# The LIFA software is not connecting to the frame grabber

If the software keeps indicating that it is trying to connect to the frame grabber (see bottom left corner of the screen), then please follow these steps:

1. Right-click the Start menu and click Device Manager.
2. Look for the section Imaging Devices and and expand it.
3. Find the device named X64 Xcelera-CL LX1 Board and check if there is a yellow triangle with an exclamation mark shown on its icon.
4. Run the installer of the frame grabber drivers and choose the option Repair.
5. Restart your computer and go back to the Device Manager to make sure the yellow triangle with the exclamation mark has disappeared.
6. Start the LIFA software to see if the software can connect to the frame grabber.

# Where do I find the log files of LI-Capture?

In the bottom left corner of the about screen, click the 'Explore .ini and log files' button:

This will open the LI-Capture folder on your computer that contains the log files. The filename of a log file can be 'li-capture.log' or 'li-capture.log.x' where x is a number.

When asked to send log files for support purposes, it is recommended to send all log files that are available.

# Where do I find the log files of LI-FLIM?

In the bottom left corner of the about screen, click the 'Explore .ini and log files' button:

This will open the LI-FLIM folder on your computer that contains the log files. The filename of a log file can be 'li-flim.log' or 'li-flim.log.x' where x is a number.

When asked to send log files for support purposes, it is recommended to send all log files that are available.

# Can I install LI-FLIM on my Mac?

LI-FLIM was designed to run on Windows. There is no Mac version of LI-FLIM available that works with the LIFA hardware to record FLIM data.

If you just want to analyze existing FLIM data on a Mac, we recommend installing Windows in a virtual machine (like VirtualBox) on your Mac. Then you can install LI-FLIM and analyze your data.

# Which factors can influence my FLIM measurements?

Lifetime imaging microscopy often involves performing delicate experiments, such as the detection of protein-protein interactions inside cells, or dynamics of certain proteins. Fluorescence decay can be influenced by many factors in the micro-environment, and each factor should be considered carefully.

### CONCENTRATION

For diluted, non-interacting chromophores, fluorescence lifetimes are independent of the chromophore concentration, and this is actually one of the major advantages of the FLIM techniques in quantification of e.g. FRET. [1] and oxygen imaging.

However, significant concentration-dependent quenching is possible. For example, a shortening of the CFP fluorescence lifetime was observed in cells expressing simultaneously the YFP acceptor. This probably reflects primarily intermolecular energy transfer between unlinked CFPs and YFPs. But also in the absence of YFP acceptors this quenching was observed, which might be attributed to pseudo-homo-FRET. [1]

Homo-FRET may lead to significant perturbations of the measured fluorescence decay, if different sub-populations of the chromophore are associated to slightly shifted emission spectra, which we know is actually the case for purified CFP. Therefore, it is called pseudo-homo-FRET. In this situation, energy transfer will preferably take place from the blue emitting sub-populations, and will lead to a specific decrease in the apparent fluorescent lifetimes determined on the blue edge of the emission spectrum (<495nm emission). This decrease in fluorescence lifetime will be maximum if the collected fraction of the emission spectrum does not include any contribution from the "red" acceptors emission. [1]

### PHOTOBLEACHING

Photobleaching of the fluorophores used in an experiment may affect the fluorescence lifetime results. However, as long as the acquisition time scale for measuring the fluorescence lifetime is small compared to the characteristic timescale for photobleaching, the effect of photobleaching on the lifetime determination can be neglected.

Acceptor photo-bleaching is a method widely used for quantifying energy transfer in intensity-based FRET imaging, although it cannot be used for time lapses. In FLIM-based FRET imaging it is very hard to obtain a full recovery of the donor lifetime only after photobleaching the acceptor. In next example [1], the CFP-YFP FRET pair was used; the lifetime of CFP only was 2,5ns and the lifetime of CFP in presence of YFP was1,68ns. After 98% photodestruction of YFP, the lifetime of CFP only increased up to 1,83ns. However, irradiating YFP in the absence of CFP leads to a significant increase in the fluorescence detected in the "CFP" detection channel (up to 28% above background after an 80% photobleaching of YFP). Therefore, it seemed that some photoproducts were created during YFP irradiation, which contributed to fluorescence in the same wavelength ranges than CFP, leading to a contamination of the CFP signal. [1]

### AUTOFLUORESCENCE

Media and cells often contain substances that are autofluorescent. Examples are NADH, riboflavins, collagen and lipofuscins [5]. This means that those substances can be excited at the same wavelength as used to excite the fluorophore of interest. Every substance that is excited and emits photons (thus fluorescence) has its own characteristic fluorescence lifetime. The emission of the autofluorescence material mixes with the emission of the fluorophore of interest. If it is not filtered out, it will influence the measured lifetime. To suppress autofluorescence FLIM users generally use phosphate buffered saline (PBS) as medium for the lifetime measurements. One can also use phenol red-free medium.

Frequency domain FLIM can be used to discriminate against autofluorescence, using either the polar (or phasor) plot [6] or a multi-frequency or multi-harmonic FLIM acquisition.

### FIXATION

Fixation can also affect fluorescence lifetimes. The important thing is that you need to use the appropriate controls for your experiments. If you are looking for FRET in fixed cells then you need to have donor only and donor + acceptor cells treated under the same conditions. [3]

Various labs use paraformaldehyde (PFA) fixation protocols, methanol fixation and combined methanol/PFA fixation protocols; which protocol to use is mostly determined upon the protein of interest. You should check that the protein localisation is not perturbed by the fixation method and also that the fluorescence is not completely diminished. [3]

### MORE FACTORS

There are more factors that should be taken into account. For example, the work of Klaus Suhling [4] shows that the refractive index of the medium can influence the fluorescence lifetime of GFP. Also GFP expressed alone can have a different lifetime to when it is part of a protein construct.

Even temperature can have severe influence on lifetime measurements. Therefore, the temperature of the sample should be kept as stable as possible, e.g. by use of a climate control chamber.

### REFERENCES

1. Regis Grailhe, Fabienne Merola, Jacqueline Ridard, Stephen Couvignou, Chantal Le Poupon, Jean-Pierre Changeux, Helene Laguitton-Pasquier. "Monitoring protein interactions in the living cell through the fluorescence decays of the Cyan Fluorescent Protein", Chemphyschem 7:1442-1454 (2006).
2. D. A. Zacharias, J. D. Violin, A. C. Newton, R. Y. Tsien, Science 296:913-916 (2002).
3. Roland Brock, Irene H.L. Hamelers, and Thomas M. Jovin. "Comparison of Fixation Protocols for Adherent Cultured Cells Applied to a GFP Fusion Protein of the Epidermal Growth Factor Receptor". Cytometry 35:353-362 (1999).
4. Klaus Suhling, Jan Siegel, David Phillips, Paul M. W. French, Sandrine Leveque-Fort, Stephen E. D. Webb, and Daniel M. Davis. "Imaging the environment of green fluorescent protein". Biophysical Journal 83:3589-3595 (2002).
5. Billinton N, Knight AW. "Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence". Anal Biochem 291(2):175-97 (2001).
6. Stringari, C. et al., PNAS, 108, 33, 13582"Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue" (2011).

# LI-FLIM reports that the background is incompatible or missing

If the software reports that it is impossible to calculate a lifetime image due to incompatible or missing background. Please check if LI-FLIM automatically takes a background recording by going to the options, upper menu bar, "View --> LI-FLIM options --> Acquisition Tab" and enable "Automatically record background..."

# LI-FLIM reports LED failure

The software reports that a LED failure is detected. Please disconnect the device by pressing the X in the upper right corner of the device window. Turn of the control unit and inspect the LED cable at the back entrance of the control unit. In case you have a single LED, please inspect the cable connected to the LED. If everything looks to be fine, power on the control unit, reconnect the device in the software and try to press FLIM. If the failure persists, please contact Lambert Instruments.

# My LIFA control unit has detected overexposure

• When an overexposure is reported, a red LED on the LIFA control unit switches on, and the device will be disabled in the LI-FLIM software. Camera and light source are turned off. The overexposure is normally triggered by high fluorescence emission from your sample and/or using high value for the MCP voltage. Be careful when using a third party light source for illumination of your sample. If the LIFA camera is used and this causes an overexposure, the LIFA control unit cannot turn off this light source and the risk of damaging the camera persists. Once an overexposure is detected, one should switch back to eye piece and investigate the cause of the overexposure (type of illumination, epi-fluorescence or transmitted light, ND filters, high MCP value, etcetera).
• Close the device by pressing the X in the upper right corner of the device window, and press the reset button on the LIFA. You will hear that the fans will be turned off and powers on a gain, the control unit has been reset and can be reconnected by the software (Devices --> LIFA). The MCP level will be set back to its minimum.

# The red light on my LIFA control unit is on

• Is there an overexposure detected? There is an anode current limiter inside the control unit; an error is reported when the current of the anode of the image intensifier is above a certain threshold preset in the factory. A high anode current normally indicates that a high instantaneous light level is entering the camera. Please make sure there is no light illuminating the camera and press the 'Reset' button. The photocathode of an image intensifier becomes less sensitive with increased total light dose. The anode current limiter protects against the highest instantaneous light doses. Note that there is no automated protection against somewhat lower light levels.
• Is there a LED failure reported? If the control unit detects a LED failure, the LIFA will also report an error. Is the LED cable connected correctly to the LED? If so, the LED could be broken.
• When using LI-FLIM through Active-X there is a watchdog function which can be used inside your scripts. It is a timer that can help to protect your image intensifier when running long series of automated measurements. To prevent entering an error mode one needs to reset the watchdog timer or set it to zero before it is zero, to turn it off.
• Is there a high temperature and humidity in the operating room? Sometimes this can give a false positive overexposure.

# The image intensity drops in part of the image

• In your images, is there a clear area with lower intensity with respect to its surroundings?
• Have you checked the alignment of your illumination, is it centered and uniform?
• Is the spot visible when you look through the eyepiece?
• There can be dust on the entrance window of the FLIM camera. To clean the camera entrance window, make sure you use (compressed) air and that the camera and control unit are turned off. Please make sure that no direct sunlight or other high intensity light entering the camera when unscrewing the camera from the microscope.
• If you turn the camera when connected, is the area turning with the image or is it staying on the same place? If it turns, the area is part of the image that is being projected to the camera. If it stays at the same position, the camera sensitivity may be reduced. Please contact Lambert Instruments.

# LI-FLIM doesn't detect one of my devices

• Is the hardware turned on and are all USB cables and/or RS232 cables connected?
• Is the device listed in the devices menu (third dropdown menu in upper menu bar in LI-FLIM)? If listed, please click 'enable' to connect. If not, please perform a search for devices by pressing "Find available Devices".
• Is the device plugin loaded (View > LI-FLIM Options > Plugins). If listed, please click 'enable' to connect.
• If searching for devices did not work out, please check the COM port settings of Windows. Most of the LIFA devices use a Virtual COM port. Windows automatically assigns COM port numbers to devices and sometimes two different devices get the same COM port. See MS Windows Control Panel > System > Devices manager. There you can change the serial port number. Please restart the computer to make sure it has been changed correctly.

# The calculated lifetimes are different from what I expected

• Are you using the right fluorescence filter cube?
• Are you using the right reference solution and reference lifetime for calibrating the system? Did you change anything in the optical setup between taking a Reference and a Sample? Please note that each optical setup requires its own reference calibration; i.e. switching dichroics, filters, objectives, light source, magnifications, or power light levels changes the path lengths inside the system. The same holds when using a different MCP gain for the intensified CCD camera. Changing ND filters or the exposure time does not require a different reference calibration.
• Is there reason to believe that the (photophysics of) your sample is as expected?
• Is there auto-fluorescence influencing the fluorescence lifetime distribution?
• In case your application is FRET; is there leak-through from your acceptor in the donor channel?

# I switched on the camera but I don't see anything

### THERE IS NO SIGNAL TO THE CAMERA

• Is the microscope switched to the camera port?
• Do you see light through the eyepiece when switching to eyepiece?
• Are you using the right fluorescence filter cube?
• Is there a shutter, neutral density (ND) filter or field stop in the light path? (the LIFA Multi-LED has a shutter, is it open?)
• Did you press the 'FLIM' button to put the system into live mode?
• Is the LED/laser on?

### THE SIGNAL IS LOW

• What is your MCP setting? Please test with MCP values up to around 800V.
• Did you use a long enough exposure time (i.e. >100 milliseconds)?
• Are you using the right fluorescence filter cube?
• Are you using ND filters, please put the system in 'IDLE' mode and remove them, if any.

### THERE IS NO LIGHT ON MY SAMPLE

• Are you using the right excitation filter?
• Is there a shutter, neutral density (ND) filter or field stop in the light path? (the LIFA Multi-LED has a shutter, is it open?)
• Did you press the 'FLIM' button to put the system into live mode?
• Is the LED/laser on?

# How should I analyze my data?

Analyzing image data and also lifetime data is often an arduous task. Always make sure that you have taken enough samples to be able to obtain data for a large number of cells. In LI-FLIM you can draw regions of interest (ROIs) in the image to obtain averages of the lifetime in that particular ROI. The STD given is the standard deviation of the lifetime values of all of the pixels in that ROI. Therefore one should always take multiple samples of different regions in the sample. Please see the LI-FLIM manual for more information (Help > Documentation).

# Do the quantum efficiency, absorption coefficient and concentration of my sample affect the calculated lifetimes?

There are two ways to calculate the fluorescence lifetime. The quantum efficiency (Q), absorption coefficient (A) and concentration (C) of your sample will not affect the outcome of either method. This is because these characteristics alter the light intensity, which is not a factor in the lifetime calculations.

Schematic overview of factors that are important for calculating the fluorescence lifetime.

When calculating the fluorescence lifetime from the phase, only the shift in phase between the excitation and emission light is taken into account. The amplitude of the modulated light intensity does not affect the phase shift.

The fluorescence lifetime calculated from the demodulation (M) is also not influenced by the quantum efficiency, the absorption coefficient or concentration of your sample. These factors determine the light intensity emitted by the sample:

$$I_{\text{emission}} = Q\cdot A\cdot C\cdot I_{\text{excitation}}.$$

A lower quantum efficiency, absorption coefficient or concentration results in a lower intensity of the emitted fluorescence light. This affects the average light intensity of the modulated light as well as the amplitude of its variation by the same amount. The modulation (m) is the ratio of the variation of the light intensity (a and b in the figure above) to its average (c and d in the figure):

$$m = \frac{Q\cdot A\cdot C\cdot a}{Q\cdot A\cdot C\cdot c} = \frac{a}{c}.$$

The quantum efficiency, the absorption coefficient and the concentration affect the variation and the average by the same amount. These factors cancel in the calculation of m and are therefore of no influence to the lifetime calculated from the demodulation.

# What frequency should I use?

In frequency domain FLIM the lifetime sensitivity depends on the used modulation frequency. The shorter the expected lifetime, the higher the required modulation frequency to obtain an accurate measurement. A rule of thumb is: $f = \frac{100}{\tau}.$ With $$f$$ in megahertz and $$\tau$$ in nanoseconds. For example for GFP transfected cells the lifetime is around 2.5 ns, so the required frequency is about 40 MHz ($$\frac{100}{2.5 ns} = 40$$ MHz). Please note that you do not have to change the frequency for slightly different lifetimes, as for a particular frequency the system is sensitive to a broad lifetime range (several nanoseconds). Further note that the above formula is a rule of thumb: it is likely that a frequency slightly different from the rule-of-thumb value gives more accurate results. This is easily verified by measuring at a series of frequencies and inspecting the statistics: the lower the standard deviation, the better the used frequency.

# What do DC and AC stand for?

DC: depending on the type of light source you are using, it stands either for direct current (Multi-LED: LED DC, in mA) or for the mean voltage (Multi-LASER MOD DC level in V).

AC: depending on the type of light source you are using, it stands either for alternating current/modulation (Multi-LED: LED AC) or how much voltage peak-peak is being supplied to the MOD OUT port of the control unit to drive the Multi-LASER (MOD DC in V peak - peak).

# Which settings should I use?

Typical settings are: 100 milliseconds of exposure time per phase frame, 100 mA LED DC for LED power. In 'Expert mode' it is possible to change the modulation properties of the camera (Cathode DC and AC level) and of the light source (LED DC and AC level), however for normal use these values do not need to be changed. During the LIFA support or LIFA advanced training session the modulation settings are checked.

It is always good to start with a color map of the camera in absolute scale (by pressing the 'A' in the menu bar of the imaging window). Subsequently increase the MCP with 50-100 Volts and check if there is signal on the camera. If signal does not appear please use the '98' color scale and play with the phase slider (LIFA Control window). Once there is signal one should move the phase slider to the setting producing the highest intensity (the "maximum phase"), subsequently one can scale back to absolute color scale and increase exposure time. For the best possible lifetime accuracy please make sure that the intensity in the 'maximal phase' image reaches a value close to the maximum of the 16 bit range.

Note that when the exposure time is set higher than 330 milliseconds, the intensity will not increase accordingly in live video mode as live mode acquisition is limited to 330 ms. To check the result of the requested integration time, press snapshot (in Expert mode). Please know that once you have pressed snapshot, the camera is still active, thus to turn it off one should press the Idle button.

Typically 12 phase images are taken for acquisition of a phase stack, without averaging. Averaging yields a lower standard deviation. A lower MCP can also yield a somewhat lower standard deviation.

# How should I start up and shut down the setup?

On startup, first make sure all the hardware cables (power and USB) are connected. Then turn on the LIFA control unit, and start up the LI-FLIM software.

For shutdown first close the LI-FLIM software and then turn off the LIFA control unit. Make sure that when you are done, you switch the microscope port from camera port back to eyepiece, to prevent unwanted light reaching the intensified camera. Turn off the mains power.