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FLIM

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.

Fluorescence Lifetime Imaging Microscopy

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.

Why measure fluorescence lifetime?

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.

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.

Spectrally Resolved FLIM

The spectrally-resolved Lambert Instruments FLIM Attachment (LIFA) is an imaging system for fluorescence microscopy that preserves the information required to determine the position, spectrum, and lifetime of the observed fluorescence. This is done by combining several modular components. These consist of the typical LIFA (modulated intensified CCD camera, modulated LED excitation) and a prism-based imaging spectrograph.

2D image

The spectrally resolved images show one line (y-axis) out of the 2D intensity image of typical dicot root. The emission wavelengths at the x-axis start at 515 nm. The fluorescence lifetime is shown in pseudo colors. The higher wavelength components have a shorter lifetime (blue) than the short wavelength components (red).

Spectrally resolved GFP-RFP transfected cell (FRET)

The spectrally resolved images show one line (y-axis) out of a sample of GFP transfected cells or GFP-RFP transfected cells. The emission wavelengths at the x-axis start at 515 nm; the first peak is the GFP emission peak and the second the RFP emission peak. The fluorescence lifetime is shown in pseudo colors. In the FRET sample (GFP-RFP) the fluorescence lifetime of the GFP has decreased from 2.3 ns (red) to 2.0 ns (yellow).

Intensified Cameras for Lifetime Imaging

Intensified cameras enable full-field frequency-domain and time-domain FLIM. The image intensifier becomes an ultra-fast electro-optical shutter by operating it at radio frequencies allowing time-resolved imaging. The high-resolution image intensifier is the key component of the TRiCAM (part of the LIFA) and the TRiCATT camera attachment. Its photon gain is typically in the range of 100 to 10000. Lambert Instruments provides different image intensifiers based on photocathodes with different spectral sensitivity to match a range of applications in the UV, visible and NIR.

For FLIM in the lifetime range of 0 ps to 1 ms we provide S20 (UV) and SuperS25 (visual) image intensifiers. For increased quantum efficiency of the photocathode in the visual part of the spectrum in this lifetime range, a GaAs intensifier is available. For near-infrared applications up to about 1100 nm an InGaAs photocathode is available.

The graph below shows the spectral sensitivity of these photocathodes.