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Label-free FLIM

Microscopy under Physiological Conditions

Many biological samples exhibit autofluorescence. Its often broad spectra can interfere with fluorescent labeling strategies. This application letter demonstrates how autofluorescence can serve as an intrinsic contrast in fluorescence lifetime imaging microscopy (FLIM) resulting in multi-color image stacks. It is also outlined how to combine spectral imaging with fluorescence lifetime information to distinguish and potentially identify different fluorescent species in biological samples.


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Microscopy meets autofluorescence

In fluorescence microscopy generally autofluorescence is viewed as a detrimental side-effect inherent to many biological samples. It tends to overlap with endogenous fluorescence labels sometimes masking its intensity. It can cause difficulties with spectral channel separation and with quantitation of intensities for ratiometric analysis. The often very broad emission spectra of autofluorescence can render it intractable by conventional spectral image recording.

However, autofluorescence as such has its merits, too. It is an intrinsic form of fluorescent label which comes for free and is completely non-invasive. The purpose of this application letter is to identify several autofluorescent components by means of fluorescence lifetime imaging microscopy (FLIM) and to demonstrate their utility in biomedical research.

Note: For a basic treatment of the FLIM techniquw please refer to the application letter FRET with Flim - Quantitative in-vivo Biochemistry

Autofluorescence meets science

Most biological samples contain biochemical species which can give rise to autofluorescence. For example a cell’s redox potential is reflected in its concentration of NAD(P)H and flavins. The former are better excited with IR sources, the latter are accessible with 405 nm as well. Quite a few structural proteins can deliver lifetime contrast as well, such as collagen, elastin and fibrillin.

Plant cells are particularly rich in a multitude of autofluorescent molecules. Very common are chlorophyll, carotenoids, polyphenols to name but a few. Often, these compounds can not only provide label-free contrast, but also provide information on the metabolic state or pathogenic alterations in cells or tissues. Thus, one can draw functional conclusions by combining FLIM and autofluorescence.

Label-free contrast

To exemplify the utility of autofluorescence as a contrasting strategy in biological samples we shall examine an example from the animal and the plant kingdom, respectively.

Let us begin with a scale insect. Different developmental stages of this plant parasite were collected and immediately mounted. An overview image of the autofluorescence present at the excitation wavelength 470 nm was recorded using a 10x objective (Figure 1). The intensity image was created from a FLIM data set and yields a good representation of the structure of the specimen. Intensity here encodes counted photons per pixel instead of arbitrary units as customary for standard intensity images. It is, however, unable to distinguish any autofluorescent species (Figure 1A). This information is contained in the lifetime map (Figure 1B) which renders the spatially resolved lifetime distribution. It gives a good impression of the different fluorescent lifetime components.

In terms of autofluorescence this often represents different molecular species or combinations thereof. We can distinguish very well the chitinized exoskeleton around the antennae and the legs (green to blue). Chitin displays a very short lifetime. The surrounding tissue appears relatively homogenous close to 3 ns (yellow). Some interspersed regions with longer lifetimes (orange) could represent other species, but here, they more likely represent darker regions which simply have a lower precision in determining the lifetime.

What is typically perceived and communicated as a FLIM image is in fact an intensity modulated lifetime map (Figure 1C). The lifetime information is multiplied by the intensity image. This rendition tends to de-emphasize the darker regions with poor photon statistics and it retains more of the structural information contained in the intensity image. The latter often facilitates the interpretation of lifetimes as in the case of the orange regions in the lifetime map. We can conclude that FLIM allows us to identify different molecular species in an unperturbed sample and to study their spatial interrelation.

Fig. 1: Autofluorescence image of scale insect (ventral view). Intensity image (A), lifetime map (B, background cropped) and intensity modulated lifetime image (C). The lifetime map shows the spatial resolution of at least three different colorcoded lifetimes. The length is 1.5 mm, the thickness of the section represents 1.3 μm. Scan format 280 x 512, objective lens 10x NA 0.4, excitation with 470 nm, detection of emission from 478 nm to 703 nm (sample courtesy of Kees Jalink, NCI, Amsterdam, Netherlands).

In depth – 3D FLIM stacks

Taking a closer look at the autofluorescence image (Figure 1) we recognize that the antennae, for example, have blue regions outside and green regions inside. So, does the green region represent a distinct species or is it rather an (optical) mixture of chitin fluorescence (encoded in blue) and the surrounding tissue (encoded in yellow)? To address this question we can use a higher NA objective on a smaller region. We also have to take into account the z-extension of the image. To better visualize the interior of the structure we can record a FLIM z-stack (Figure 2). It turns out that the smaller lifetime is caused by chitin, since the interior of the antenna has the same lifetime as the surrounding tissue revealed by a middle section (Figure 2A, white box). Thick structures can now be viewed in extended focus using a maximum projection of the z-stack (Figure 2B). An even larger appreciation of the 3D topology is facilitated by an isosurface rendering (Figure 3). Note, the joints are free of chitin, which is clearly visible in the FLIM data. Important taxonomic features become visible, such as the hairlike setae scattered across all body segments. They clearly also contain a chitin species.

Fig. 2: FLIM z-stack of antenna of scale insect. 40 z-slices of about 1 μm depth have been recorded using a 40x NA 1.25 lens (A). A maximum projection of the organ shows the antenna and some chitinized hairs (B). Maximum projection was done using external software. The Field-Of-View is 388 x 151 μm.

Multi-color autofluorescence

Fluorescence imaging in plants is often somewhat impeded by the presence of a multitude of fluorescent species, such as chlorophyll and several cell wall constituents as well as a multitude of endogenous pigments. Here we make use of these intrinsic autofluorophores to lend contrast to otherwise poorly distinguishable structures. We recorded a z-stack of a pollen grain (microspore) taken from a lily flower (Figure 4). The anthers of the pollen grains have a yelloworange color visible by eye as well as in transmission light (not shown). Possible candidates for such yellow fluorochromes in the plant kingdom are, for example, carotinoids. We obtained lifetimes ranging from 0.5 ns to 1.2 ns. Two main structural features were clearly contrasted by lifetime. In green we recognize the net-like outer layer of the (putative) tube cell, which surrounds the core rendered in blue (0.5 ns). Z-sectioning reveals that the core is not filled with the short lifetime species, but it rather forms a thin inner layer around the tube cell (Figure 4A). Extended focus (Figure 4B) and 3D visualization (Figure 4C) help to clarify the relative topology of both layers to one another.

Fig. 4: Microspore (pollen grain) from Lilium sp. (lily flower). Autofluorescence excitable with 470 nm reveals two structurally distinct fluorescent species in the outer layers of the tube cell. 45 z-slices taken recorded with a 20x NA 0.7 lens detecting emission from 482 nm to 744 nm (A), maximum projection using external software (B) and 3D isosurface rendering using external software (C). The longitudinal extension of the spore is 130 μm.

Read more about the implementation of an FLIM experiment, curve fitting and how to interpret the data

Detailed information on:

  1. Step-by-step guide for the implementation of an FLIM experiment using a Leica TCS SMD system,
  2. Step-by-step guide for curve fitting using Picoquant's SymPhoTime software,
  3. How to interpret the data

you can find here:

Confocal Application Letter No. 34: Label-Free FLIM Microscopy under Physiological Conditions