FLIM – an Extra Dimension to Fluorescence Microscopy

Abstract

The use of imaging techniques is of fundamental importance in uncovering the structure and dynamics of both biological substances and advanced materials. Fluorescence microscopy in particular is well suited to visualise cellular structures and interactions. This can use fluorescent labels or the measurement of endogenous fluorescence. Techniques, such as FRET (Förster resonance energy transfer) also enable the elucidation of interactions below the diffraction limit to be achieved. The use of the fluorescence lifetime is advantageous as it is an absolute measure and the best way to observe FRET. Fluorescence lifetime imaging (FLIM) products such as the FLIMera and InverTau harness this power in the area of biological and material sciences.

Dr. Philip Yip

Fluorescence Engineer
HORIBA Jobin Yvon IBH Ltd.
Glasgow UK
hilip.yip@horiba.com



Dr. David McLoskey

Managing Director
HORIBA Jobin Yvon IBH Ltd.
Glasgow UK
david.mcloskey(at)horiba.com

Dr. Graham Hungerford

Principal Scientist
HORIBA Jobin Yvon IBH Ltd.
Glasgow UK
raham.hungerford@horiba.co

► Click Here  for original paper (PDF file)

Introduction

Fluorescence microscopy is an illuminating method to shed light on molecular interactions. It has the potential to work on the nanoscale up to the imaging of macroscale objects. It is a very sensitive technique, capable of detecting single molecules and makes use of either the intrinsic fluorescence properties of the sample or that of an introduced “tag” molecule. The fluorescence signal is multiparameter; depending on the wavelengths of excitation and emission, excitation intensity, polarisation and the fluorescence lifetime ^[1]. The lifetime is a measure of how long a molecule spends in the excited state (stores the excitation energy) and is typically on the ps to ns timescale. This enables a high degree of specificity, along with sensitivity. The fluorescence signal is highly dependent on the local microenvironment and molecular interactions which makes it well suited to the study of biomolecules. Thus, fluorescence microscopes are extensively used in the biological field ^[2,3] ; in the study of cell biology, medical diagnostics, drug discovery, cancer research and neuroscience, for example. In addition, they have application in materials science in the characterisation of various substances, such as nanomaterials and polymers.

Fluorescence microscopy is optical microscopy based on the imaging of light, which means that it is diffraction limited in resolution (~250nm). However, by using Förster resonance energy transfer (FRET) ^[1] and interpretation of the fluorescence signal, interactions on the nanometre (~1-10nm) scale can be inferred. The use of the fluorescence lifetime parameter is particularly elucidating and greatly simplifies the measurement of FRET. Fluorescence lifetime imaging (FLIM) ^[4] is therefore a powerful extension to fluorescence microscopy, especially as the lifetime parameter is an absolute measure, independent of concentration This enhances the ability to provide image contrast and elucidate molecular and environmental interactions.

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Figure 1 　
An inverted microscope equipped with an InverTau (left) and a FLIMera (right).

Time-correlated Single-Photon Counting (TCSPC) ^[1] is often considered the “gold standard” in obtaining the fluorescence lifetime and it is the basis of two FLIM products from HORIBA; the FLIMera widefield TCSPC camera and the InverTau – Fluorescence Lifetime Imaging Platform. The FLIMera is based on a SPAD (single-photon avalanche photodiode) array with in-pixel timing, whilst the InverTau is laser scanning making use of galvanometers. This is advantageous since the sample is not moved (no scan stage required). These two technologies (shown on an inverted microscope, Figure 1) will be explained and examples of their use given.

Widefield imaging with a SPAD array with in-pixel timing

In the case of widefield imaging on a microscope the whole of the sample is illuminated and measurement of the image data can be done relatively quickly. Typically, a camera would be employed to record the image. Replacing the intensity camera with a FLIMera and the light source with a pulsed laser enables a simple transformation to a FLIM measurement system. Since it is a widefield approach no scanning is required. In fact, as well as usage on a widefield microscope the FLIMera can give lifetime functionality to light sheet microscopes used in volume imaging and also enables the possibility of macroFLIM measurements.

Figure 2 
A representation of the FLIMera and one of its 24, 192 pixel

The principal feature of the FLIMera is its 192 x 126 pixel array based on CMOS technology (See Figure 2), where each pixel contains a detection element (SPAD) and its associated TCSPC timing electronics. Thus, it has the ability to record up to 24,192 fluorescence decays simultaneously. The FLIMera can measure fluorescence over wavelengths from ~400nm to 900nm and is optimised for measuring the lifetimes of common biological probes and endogenous fluorescence (range 200ps to 20ns).

The in-pixel timing means that an average fluorescence lifetime can be visualised in real time (up to ~30fps), whilst post processed data can be temporally resolved down to the readout rate of the sensor (~80μs). This response speed means that moving samples or dynamic events can imaged using the fluorescence lifetime. Thus, SPAD arrays based of CMOS technology exhibit a great deal of promise in bioimaging ^[5].

The FLIMera in addition to microscope based measurements shows promise for macroFLIM in areas ranging from cancer margin identification to art conservation, where a forerunner of the FLIMera sensor was used to monitor vanish removal ^[6]. Figure 3 shows a macroFLIM measurement of a whole variegated rubber tree leaf. The green chlorophyll containing areas are clearly discerned in the lifetime image, but harder to make out in the intensity image. Further processing of the lifetime data could then give insight into the photosynthetic mechanism.

Figure 3 
Images of a whole leaf from ficuselasticavariegata taken with a phone camera (left) and a FLIMera (centre and right).

Although the FLIMera can provide simple non scanning FLIM, by replacing intensity based cameras on a microscope to enable real time determination of the average fluorescence lifetime, the widefield configuration does have some limitations. The major one is that of out of focus fluorescence, which can cause blurring and loss of resolution. This is common when using a widefield camera. Also, the number of pixels in the FLIMera can mean that a trade off maybe required between field of view (FoV) and resolution, although researchers have published work to enhance the resolution ^[7]. Where higher resolution FLIM is required, scanning the laser across the sample with a pinhole in the emission path can be used. This is the principle underpinning the InverTau, which is capable of collecting images up to a 4K x 4K spatial resolution. The use of a pinhole enables optical section by mitigating out of focus blur. However, galvanometer scanning of the laser beam over the sample is a slower approach compared to that of widefield. Thus, the FLIMera and InverTau maybe considered complementary in imaging capability.

Laser scanning applied to cellular structure

As with all light microscopy the resolution that can be achieved is determined by the diffraction limit. This is, in turn, dependent on the wavelength of light (fluorescence emission) and the numerical aperture of the microscope objective. Super-resolution techniques do exist in fluorescence, but these are complex and the InverTau has been optimised to make FLIM measurements as simple as possible by using EzTime Image software (also used with the FLIMera) and computer controlled optics. It is designed to attach a side port (C-mount) of an inverted microscope, as shown in Figure 1. To allow for future flexibility of use the InverTau has two excitation ports and two emission ports. Typically a pulsed laser (eg DeltaDiode) is fibre coupled into the source one port. The other excitation port is optimised for use with two-photon excitation sources. Typically, only one HPPD detector is employed. A schematic of the optical layout is shown in Figure 4 and shows the path through the InverTau and its independent computer controlled motorised components.

Figure 4  
Schematic of the light path through the InverTau

A major use of FLIM is that in the Bio- and life sciences, where many applications involve the study of cellular structure and the interaction between cellular components. Examples are in cellular biochemistry and cancer research. Fluorescent tags can be introduced to label specific components or endogenous fluorescence, for example from NADH which is indicative of cell metabolism, can be used. An example of labelling cell structure is shown in Figure 5, in which different tags (emitting at different wavelengths, with different lifetimes) are used to label parts of the cytoskeleton of a BPAE cell line.

For simplicity the emission wavelengths are often referred to in terms of colour. DAPI, a DNA stain, is used to label the nucleus (blue), a BODIPY derivative is used to label the microtubules (green) and a Texas red derivative the actin fibres (red). Independent measurements using different lasers and emission filters can be made for each and the images exported and merged in open source software (eg Fiji / ImageJ). Also discernible in the images shown in Figure 5 are cells undergoing division (mitosis). This is an important part of the cell lifecycle and its study is of great importance in both genetic and disease research areas.

Figure 5 
InverTau data showing multi-labelled BPAE cells, showing different parts of the cytoskeleton and nuclei (A-C, scale bar 50μm) with a merged image (D). E shows a cell, seen in A, undergoing cell division (mitosis). Images F to H show other cells (merged “blue” and “green” emissions) in different stages of division.

The information that can be gained from FLIM is very dependent on the probe molecules used and measuring fluorescence lifetimes are advantageous when looking at FRET interactions, for example to investigate interactions between genetically encoded proteins, or using tension probes (eg Flipper T) for monitoring cell membrane rigidity for cancer research.

In plant science measurements an important area making use of fluorescence is that of photosynthesis. This has implications for plant health and in research applying photosynthesis as a model for artificial systems for light harvesting. An example of a whole leaf FLIM measurement is illustrated in Figure 6, along with a schematic for the process involving photosystems I and II showing indicative lifetimes. Note for exact details and lifetime values research literature should be consulted.

Figure 6 
A – schematic of the process involving hotosystems I and II. B – Lifetime image for the abaxial surface of a variegated ivy (hedra) leaf obtained using two-photon excitation. C – SHG image using 920nm excitation from a femtosecond fibre laser (also used for two photon excitation).

The data shown in Figure 6 was measured using a fibre coupled two photon excitation source (Femtosecond laser coupled to the second excitation port). The average fluorescence lifetime image (Figure 6B) shows emission principally emanating from the chloroplasts, which contain the photosynthetic reaction centres. Measurement of the fluorescence lifetime can be very informative concerning the state of the photosynthetic process. The position of the stomata can also be inferred. Use of a two photon excitation laser also enables a SHG (second harmonic generation) image to be obtained. Certain biological substances exhibit non linear optical effects and this can be used to provide structural information. In this case the stomata, for example, are more clearly defined.

Conclusion

FLIM is a powerful tool for use in the Bio- and life science fields. The fluorescence lifetime parameter provides an additional means to add contrast to images and is fundamental to in uncovering molecular processes at the cellular level. Both the InverTau and FLIMera use TCSPC, the “gold standard” in fluorescence lifetime determination, to add this measurement modality to fluorescence microscopes. Simple to use software (EzTime Image) moves FLIM from specialist centres to common research laboratories.

References

[1]  Lakowicz JR Principles of Fluorescence  Spectroscopy: Third Edition, 2006, Springer, New York.

[2]  Hickey SM et al, Cells, 2022, 11, 35.

[3]  Scheneckenburger H; Richter V, Appl. Sci., 2021, 11, 733.

[4]  Datta R et al, J. Biomed. Opt., 2020, 25, 07203.

[5]  Bruschini C et al, Light: Sci. Appl., 2019, 8, 87.

[6]  Wilda CB et al, Herit. Sci., 2023, 11, 127.

[7]  Kapitany V et al, Sci. Adv., 2024, 10, eadn0139.

About the InverTau and FLIMera

Both the FLIMera and InverTau can be sold individually or incorporated with an inverted microscope (Nikon Ti2-U), with the possibility of both being coupled on the same microscope. The InverTau comes with FiPho timing electronics and our DeltaDiode range of lasers provide the ideal excitation sources for both products.

Information can be obtained from the HORIBA website

Fluorescence Microscopy - HORIBA

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