space such as vesicle release and transport, cell adhesion, secretion, membrane protein dynamics and distribution or receptor-ligand interactions. The  combination of TIRF and frequency domain FLIM makes it possible to measure fluorescence lifetimes of for instance small focal adhesions near the cover glass.

High NA TIRF objectives (up to 1.49) make it possible to introduce illumination at incident angles greater than the critical angle ($$\theta$$) resulting in TIR (Total Internal Reflection) accompanied by the formation of an evanescent wave immediately adjacent to the coverglass-specimen interface. The evanescent wave energy drops off exponentially with distance from the coverglass and reaches about a hundred nanometers into the specimen. For TIR to occur, the refractive index of the coverglass should be higher than the refractive index of the specimen (which is e.g. the case when using buffered saline solution).

The white-TIRF as well as the laser-TIRF system utilises this evanescent wave to excite fluorescent molecules in a very thin section in contact with the coverglass (here: green dots). Because the specimen is not excited beyond the evanescent wave (here: white dots), this imaging system can produce fluorescence images with an extremely high signal-to-noise (S/N) ratio and z-resolution.

# Confocal FLIM

Confocal microscopy is a technique for high-resolution three-dimensional imaging which uses a pinhole to increase resolution in the image plane and eliminate out-of-focus light in thick specimens. The thickness of the focal plane is generally defined mostly by the objective lens and also by the optical properties of the specimen and the ambient conditions. With an imaging confocal only the light within the focal plane is detected, so that the resulting confocal images appear crisper than widefield images [read more at the cell biology wiki]. Typical applications occur within the life sciences, e.g. in cell biology. In essence there are two classes of confocal systems: single beam and multi-beam.

## Confocal Scanning with CSU Spinning Disk

A Nipkow spinning disk is a multi-beam confocal scanner. The main advantage of this type of confocal imaging is the relatively fast imaging acquisition making it useful for live cell imaging applications.

The operating principle of the Yokogawa CSU spinning disk is explained here. Briefly, the disk has a spiral pattern of pinholes that is illuminated by an expanded laser beam. This generates a multi-beam illumination pattern which which the sample is illuminated. By rapidly rotating the disk this multi-beam visits all positions in the sample plane near simultaneously. The part of the fluorescence that travels back through the pinholes generates a full field confocal image at the camera detector.

Lifetime images of mixed polled grains, at three different z-positions.

Being a camera-based system, the Lambert Instruments LIFA system for frequency domain FLIM is compatible with multi-beam confocal microscopy techniques, most notably the Yokogawa CSU spinning disk series (based on the Nipkow disk scanner), and the VTInfinity series by Visitech International Ltd.

# Frequency-Domain FLIM for Beginners

Fluorescence lifetime imaging microscopy (FLIM) can be performed in the time domain and in the frequency domain. Scanning single point lifetime detection units on confocal laser scanning microscopes mainly operate in the time domain. Camera-based lifetime detection on widefield, multi-beam confocal and total internal reflection fluorescence (TIRF) microscopes operate both in time domain and frequency domain. The Lambert Instruments LIFA for example is a fast frequency-domain system, whereas the Lambert Instruments TRiCAM can be operated both in the frequency and time domains.

### Time Domain

In the time domain the fluorescence decay can be measured by using time-correlated single photon counting (TCSPC) or fast-gated image intensifiers. A measurement requires short excitation pulses of high intensity and fast detection circuits. Each point in the sample is excited sequentially. TCSPC records a histogram of photon arrival times at each spatial location using Photo Multiplier Tubes (PMTs) or comparable single photon counting detectors. Fast-gated image intensifiers measure fluorescence intensity in a series of different time windows. With both time domain techniques lifetimes are derived from exponential fits to the decay data. When sufficient channels (time windows) are used, multi-exponential lifetimes can be extracted.

### Frequency Domain

The frequency-domain FLIM technique requires a modulated light source and a modulated detector. The excitation light is modulated or pulsed in intensity at a certain radio frequency (the blue curve in the figure below). The induced fluorescence emission will mirror this modulation pattern and show, due to the fluorescence decay, a delay in time in the form of a phase-shift (the red curve). In addition, the modulation depth will decrease with respect to the excitation light, while the average intensity remains the same. The phase-shift and modulation-depth directly depend on the fluorescence lifetime and the known modulation frequency (see figure).

To extract the phase shift and modulation depth from the fluorescence emission signal, a homodyne detection method is often used. In this method the sensitivity of the detector - often an intensified camera - is modulated (or gated) with the same radio frequency as the light source (the green curve in the figure on the right). For a camera detector the result is an intensity image with a fixed brightness. By shifting the phase of the image intensifier with respect to the light source in a series of fixed steps a low-pass signal is generated for each pixel: the output image will be brighter or dimmer depending on whether the detector sensitivity is in or out of phase with the fluorescence emission. The result is a frequency-domain FLIM signal as a function of the phase difference between light source and camera (purple curve in the figure on the right) for each pixel in the image.

The key is that this frequency-domain signal (purple curve) exactly mirrors the phase shift and demodulation in the time domain. The phase and modulation depth can be directly extracted from the measurements and are the fundamental data in a homodyne FD FLIM measurement.

From the acquired modulation depth and phase shifts, two independent determinations of the fluorescence lifetime can be calculated. For an absolute determination the system needs to be calibrated at the pixel level with a reference fluorophore of known lifetime. For this calibration the only requirement is a FLIM acquisition of the reference fluorophore with known lifetime (figure on the right).

### Multi-Exponential Decay

Some fluorophores have a multi-exponential decay, consisting of two or more lifetime components. For example, the decay of CFP is bi-exponential. These multiple lifetime components can be separated and extracted using multiple frequency measurements and the polar (or phasor) plot.

The key advantage of frequency-domain FLIM is its fast lifetime image acquisition making it suitable for dynamic applications such as live cell research: the entire field of view is excited semi-continously - using relatively broad excitation pulses - and read out simultaneously. Hence frequency domain lifetime imaging can be near instantaneous. Another advantage of a camera-based FLIM setup, such as the Lambert Instruments LIFA, is its ease of use and its low maintenance requirements. For more information about our products, please visit the FLIM product pages and our FLIM software page.

Time-lapse of EPAC sensor courtesy of Netherlands Cancer Institute

Principles of Fluorescence Spectroscopy, Springer 3rd ed., J.R. Lakowicz (2006)

Advances in Biochemical Engineering/Biotechnology, chapter Fluorescence Lifetime Imaging Microscopy (FLIM) by E.B. van Munster & T.W.J. Gadella (2005) 95:143-175

FLIM microscopy in Biology and Medicine, CRC Press, by A. Periasamy and R.M. Clegg, editors (2010)

FRET & FLIM Techniques, Elsevier, by T.W.J. Gadella, editor (2009)

### What is the fluorescence lifetime?

The fluorescence lifetime - the average decay time of a fluorescence molecule's excited state - is a quantitative signature which can be used to probe structure and dynamics at micro- and nano scales. FLIM (Fluorescence Lifetime Imaging Microscopy) is used as a routine technique in cell biology to map the lifetime within living cells, tissues and whole organisms. The fluorescence lifetime is affected by a range of biophysical phenomena and hence the applications of FLIM are many: from ion imaging and oxygen imaging to studying cell function and cell disease in quantitative cell biology using FRET.

For fluorescent molecules the temporal decay can be assumed as an exponential decay probability function:

$P_{decay}(t)={1/\tau} e^{-{t/\tau}},\quad t\gt 0$

where $$t$$ is time and $$\tau$$ is the excited state lifetime.

More complex fluorophores can be described using a multiple exponential probability density function:

$P_{multiple-decay}(t)={\sum_{i=1}^{N}}\alpha_i\cdot P_{\tau_i}(t),\quad t\gt 0$

where $$t$$ is time, $$\tau_i$$ is the lifetime of each component and $$\alpha_i$$ is the relative contribution of each component.

A key advantage of the fluorescence lifetime is that it is a basic physical parameter that does not change with variations in local fluorophore concentration and is independent of the fluorescence excitation. Hence the lifetime is a direct quantitative measure, and its measurement - in contrast to e.g. the recorded fluorescence intensity - does not require detailed calibrations. Excited state lifetimes are also independent of the optical path of the microscope, photobleaching (at least to first order), and the local fluorescence detection efficiency.

The fluorescence lifetime does change when the molecules undergo de-excitation through other processes than fluorescence such as dynamic quenching through molecular collisions with small soluble molecules like ions or oxygen (Stern-Volmer quenching) or energy transfer to a nearby molecule through FRET. As a result the fluorophores (in the excited state) lose their energy at a higher rate, causing a distinct decrease in the fluorescence lifetime. The measured rate of fluorescence is actually a summation of all of the rates of de-excitation. In this way the fluorescent lifetime mirrors any process in the micro-environment that quenches the fluorophores; and spatial differences in the amount of quenching reveals itself as contrast in a lifetime image.

# FLIM Papers and Reviews

A selection of papers papers based on Lambert Instruments FLIM systems is maintained here.

The following is a selection of books and papers on FLIM technology:

BOOKS / REVIEWS:

• Gadella TW Jr., FRET and FLIM techniques, 33. Elsevier, ISBN-13: 978-0080549583. (Dec 2008) 560 pages. Elsevier link
• Periasamy, A & Clegg RM, FLIM Microscopy in Biology and Medicine. Chapman & Hall/CRC, 1st edition, ISBN-13: 978-1420078909. (Jul 2009) 368 pages. Amazon link
• Lakowicz JR. Principles of fluorescence spectroscopy, 3rd edition, ISBN-13: 978-0387312781 . Springer, 3rd edition (Sep 2006) 954 pages. Amazon link

BOOKS; FLUORESCENT PROTEINS / CELL BIOLOGY:

• Sullivan KF, Fluorescent Proteins, 2nd Edition Volume 85. Academic Press, ISBN-13: 978-0123725585. (Dec 2007) 660 pages. Elsevier link
• Sullivan KF, Kay SA, Wilson L, Matsudaira PT,Green Fluorescent Proteins, Volume 58. Academic Press, ISBN-13: 978-0125441605. (1998) 386 pages.

PAPER MULTIFREQUENCY:

• Squire A, Verveer PJ, Bastiaens PIH, Multiple frequency Fluorescence lifetime imaging microscopy. Journal of Microscopy, (2000) 197(2):136-149. Pubmed link

PAPER PHASE STEP ORDER:

• Van Munster EB, Gadella TW Jr, Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order. Cytometry A. (2004) 58(2):185-94. Pubmed link

PAPERS POLAR PLOT:

• Redford GI, Clegg RM, Polar plot representation for frequency-domain analysis of fluorescence lifetimes. Journal of Fluorescence (2005) 15(5):805-815. Pubmed link
• Clayton AHA, Hanley QS, Verveer PJ. Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data. Journal of Microscopy (2004) 213(1):1-5. Pubmed link

PAPERS TIRF-FLIM:

• Valdembri D, Caswell PT, Anderson KI, Schwarz JP, König I, Astanina E, Caccavari F, Norman JC, Humphries MJ, Bussolino F, Serini G, Neuropilin-1/GIPC1 signaling regulates alpha5beta1 integrin traffic and function in endothelial cells. PLoS Biol. (2009) 27:7(1):e25. Full text

# Modulated Intensifiers for Lifetime Imaging

As part of its portfolio, Lambert Instruments designs and manufactures ICCD cameras based on modulated image intensifiers for frequency domain imaging techniques such as FLIM. Standard products such as the new TRiCAM M ICCD camera, the TRiCATT M modulated intensifier attachment, and the LIFA system are all based on a proximity-focused modulated Gen II or Gen III image intensifier.

Figure 1. Schematic representation of a proximity-focused image intensifier, showing its photocathode, micro-channel plate and anode, fiber-optically coupled to a sensor.

Figure 2. Modulated detector gain (green), pulsed excitation (blue) and fluorescence emission (red) in a homodyne FLIM system. The phase and duty cycle of the detector gain is controllable.

The modulated sensitivity of an image intensifier is produced by high-frequency switching of its photocathode voltage. In this modulation mode the temporal and also the spatial characteristics of the image intensifier are different from the nominal characteristics under continuous operation.

The following table lists the nominal characteristics for the Gen II and Gen III intensifiers provided.

### Gen III GaAsP

Spatial resolution
Photocathode QE at 550 nm

28 lp/mm
14%

17 lp/mm
28%

7 lp/mm
48%

The increased sensitivity of the Gen III image intensifier results in a better lifetime accuracy, although the amount further depends on the model and the modulated light source used. For a LIFA Gen III GaAs compared to a LIFA Gen II S25 at 550nm, both using the same Multi-LED light source, the increased sensitivity results in an increased lifetime accuracy of 40% at the same acquisition speed and fluorescence intensity.

For phosphorescence lifetime imaging with the TRiCAM GM and the LIFA-P the image intensifier is operated at lower frequencies spatial resolution is higher, reaching values in excess of 35 lp/mm for a Gen III GaAs image intensifier.

Please note that the characteristics of an image intensifier are unique and are known to vary between units.

Figure 3. Image intensifier spectral response.

# Intensified High-Speed Imaging

## Related Posts

Clever use of digital cameras in combination with intensifiers and boosters allow us to create images of high-speed events, even when light is failing. In addition, fast gating offers possibilities to use extremely short exposures and to record multiple images in one frame. For creating images of events that are invisible to the human eye, like near-infrared (NIR) and ultraviolet (UV), radiation conversion techniques can be used. This technology note will review the techniques that make this possible.

### Issues concerning high-speed imaging at low light levels

Ever since the invention of the digital camera, new imaging applications have been explored. The increasing possibilities of fast digital cameras have resulted in applications that were unthinkable only twenty years ago. High-speed cameras nowadays are widely used for recording of dynamic events at high frame rates (e.g. 10000 fps). The results can then be inspected by playing individual frames at a lower speed.

High-speed imaging up to 100000 fps is easily feasible with current technology. But what if you need to create high-speed images when light conditions are far from optimal? Your high-speed camera will be no good under these circumstances, as a certain brightness of the object is required for the high frame rates that are used. The lack of light in combination with short exposure times will result in underexposed and noisy images. The obvious solution would be to increase the illumination level of the object. However, in some cases it is just not possible to add more light, for example because:

• The object to be recorded generates light by itself. This may be the case for phenomena like the combustion process (flames and turbines), or in living cells that emit fluorescent light.
• The radiation level corresponding to the required brightness would cause an unacceptable temperature rise of the object.

And what if the image signal has become too low because of the high frame rates? Camera noise will be an additional problem then.

Fortunately, there is a high-tech solution for these problems: the image intensifier. It is used to intensify the image before it is projected onto the image sensor of the high-speed camera. The intensified image results in a sensor signal that is typically 10000 times higher than without using an image intensifier - in the process elevating the signal above camera noise level.

### How does an intensifier work?

Figure 1. Photons are converted to electrons, accelerated and then multiplied in the MCP.

The image intensifier is a vacuum tube with a photocathode at the input, a micro-channel plate (MCP) in the middle and a phosphorescent screen at the output, as shown in figure 1. Photons are processed as follows:

1. The image is projected onto the photocathode. The photocathode converts the incoming light (photons) into electrons. The electrons are emitted in the vacuum tube and accelerated towards the MCP by an electric field.
2. The MCP is a thin plate consisting of many parallel micro channels; each channel works as an electron multiplier by secondary emission from the channel wall. The gain of this multiplier depends on the voltage that is applied between the input and the output of the MCP. Typical electron gain is in the order of 10,000. At the end of the channel, the electrons are accelerated by an electric field towards the anode screen.
3. The anode screen is a phosphor layer deposited at the vacuum interface of the output window; it is covered by a thin aluminum film to prevent light feedback. The anode screen has a potential of 6 kV with respect to the MCP. The electron energy is absorbed by the phosphor material and converted into light. The result is a visibly intensified image at the output of the intensifier.

The output window of the intensifier is usually fiber-optically coupled to the next component. This can either be the image sensor or to a next stage of the intensifier.

### What if an intensifier is not enough?

The intensifier in combination with a high-speed camera offers great possibilities, but sometimes the quality of the resulting images is still insufficient. Light output limits the maximum frame rate that can be obtained when using an image intensifier. The light output increases linearly with the input as long as the gain is constant. However, the gain of the MCP is only constant up to a certain output level, even in the case of special lower-resistance MCPs that are used in high-speed applications. Above a certain level, the MCP becomes saturated and the number of electrons at the end of the MCP will no longer increase. This will result in a maximum output brightness that is insufficient for many high-speed applications.

Increasing the gain of the intensifier by applying multiple MCPs will not help either: the maximum output is limited by the same maximum output current of a single MCP.

What happens if we add another intensifier, but without the MCP, as a second stage? This is what we call a booster. There will be no saturation in the second stage, but the extra gain factor results in more light at the output of the second stage. If there is more light, a higher frame rate can be used.

Figure 2. A booster is a second stage intensifier, but without the MCP. The advantage is that no saturation will take place in the second stage.

Figure 3 shows a comparison of three images of a blue gas flame, recorded with different techniques. The first recording (figure 3a) shows the gas flame to be studied in  detail. The light intensity of the flame is not very high. To see any details, especially in close-ups, very short exposure times are required.

Figure 3. Combustion research - three recordings compared.

Figure 3b was made with a standard high-speed camera at 1000 fps and a 1 ms exposure time. On the one hand a longer exposure time is needed to increase the sensitivity of the camera; on the other hand, a shorter exposure time is needed to prevent motion blur.

The third recording (figure 3c) was made with an intensified high-speed camera system at 2000 fps and a 15 us exposure time. The intensified high-speed camera is sensitive enough to image the flames at frame rates up to 100000 fps. By using gating (fast electro-optical shutter function of the image intensifier), the exposure time can be limited to a value at which motion blur is no longer an issue.

### Boosting image brightness

So the output brightness of an image can be increased by adding a booster as the second stage intensifier. A booster is a so-called first generation (Gen1) image intensifier. It offers a relatively low but constant gain (ca. 10x) up to very high light levels.

Gen1 image intensifiers are available as proximity-focused diodes. This is the type of intensifier schematically displayed in figure 2 as second stage. Gen 1 image intensifiers can also be used in electrostatic inverter tubes. This type offers electron-optical demagnification; it can be used to match the intensifier better to the image sensor format of the camera. The demagnification also increases the output brightness with a factor $$1/M^2$$ ($$M$$ is the magnification).

A demagnifying intensifier may be the ideal solution if either a booster is needed as a second stage intensifier or the format of the image sensor is considerably smaller than the 18 or 25 mm intensifier.

An extra brightness gain is achieved that increases the sensitivity of the intensified high-speed camera. No tapered fiber optics nor demagnifying relay lens, both cutting down the coupling efficiency, are needed. The output of the demagnifying image intensifier is 1:1 imaged onto the image sensor, either by a straight fiber optic face plate or a 1:1 relay lens.

### Enhanced intensifier techniques

Apart from the obvious advantages of gain and intensification, the intensifier offers additional possibilities. It can serve as a fast shutter, by use of gating. At a negative cathode voltage, the intensifier is open. It closes at a positive voltage. Switching can be done very quickly and at high repetition rates, resulting in very short exposures (down to nanoseconds), synchronized with a camera that can operate at very high frame rates. Ultrashort exposure will reduce any motion smear to a minimum. Figure 4 shows a recording sequence of a combustion cycle of a fuel injection engine at 22000 fps, made with a gated intensified high-speed camera.

Figure 4. Combustion cycle of a fuel injection engine.

Figure 5. Sensitivity of photocathodes. GaAs and GaAsP cathodes are usually offered in Gen3 intensifiers, while S20, S25 and broadband are common in Gen2 intensifiers.

An image intensifier can also serve as a radiation converter. Images in the part of the spectrum that are invisible to the human eye (for example UV or NIR) can be converted to a different part of the spectrum that can be detected by an image sensor. The spectral sensitivity of the image intensifier is determined by the type of photocathode that is chosen. Figure 5 shows the spectral sensitivity curves of various photocathode types.

### When not to use a booster

As explained earlier, adding a booster to your intensifier will allow for higher frame rates. However, as the booster is an extra component in the imaging system, it will also lower the resolution of the total system. For comparison: an intensifier has a typical resolution of 45 line pairs per mm while an intensifier combined with a booster will bring the resolution down to 25 line pairs per mm.

This is when camera pixel size becomes important: when the camera has large pixels (e.g. the 20 um typical for a high-speed camera), the change from a 45 to a 25 lp/mm resolution will be negligible. When a camera with small pixels is used, however, the change in quality of the image will be significant.

### Fiber-optic coupling or lens coupling?

Obviously, it is important that image quality is maintained as much as possible when using intensifiers. At the same time, light efficiency should be maximized. This can be achieved by using a fiber-optic window as the output of the first stage and as the input of the second stage.

A fiber-optic window is a solid piece of glass consisting of millions of parallel glass fibers sealed together. Each fiber acts as an independent light conductor. The shape of the window can either be flat (parallel input and output faces), or concave. Fibers with a concave surface are used for distortion correction in electrostatic image inverters.

Often the second stage will also have a fiber-optic output to allow coupling to a third stage, or to the image sensor of the camera. In the latter case the image sensor of the camera should be equipped with a fiber optic input window. In addition, take the following into consideration when you need to make a choice for either fiber-optic coupling or lens coupling:

• Fiber-optic coupling is a permanent connection; the connection is made during the manufacture of the integrated intensified camera.
• A fiber-optic window transfers an image from one face to the other. If the fiber optic has a tapered form, the image is reduced or enlarged. This characteristic can be used to match it to the format of a coupled imaging component.
• While fiber-optic coupling between intensifiers is the standard technique, coupling to the camera can also be done by lens optics. Disadvantages of lens coupling are the greater loss in efficiency (compared to fiber optics) and the lenses are more bulky.
• Lens coupling offers the flexibility of easy decoupling, allowing you a choice to make camera recordings with or without the use of an intensifier.
• Governed by the laws of optics no increase in brightness is achieved when demagnifying the image either by use of fiber optics or by lenses.

### How to choose the correct image intensification solution

Before you start to look for a solution for a high-speed imaging problem, you will need to determine the following:

• What exactly would you like to record?
• How many frames per second (fps) do you need to record?
• What is the required gating repetition rate?
• Do you wish to use a camera that you already have? If yes, what type of camera is it and what type of lens mounting is required.
• Which photocathode will be suitable? You may want to consult a specialist in intensified high-speed imaging for help.
• Will you need a booster? Again, you may want to consult a specialist in intensified high-speed imaging for help.

### Dual-stage intensifier

Max. gain
Effective diameter (mm)
Max. gating (ns)
Gate repetition rate (kHz)
Max. sensor framerate (fps)

10000
18 or 25
40 (fast gating: < 3)
100 (gast gating: 200)
10000

100000
18 or 25
40 (fast gating: < 3)
100 (fast gating: 200)
100000

Generally, a two-stage intensifier (with the booster as second stage) is recommended at frame rates of 1000 fps or more. If the object to be imaged consists of individual light emitting events against a dark background, a single stage might be adequate up to much higher frame rates, as the charge capacity of the MCP allows a temporal high-brightness output when such an event is imaged.

Depending on the sensitivity of the high-speed camera and the light-coupling efficiency between intensifier and camera, frame rates of up to 200000 fps are feasible. If higher frame rates are required, more than one booster stage can be used.

### Looking for a specialist in intensified high-speed imaging?

When looking for an intensified high-speed imaging specialist, be sure to consider the following requirements:

• At least 20 years market experience: make sure you contact a company that has experience with intensified high-speed imaging, has a variety of customers and extensive market knowledge.
• Extensive support: you will want a company that provides consulting, implementation and support.
• Variety of solutions: the ideal company offers a range of practical solutions for high-tech imaging issues.
• Integration with current equipment: you will want a company that offers options for integration of high-speed cameras, intensifiers and/or boosters with existing equipment.

### Lambert Instruments

Lambert Instruments is a company devoted to development, production and worldwide sales of products for high-speed imaging at low light levels. The aim of Lambert Instruments is to provide a well-defined product that fits budget and planning.

Lambert Instruments has successfully provided solutions with intensified high-speed imaging for a wide range of applications:

• Combustion research
• Plasma physics
• Time-Resolved Fluorescence
• Dynamic phenomena in microscopy (imaging of rotation of single molecules of ATPase, detection of Brownian motion)
• Laser-induced fluorescence (LIF)
• Particle image velocimetry (PIV)
• Microfluidics

We welcome feedback on this technology note and we encourage discussion about the principles of high-speed imaging at low light levels. Also, we consider it a challenge to solve any problem you may have concerning high-speed imaging at low light levels. Please feel free to contact us.

# Dual-Stage Image Intensifier

In very low-light situations or when a very short exposure time is required, a dual-stage image intensifier may be required. The first stage is the same as a single-stage image intensifier; it has a micro-channel plate that multiplies the electrons emitted by the photocathode. The second stage is often referred to as a booster. This stage does not have a micro-channel plate, it multiplies the incoming photons without the saturation characteristics of a micro-channel plate.

### Dual-stage image intensifier with fiber-optic coupling

The figure above shows a schematic representation of a dual-stage image intensifier that is fiber-optically coupled to the image sensor. The first stage is similar to a single-stage image intensifier.

# Intensified High-Speed Cameras

Normal consumer cameras operate very well in day-light, or room ambient lighting conditions. However, when you want to make a snapshot of a fast moving object, exposure-time has to be shortened to obtain a sharp image. This comes with a cost; images are much darker when using a short exposure time. At a certain threshold, the attenuation has to be compensated. This could be done by increasing the light (by using a flash), or by improving the photo-sensitivity of the camera. In high-speed cameras this effect is even stronger.

To get clear images in high-speed cameras, an object has to be illuminated with a high intensity light-source. The higher the frame rates the shorter the exposure time per frame, the higher the intensity of the light-source must be. In many applications increase in illumination is an adequate method to compensate the shorter exposure times. However, in some applications the object itself is emitting light, or is influenced by the light-source. In combustion research, for example, or imaging of dynamic phenomena in fluorescent biological cells, or low intensity PIV, light intensities are too low to record with conventional high-speed cameras. In applications like microfluidics, the heat generated by a powerful light source can have a tremendous effect on liquid flows.

To apply high-speed imaging in the forementioned situations, Lambert Instruments has developed intensified high-speed cameras and high-speed intensifying camera attachments. The special two stage high-speed image intensifiers in these products amplify the input light to a typically 10000 times higher level on the output. This makes it much easier to distinguish an image from the noise. Furthermore, the gating feature of the image intensifier makes it possible to capture even the fastest objects without motion blur.

# Spatial Resolution of Image Intensifiers

The limiting spatial resolution of an intensified imaging system depends on several factors, including (but not limited to)

• Image intensifier type
• Image intensifier gain
• Pixel size

Before we can discuss each of these factors, we need to define what limiting spatial resolution means. When characterizing an imaging system, the limiting spatial resolution describes the smallest features that can be distinguished. There are several ways of characterizing the spatial resolotion, most of them use a test chart like the USAF resolution test chart. Such charts have a series of lines on them, the smaller the lines an imaging system can distinguish, the better the spatial resolution.

SilverFast Resolution Target USAF 1951 (Creative Commons 3.0)

Spatial resolution is quantified in the number of line pairs that can be distinguised per millimeter (lp/mm). A line pair consists of a dark line and a bright line. So if one line is 5 microns wide, then a line pair will be 10 microns wide and there would be 1 mm/10 microns = 100 line pairs per millimeter.

### Image intensifier type

There is a wide range of image intensifiers available. We advise our customers on the type of intensifier they need for their application based on the wavelengths that are important for our customers, and the frame rates they need. High-speed intensifiers usually have a lower spatial resolution than image intensifiers that are optimized for lower frame rates.

### Image intensifier gain

We can increase the MCP voltage of an image intensifier to increase its gain. But MCP noise and the size of the electron cloud at the exit of the MCP also depend on the MCP voltage, so the spatial resolution will be slightly reduced as the MCP voltage is increased. You can learn more about how an image intensifier works on our image intensifier page.

### Pixel size

Finally, the limiting spatial resolution of an imaging system is determined by the size of the pixels that collect the light from the image intensifier. You can use our intensifier-sensor matching calculator to find the theoretical maximum sensor resolution. It is calculated using the size of the pixels.

25 and 50 lp/mm (not to scale)

For example: If the pixels are 20 microns wide, we would need two adjacent pixels to distinguish a bright line and a dark line of a test chart. Those two pixels would have a total width of 40 microns, so the theoretical spatial resolution would be 1 mm/40 microns = 25 lp/mm.

The element of the imaging system with the lowest spatial resolution determines the limiting spatial resolution of the whole system. In our example, we have a sensor that has a limiting resolution of 25 lp/mm. If we have an image intensifier with a 50 lp/mm resolution, the size of the pixels would limit the resolution of the imaging system to 25 lp/mm.

However, if the pixels are smaller, 2 microns for instance, then the theoretical resolution of the sensor would be 250 lp/mm. In that case, the resolution of the image intensifier would determine the resolution of the total system.

# Low-Light Imaging

Ever since the invention of the digital camera, new imaging applications have been explored. The increasing possibilities of fast digital cameras have resulted in applications that were unthinkable only twenty years ago. High-speed cameras nowadays are widely used for recording of dynamic events at high frame rates (e.g. 10000 fps). The results can then be inspected by playing individual frames at a lower speed.

High-speed imaging up to 100000 fps is easily feasible with current technology. But what if you need to create high-speed images when light conditions are far from optimal? Your high-speed camera will be no good under these circumstances, as a certain brightness of the object is required for the high frame rates that are used. The lack of light in combination with short exposure times will result in underexposed and noisy images. The obvious solution would be to increase the illumination level of the object. However, in some cases it is just not possible to add more light, for example because:

• The object to be recorded generates light by itself. This may be the case for phenomena like the combustion process (flames and turbines), or in living cells that emit fluorescent light.
• The radiation level corresponding to the required brightness would cause an unacceptable temperature rise of the object.

And what if the image signal has become too low because of the high frame rates? Camera noise will be an additional problem then. Fortunately, there is a high-tech solution for these problems: the image intensifier. It is used to intensify the image before it is projected onto the image sensor of the high-speed camera. The intensified image results in a sensor signal that is typically 10 000 times higher than without using an image intensifier—in the process elevating the signal above camera noise level.