Microscopy > Components > FastFLIM

FastFLIM

FastFLIM is the data acquisition card for your FLIM acquisition when data acquisition time is of the essence. The card has been developed using Digital Frequency Domain (DFD) technique that allows for the acquisition of Time-Tagged-Time-Resolved data without the dead time typical of TCSPC approach. The 4 independent input channels can be configured for accepting signals from PMTs, APDs with TTL output, or a combination of the two types of detectors.
The design allows for maximum acquisition of up to 15 million counts per second on each channel. Data are acquired as photon counts or in time-tagged mode. Decay times from 1 ms to 50 ps can be resolved. The card is supported by drivers in Windows 7 and Windows 10 operating system. The connection to the computer is through USB2.

Measurements:
  • Time-Tagged-Phase-Resolved lifetime measurements
  • Single-wavelength FLIM, multi-wavelength FLIM
  • Single-wavelength PLIM, multi-wavelength PLIM
  • Confocal images
  • Polarization images (steady-state and time-resolved)
  • FCS, FCCS, PCH
  • Scanning FCS, RICS, N&B
  • Stoichiometry
  • Single Molecule FRET
  • PIE measurements
Features:
  • 4-channel simultaneous acquisition
  • Direct input from PMTs and APDs (or a combination of the two)
  • Photon count rate up to 15 million/second
  • Dead time 3.125 ns
  • Trigger out to synchronize external devices
  • Trigger input from external source
  • Line and Frame CLK synchronization
  • USB communication
  • Drivers for Windows 10 OS

How It Works in an Instrument
A schematic of the instrument is reported in the Figure below. The signal from the photomultiplier tubes (PMTs) is directed to the FastFLIM unit. Also, the FRAME CLK, or LINE CLK signal, comes from the scanning mirror unit of the confocal microscope.

Applications
The FastFLIM unit is utilized as an upgrade for commercial Laser Scanning Microscopes (LSM) by Nikon, Olympus, Zeiss and Leica. Also, it can be utilized as a stand-alone unit for assembling a custom-built FLIM instrument.

The Measurements of Fluorescence Decay Times
Fluorescence is the light emitted by molecules in solution (or in a solid or gaseous state) following the absorption of radiation. Upon excitation with a short pulse of light of very short duration, the fluorescence emitted by the sample is described by the relationship:

[1]

where I0 is the intensity of the fluorescence at time t=0 and is the time it takes the intensity to decrease to a value its original value. The value is called the "fluorescence decay time". In a multi-components environment, the fluorescence is described by the relationship:

[2]

where the coefficients αii), called the pre-exponential factors and the decay times τi characterize fluorescence decay of the i component of the mixture. These parameters can be related to the fractional contributions, defined as the fraction of the total fluorescence emitted by the i component of the mixture:

[3]

In specific measurement situations the decay time of fluorescence is best described by non-exponential relationships. In any experimental case, devices measuring the fluorescence decay times provide the values i, τi) and any other parameter that describes the fluorescence decay times of each component in a mixture.

Frequency-domain and Time-domain Techniques
The instrumentation for the measurement of fluorescence decays times is broadly classified in two groups, time-domain and frequency-domain techniques.

The time-domain technique uses time correlated single photon counting (TCSPC). Usually, a laser emitting short pulses which repeat with a period slightly longer than the common fluorescence lifetime is used as the excitation light source, although other light sources (LEDs, synchrotron radiation, pulsed lamps) can be utilized as well. At the arrival of each pulse, a high precision timer is triggered which records how much time has passed between the arrival of the excitation pulse and the emitted photon. The precision of the technique is determined by the accuracy of the clock. Either a time-to-amplitude converter (TAC) or a GHz digital clock can be employed.

To interpret the lifetime time information obtained by TCSPC device a histogram of such arrival times is built. For a single exponential decay, a curve similar to the one of Equation [1] is collected and the decay time τ is determined using a minimization technique to fit the experimental data to the theoretical decay model. For multiple exponential decays, a curve similar to Equation [2] is built; the decay times of the components are determined using a minimization technique to fit the theoretical decay model to the experimental data.

For microscopy applications, the TCSPC acquisition electronics is synchronized to the scanning device (usually galvo-controlled mirrors or piezo-controlled stages), and the histogram acquisition restarts for each pixel of the image.

Frequency domain technique requires the modulation of the excitation light source. The modulated excitation results in a modulated fluorescence with a phase and modulation which is dependent on the lifetime of the excited fluorophores (see Figure below).

From the phase and modulation of the Δω frequency, the phase and the modulation of the fluorescence can be calculated relative to that of a reference lifetime. The lifetime is deduced from the phase and modulation:

[4]


[5]



Representation of the emission light (EM) following the excitation (EX) with a modulated beam. DC and AC are the direct and alternate components of the light.

The instruments utilized in frequency domain technique are called multifrequency phase fluorometers (MPF) or, simply, frequency domain fluorometers. When using a MPF to determine the characteristic decay times of the fluorescence, the excitation light source is modulated at a frequency ω; the phase shift Φ and the modulation m are measured. Such measurements are repeated at several different values of the modulation frequency, ω ranging typically from two or three for a single exponential decay, to up to about twenty five for multiple exponential decays. The decay times τi are determined using a minimization technique to fit the experimental data.

What is the Digital Frequency Domain (DFD)?
In digital heterodyning the cross-correlation frequency ƒcc is the difference between the sampling frequency utilized to probe the collect the data and the excitation frequency of the light source, that is:

[7]

It is convenient to have for ƒcc a value that is an integer fraction of the sampling frequency; in our implementation we use:

[8]

The advantages of DFD over AFD are multiple:
• There is no need to modulate the light detector
• The duty cycle is 100% therefore improving the sensitivity of the measurement.

Input Channels

No. of channels 4 independent channels
Input voltage range Directly from PMTs and/or APDs (TTL)
Decay times measurement From 100 ms to 100 ps
Dead Time (at 80MHz internal or external clock) 3.125 ns
External Reference CLK
External CLK IN 80 MHz, 40 MHz, 20 MHz, 10 MHz
TTL
Reference CLK OUT 0.02Hz - 80 MHz
Amplitude: +2.5 V, +1.8 V, +1.2 V on 50 Ohm load
Synchronization with galvano-scanner
LINE Scan Connects to the Line Scan signal from the LSM system
FRAME Scan Connects to the Frame Scan signal from the LSM system

Data Acquisition (Counts Mode, or Histogramming)

Max windows at 80MHz 4
Raw data size 32 bits
Raw data file structure Binary File with a header of 256 bytes
Data Incoming Stream Up to 140 x 106 counts/sec (35 million counts/sec per channel).
Sampling rate Up to 5MHz
Data handling and storage Acquisition of raw data for FCS, FCCS, PCH, smFRET.
On-line processing or post-processing
Max signal 35 x 106 counts/sec

Data Acquisition (Photon Mode or Time-Tagged)

Max windows at 80MHz 4
Raw data size 32 bits
Raw data file structure Binary File with a header of 256 bytes
Data Incoming Stream 60 x 106 counts/sec (15 x 106 counts/sec per channel)
Data handling and storage Acquisition of raw data for FCS, FCCS, PCH, smFRET.
On-line processing or post-processing
Sampling rate Up to 80MHz
Max signal in FCS 60 x 106 counts/sec (15 x 106 counts/sec per channel)
Max signal in FLIM 60 x 106 counts/sec (15 x 106 counts/sec per channel)

Data Acquisition (Photon Mode or Time-Tagged-Phase-Resolved, TTPR)

Max windows at 80MHz 4
Raw data size 32 bits
Raw data file structure Binary File with a header of 256 bytes
Data Incoming Stream 60 x 106 counts/sec (15 x 106 counts/sec per channel)
Data handling and storage Acquisition of raw data for FCS, FCCS, PCH, smFRET.
On-line processing or post-processing
Sampling rate Up to 80MHz
Max signal in FCS 60 x 106 counts/sec (15 x 106 counts/sec per channel)
Max signal in FLIM 60 x 106 counts/sec (15 x 106 counts/sec per channel)

Operation

Architecture USB2 (Windows 10)
CLK frequency 320 MHz
Clock Managers 4
Power 120/240 V, 40 W
Dimensions (cm) 42.5 (W) x 36 (D) x 10 (H)