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FLIM

# Automated lifetime-based screening and characterization of fluorescent proteins

Lambert Instruments has provided a versatile library of interface functions (API) that allows direct communication with the LIFA software via MATLAB, in order to control the LIFA camera and the microscope. With the aid of this API, a custom made MATLAB graphical user interface (GUI) was developed that allows multi-position acquisition.

# Fluorescence Lifetime Imaging with a Time-Domain FLIM System on a Widefield Microscope

Fluorescence lifetime can be recorded for every pixel in the image simultaneously with a time-domain FLIM camera. This method requires an intensified camera, a pulsed laser and a widefield fluorescence microscope. This is typically more cost-effective than alternative methods that need a confocal set-up.

One of the most popular methods for fluorescence lifetime imaging microscopy (FLIM) is time-correlated single photon counting (TCSPC). This method requires a confocal microscope with a pulsed laser and a photomultiplier tube (PMT). The sample is briefly illuminated by a laser pulse after which the PMT counts the number of emitted fluorescence photons. The intensity I of the fluorescence emission decays exponentially after the laser pulse has excited
the sample:

I(t) = I_0\exp\left(-\frac{t}{\tau}\right).

The fluorescence lifetime $$\tau$$ quantifies the rate of decay of the fluorescence light. By scanning the sample with a focused laser beam, TCSPC systems can construct a fluorescence lifetime image of the sample one pixel at a time.

As an alternative to TCSPC on a confocal microscope, Lambert Instruments has developed a new system that brings time-domain FLIM to widefield microscopes. By carefully timing the exposure of the camera in the subnanosecond range, a light pulse profile of the fluorescence light can be captured. This method requires a pulsed laser and an intensified camera to record the raw data. Custom Lambert Instruments software then processes this data to automatically calculate the fluorescence lifetime.

### Set-up

Images were recorded with the LIFA-TD, which has a CCD camera with a fiber-optically coupled image intensifier. The image intensifier boosts the incoming light levels and it can achieve gate widths of less than 3 ns. A 485 nm pulsed laser (Picoquant LDH-D-C-485 laser head with a PDL 800-B laser driver) with a fiber-optical output was coupled into a widefield fluorescence microscope (Nikon Eclipse Ti) to provide 85 ps excitation pulses.

### Methods

The set-up was calibrated by recording the light pulse profile of the laser by placing a highly reflective material in the sample holder of the microscope. Next, the fluorescence decay profile of a convallaria (lily of the valley) sample was recorded. The fluorescence lifetime is determined by correlating the fluorescence emission to the light pulse profile.

### Results

Figure 1 shows the fluorescence lifetime of a convallaria sample overlayed on the original image. The LIFA-TD is able to detect the small variations in fluorescence lifetime between different parts of the sample, stained with different dyes.

Figure 1: Fluorescence intensity (left) recording of convallaria sample and corresponding fluorescence lifetimes (right) overlayed on the original image.

### Conclusion

Time-domain fluorescence lifetime imaging microscopy can be done on a widefield fluorescence microscope by using an intensified camera and a pulsed laser. The LIFA-TD is an entry-level FLIM system that offers an integrated solution.

# Revealing Cancer's Infrastructure

Lambert Instruments has been shipping the LIFA to cancer research facilities all over the world for years. We visited Dr. Kees Jalink of the Biophysics of Cell Signaling group at the Netherlands Cancer Institute. His research group purchased the first ever LIFA to leave the labs of Lambert Instruments. Ten years later, the LIFA is still their fluorescence lifetime imaging method of choice for studying signal transduction pathways in living cells.

# Confocal FLIM Applications

Confocal imaging on a widefield fluorescence microscope can now be done in combination with frequency-domain fluorescence lifetime imaging microscopy (FLIM). The increased spatial resolution in the z-direction results in lifetime images with enhanced contrast as the detection of out-of-focus emission is reduced significantly. This allows you to see differences in fluorescence lifetime e.g. between the cell membrane and the cytoplasm.

The data below were obtained with the Lambert Instruments FLIM Attachment (LIFA), either widefield (with LED light; 468nm peak) or confocal (with spinning disk CSU10 and 470nm-diode laser). The fluorescence lifetime images are generated at 2 different z-positions, z1 and z2. Snapshots of several z-positions are shown, as well as movies through even more.

### z2

These images show different pollen grains: the lifetime in pseudo colours and the intensity in grey scale. Because of the pinholes in the spinning disk, the exposure time is higher with confocal imaging. In this image 220 ms exposure time per phase step was taken for the confocal image versus 195 ms for the widefield (LED light). However, when a diode laser with higher power is used, the exposure time can be shortened.

### z2

These images show YFP-transfected mammalian cells and were taken with 12 phase steps of each 400 ms for the confocal, versus 70 ms for the widefield image. The calculated lifetimes are 2.34 ns (z1) and 2.42 ns (z2) in the confocal images, versus 2.58 ns (z1) and 2.57 ns (z2) in the widefield images.

The differences in lifetime could be due to the fact that the diode laser has one excitation wavelength, exactly 470 nm, while the LED has a range of wavelengths for which we used the emission band pass filter of 465-495 nm.

# Total Internal Reflection Fluorescence Lifetime Imaging Microscopy

Total Internal Reflection Fluorescence (TIRF) microscopy facilitates extremely high-contrast visualization and thereby high sensitivity of fluorescence near the cover glass. Typically, the optical section adjacent to the cover glass is about 100 nm. TIRF does not disturb cellular activity, thus enabling 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 like vesicle release and transport, cell adhesion, secretion, membrane protein dynamics and distribution or receptor-ligand interactions. The unique combination of TIRF and frequency domain FLIM makes it possible to measure lifetimes of, for instance, small focal adhesions near the cover glass.

### Widefield

These cells (kindly provided by Ms. S.E. Le Devedec, Leiden University, The Netherlands) express dSH2-GFP in small focal adhesions as well as in the nuclei as shown by widefield microscopy. However, by the use of TIRF only fluorescence close to the coverslip is obtained, thus only the focal adhesions are excited.

Fluorescence lifetime images give a more accurate measurement in TIRF mode, as out of focus light is emitted from the average lifetime in the focal adhesions.

The images shown here are taken with the Nikon TE2000-U widefield microscope with white-TIRF illuminator, combined with the Lambert Instruments Fluorescence lifetime imaging Attachment (LIFA). As light source the modulated LED of 468nm 3W was used and as demonstrated here enough intensity was generated to obtain fluorescence lifetime images with TIRF.

### Widefield

Cells (kindly provided by Ms. S.E. Le Devedec, Leiden University, The Netherlands) expressing dSH2-GFP in small focal adhesions as well as in the nuclei.

The images shown here are taken with the Olympus TIRFM (laser-TIRF), combined with the Lambert Instruments FLIM Attachment (LIFA). As light source the modulated diode laser of 473 nm 20 mW was used and as demonstrated here enough intensity was generated to obtain fluorescence lifetime images with TIRF.

### TIRF

Cells (kindly provided by Ms. S.E. Le Devedec, Leiden University, The Netherlands) expressing dSH2-GFP in small focal adhesions as well as in the nuclei.

# FLIM-FRET Experiments

Fluorescence Lifetime Imaging Microscopy Forster Resonance Energy Transfer (FLIM-FRET) has a lot of advantages over other FRET detection techniques. A major advantage is that FLIM-FRET measurements are more robust and quantitative than the FRET measurements done by, for example, sensitised emission FRET. Another advantage is that only the lifetime of the donor fluorophore has to be measured; steps to determine acceptor lifetimes are not needed. The acceptor fluorophore may therefore have an inefficient emission, or even may be a quencher, and still good quality FRET-data can be retrieved. This makes the FLIM-FRET method more versatile, faster, and easier. Furthermore, no corrections are needed for donor fluorophore emission bleed through in the acceptor emission channel.

### Acceptor Photobleaching

When photobleaching the acceptor fluorophore during FRET, the non-radiative transfer of energy from the donor to the acceptor decreases. The donor fluorophore, in its turn, loses less energy and its fluorescence lifetime, with respect to FRET without a photobleached acceptor. Only when the acceptor is bleached completely, the lifetime of the donor fluorophore will be similar to the situation of no FRET occurrence (a donor-only situation).

### Enhanced Acceptor Fluorescence (EAF)

In the case where the donor and acceptor fluorophores are both excited with the same excitation light wavelength, e.g. in the FRET pair GFP-YFP, a special kind of FRET can be detected. Namely, the average lifetime that is calculated is the contribution of both donor and acceptor fluorophores. Taking GFP and YFP as an example, GFP has a small lifetime compared to YFP. When no FRET is occurring, the average lifetime is measured of both GFP and YFP that are both excited by the 480nm wavelength light source. However, when FRET occurs, the energy of the GFP proteins transfers non-radiatively to the YFP proteins, so relatively more YFP emission (with a long lifetime) is taken into account. So, the average lifetime increases instead of decreases, as is normally the case when you measure only the lifetime of the donor fluorophore.

# Microscopy

Cell biology is the discipline that studies cells to answer scientific questions. All organisms are composed of one or more cells and all vital functions of an organism occur within cells. Cells contain the hereditary information necessary for regulating cell functions. Cells possess DNA, the hereditary material of genes, and RNA, containing the information necessary to build various proteins such as enzymes, the cell's primary machinery. There are also other kinds of biomolecules in cells, e.g. lipids, proteins, macromolecules, and more.

Cell biology research includes learning the physiological properties such as the structure and the organelles of cells, their environment, interactions, life cycle, division, function, and eventual death. This is done both on microscopic and molecular level, and includes the research of single-celled organisms like bacteria as well as specialized cells in multi-cellular organisms like humans.

Knowing the composition of cells and how cells work is fundamental to all of the biological sciences. Appreciating the similarities and differences between cell types is particularly important to the fields of cell and molecular biology. These fundamental similarities and differences provide a unifying theme, allowing the principles learned from studying one cell type to be extrapolated and generalized to other cell types.

In combination with our products, wide field fluorescence microscopy is used to measure characteristics of fluorescent proteins. Cells, originating from bacteria and insects to mammals, generally are kept in culture and plated at coverslips to do specific experiments. Microscopy enables viewing objects inside cells that are stained or fluorescently tagged. By observing the characteristics, e.g. the fluorescence lifetime, of the fluorescent compounds, not just the localization of a fluorescent protein, but also the characteristics of its local environment can be imaged. Novel multi-parameter fluorescence imaging systems are being used to study intracellular organization and inter- and intracellular signalling.

One way to observe the proteins, is by fixation of the cells to the coverslips. Before the cells are fixed, the compounds in the cells can be fluorescently tagged (see living cell-imaging). Also, the compounds inside the cells can be stained after their fixation, for example by use of antibodies. Staining is a biochemical technique of adding a class-specific (DNA, proteins, lipids or carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound. The characteristics of the dyes can give answers to specific scientific questions, like whether there is interaction between two different proteins, whether there is a conformational change of the protein after a kind of treatment (see also Fluorescence Lifetime Imaging Microscopy and Forster Resonance Energy Transfer), or whether specific ions have bound to the proteins of interest, etc.

The cells can also be analyzed in-vivo. These living cell imaging experiments seek to gain information about the localization and interaction of the desired protein. One way to do this is to replace the wild-type gene with a 'fusion' gene that has a reporting element such as GFP. That will allow easy visualization of the products of the genetic modification. More sophisticated techniques are in development that can track protein products without mitigating their function, such as the addition of small sequences which will serve as binding motifs to monoclonal antibodies.

# Frequency-Domain FLIM: From nanoseconds to milliseconds

In modern day science Fluorescence Lifetime Imaging Microscopy (FLIM) is more and more recognized as a powerful diagnostic tool. The lifetime of fluorescence is the decay time of the emission after excitation stopped.

Fluorescence lifetime can be affected by a number of effectors including pH changes and changes in oxygen concentration. One very potent application of FLIM is Forster Resonance Energy Transfer (FRET) analysis for studying protein interactions. In case of FRET, the emission energy is transferred to an acceptor molecule instead of being released as a photon (emission). FRET effectively reduces the decay time of the donor emission.

Fluorescence lifetimes fall typically within the range of 1–10 ns. Lambert Instruments offers a user friendly robust FLIM system, the Lambert Instruments FLIM Attachment, for resolving lifetimes within the range of 0 to 300 ns, with an accuracy of 80 ps. Now this lifetime resolving capacity range has been extended to 1 ms with their new product, the LIFA-X.

### Frequency-Domain FLIM

Figure 1. Modulated excitation will result in modulated emission with an identical repetition frequency. The phase shift and decrease in modulation depth are caused by the decay of the fluorescent molecule and can be used for deducing the lifetime information.

Frequency-domain FLIM is a method to acquire wide-field FLIM images. For all pixels in the image the decay times of fluorophores are collected simultaneously. In order to achieve this, a homodyne detection scheme is applied in which the intensity of the excitation light is modulated in a sinusoidal fashion. Consequently, the emission signal will have a modulated intensity with an identical repetition frequency. However, the phase of the emission signal will be delayed (= phase shift) because of the decay of the fluorophore. The decay of the emission also results in a lower amplitude of the emission signal (= decrease in modulation depth). Therefore, the emission signal contains two parameters from which the fluorescence lifetime can be deduced: the phase shift and decrease in modulation depth (figure 1).

The lifetime of a fluorescent material can be determined from the phase-shift by the following relation:

\tau_\phi = \frac{1}{\omega} \tan \phi,

where $$\omega$$ is the angular frequency of the modulation and $$\phi$$ is the phase-shift.

Equally, the lifetime can be determined from the decrease in modulation depth $$m$$. The decrease in modulation depth $$m$$ is the relative modulation depth of the emission signal as compared to the excitation:

\tau_m = \frac{1}{\omega} \sqrt{\frac{1}{m^2} - 1}.

In case of a single exponential decay it can be shown that $$\tau_\phi$$ is equal to $$\tau_m$$. A significant difference between these two calculated values indicates the presence of more components having different lifetimes. For further analysis of multi-exponential decay the software offers the Multi-Frequency method and the Polar Plot method.

### LIFA

The Lambert Instruments FLIM Attachment, or LIFA, is a complete, easy to use lifetime imaging system. It can be mounted onto any wide-field fluorescence microscope. The detector is an intensified CCD (ICCD) camera that uses an image intensifier as prime detector that is fiber optically coupled to a CCD. This ICCD allows detection of low-light levels, a typical feature of fluorescence. The high sensitivity of the detector allows the usage of a low excitation intensity, minimizing photo-bleaching of the sample. Modulated LEDs are used as the standard source of light in the LIFA system.

Figure 2. The LIFA system can be mounted onto any wide-field fluorescence microscope.

The image intensifier of the ICCD is modulated at the same frequency as the excitation signal. Here, modulation means modulation of detector sensitivity. The phase of modulation is altered through 360 phase degrees with dedicated steps. All pixels are detected simultaneously. When the detector is most sensitive at the peak of the emission signal, the measured intensity per pixel will be high; i.e. the detector modulation is in phase with the emission signal. If the detector modulation is out-of-phase with the emission signal, the detector will be least sensitive when the emission signal is most intense. The measured intensity per pixel will be low. The measured intensities per pixel are then plotted versus the detector phase and the fitted sine wave contains both the phase and the modulation depth information (figure 3).

By using a uniform material with a known lifetime (e.g. rhodamine 6G) as a reference, the unknown lifetime of a sample can be distilled when comparing the two fitted sine waves. This calculation of the lifetime is done for every pixel of the image simultaneously, which is the fundament for wide-field FLIM.

The LIFA, a set-up with modulation of both the excitation light and detector sensitivity, can be used for modulation frequencies from 100 kHz to 80 MHz with LED as a light source. Moreover, modulation frequencies up to 120 MHz are feasible with the use of modulated laser diodes. This frequency range is adequate for the measurement of lifetimes from 0.1 ns to 300 ns with a temporal resolution of 80 ps.

Figure 3. The fit through the data points when plotting single-pixel intensities versus detector phase contains information about both the phase of the modulated emission signal as well as the modulation depth in that pixel.

The theoretically optimal frequency for resolving fluorescence lifetimes is determined using Equations 3 and 4:
\begin{align}
f & = \frac{1}{2\pi\tau}\text{ for phase lifetime,} \label{eq:PhaseFrequency} \\
f & = \frac{\sqrt{2}}{2\pi\tau}\text{ for modulation lifetime,} \label{eq:ModulationFrequency}
\end{align}
with $$\tau$$ = lifetime (s) and $$f$$ = frequency (Hz). This means that for a lifetime of e.g. 4 ns the optimal frequency is approximately 40 MHz.

For much longer lifetimes like 10 $$\mu$$s the optimal frequency is approximately 15 kHz. For these long lifetimes a much lower frequency of the excitation is needed, else the modulation of the emitted signal from the sample is too low for accurate measurements. For coverage of the low frequency range Lambert Instruments developed the LIFA-X.

### The new LIFA-X

Figure 4. The new LIFA-X ICCD camera with integrated pulse generator.

The LIFA-X is Lambert Instruments' latest addition to their FLIM product range with the 'X' indicating the extended range of lifetimes this product can resolve. The LIFA-X can resolve lifetimes from 0.1 ns up to 1 ms (phosphorescence) and is well suited, e.g., for measuring oxygen concentrations using optodes or reactive oxygen species in cells.

The LIFA-X includes a modified ICCD camera in which a dual pulse generator is built in. It can be used in modulated mode in which it can be operated like the LIFA, resolving short lifetimes (< 300 ns). Additionally it can be operated in pulsed mode for the measurement of long lifetimes (> 300 ns). The design allows retro fitting onto existing LIFA systems. When the system is operated in ‘long lifetime mode’ – i.e. the image intensifier is gated - the pulse generator controls the gating of the image intensifier. The pulse generator also controls the LED or laser excitation source. The use of a pulsed signal creates a block wave modulation pattern (figure 5). Especially at low frequencies a block wave is more effective than a sinusoidal wave in relation to an efficient use of photons.

Figure 5. Frequency domain FLIM using a pulsed lightsource results in a block wave excitation and emission pattern.

In gated mode frequencies of 1 kHz to 100 kHz are supported. Data analysis comprises the same statistic strategies as with modulated excitation and detection and can be done with the same LI-FLIM software package as for the LIFA.

### Software

For data acquisition the LIFA and LIFA-X are supplied with the software program LI-FLIM. LI-FLIM is a user-friendly software package that controls the hardware components, collects phase images, and processes the intensity information per pixel. These data are analyzed instantaneously allowing visual representation of the lifetime data in a matter of seconds.

Figure 6. Screenshot of the LI-FLIM acquisition software. This software is used for analyzing the FLIM data. In the recorded sample shown, CHO cells are transfected with either GFP (donor-only, ROI with long lifetimes – red pixels) or GFP-RFP fusion protein (widely used FRET pair) of which the donor protein has a significantly shorter fluorescence lifetime due to FRET (ROI with short lifetimes – blue pixels). An overlay of the sample intensity image with lifetime data is shown on the right. The left plane of the screen shot displays the statistics window with analyzed lifetime data of the drawn regions of interest.

The data can be presented in an overlay of lifetime values over an intensity image of the sample. Polar plot presentation is also supported. Regions of interest can be selected for statistical analysis (figure 6). Other features include time-lapse recordings (minutes to days) and multi-frequency measurements for unmixing multiple lifetime components.

### Image intensifier

A second and third generation image intensifier consists of a photocathode that emits electrons when illuminated.  The emitted electrons are accelerated in the direction of the anode screen. Between cathode and anode a micro-channel plate (MCP) is positioned. A MCP is the micro-channel equivalent of a photon multiplier tube (PMT) and multiplies the number of primary electrons in each channel by secondary emission from the channel wall. The gain of the intensifier can be controlled by the voltage across the MCP and by the cathode voltage.

When the electrons hit the anode screen, their energy is converted into light which is emitted through the output window of the intensifier. The wide dynamic range allows imaging of dim and bright features within one image. The spatial resolution is determined by a number of parameters such as the individual channel diameter in the MCP and the applied voltages to cathode and anode.

Figure 6. Schematic representation of an image intensifier. p, photon.

The photocathode needs to be negatively charged in order to realize sensitivity. Namely, if positive the released electrons will remain at the photocathode; i.e. the tube is closed. Modulation/gating of the image intensifier is achieved by (rapidly) changing the voltage at the photocathode.