WO2008125855A1

WO2008125855A1 - Microscope test sample

Info

Publication number: WO2008125855A1
Application number: PCT/GB2008/001320
Authority: WO
Inventors: Kurt Anderson
Original assignee: Cancer Research Technology Ltd
Priority date: 2007-04-16
Filing date: 2008-04-15
Publication date: 2008-10-23
Also published as: GB0707291D0

Abstract

A microscope test sample comprises a transparent substrate (1), and a transparent fluorescent medium (2) interfacing with the substrate (1). The medium (2) is of lower refractive index than the transparent substrate (1). The microscope test sample further comprises fluorescent entities (4, 5) within the medium (2) at the substrate-medium interface. When the fluorescent entities (4, 5) are excited to fluoresce, they are f luorescently distinguishable from the medium (2).

Description

MICROSCOPE TEST SAMPLE

Field of the Invention

The present invention relates to a microscope test sample and uses thereof, and particularly a total internal reflection fluorescence (TIRF) microscope test sample.

Background of the Invention

Fluorescence microscopy is widely used, e.g. in the study of biological specimens. Fluorophores bound to a biological specimen surface fluoresce when excited by excitation light at a suitable wavelength. However, fluorophores present in the medium surrounding the specimen may also be excited by the excitation light and fluoresce. This background fluorescence can lead to a reduction in image contrast when the specimen is viewed through the microscope. Indeed, if the background fluorescence is sufficiently intense, the specimen fluorescence may not be discernible over the background fluorescence. The presence of background fluorescence becomes particularly problematic when the fluorescence microscopy is used to view very small or weakly fluorescent structures, such as endosomes or single microtubules, or very thin layers or surfaces of a specimen.

A solution to this problem is provided by TIRF microscopy, in which only a very thin layer of the specimen is illuminated, using an evanescent field (EF) produced by total internal reflection of an excitation light beam.

Total internal reflection occurs when a light beam travelling in a first medium is incident on an interface between the first medium and a second medium having a lower refractive index at an angle of incidence greater than the critical angle for total internal reflection. Under these conditions, an evanescent field forms on the opposite side of the interface to the incident and reflected light beams. The EF decays exponentially with distance from the interface. The EF (also known as an evanescent wave) penetrates only a very short distance into the second medium, typically of the order of a hundred nanometres, depending on the wavelength of the light and the angle of incidence .

In a TIRF microscope, the specimen is located on a substrate which is transparent to the excitation light. This may be, for example,. a coverslip or prism, or a tissue culture container. The substrate may be made, for example, of glass or quartz. The medium containing the specimen, for example an aqueous medium, has a lower refractive index than the substrate.

The excitation light beam arrives at the substrate-specimen interface, from the substrate side, at an angle of incidence greater than the critical angle for total internal reflection. The EF thus formed, on the specimen side of the interface, can excite the fluorophores located in a very thin layer of the specimen immediately adjacent to the total internal reflection interface. The fluorescence subsequently emitted by fluorophores is imaged by the microscope.

TIRF microscopy is a particularly useful technique for live cell imaging, and can be readily implemented on a standard epi- fluorescence microscope. Indeed, the light source (typically a laser) used to produce the EF illumination can generally be arranged so as to generate epi-fluorescence instead. Increasing the angle of incidence of the excitation illumination on the substrate-specimen interface gradually shifts the mode of illumination from epi to EF. One known test sample used for alignment of TIRF systems is a liquid carrying a suspension of sub-resolution fluorescent beads. Beads are illuminated throughout the liquid volume in epi-fluorescence, but are only illuminated when near the substrate surface in TIRF. The different "behaviours" of the beads under epi and EF illumination, and especially their diffusion in and out of the EF field under TIRF illumination, is used to determine when the microscope is operating under TIRF conditions .

Summary of the Invention

However, a disadvantage of the known test sample is that identification of the point at which illumination changes mode from epi to EF is subjective.

Various other difficulties also exist in setting up and evaluating the quality of illumination in TIRF microscopes. One difficulty is identifying the optimum angle of incidence of the excitation illumination. Another difficulty is obtaining a flatly illuminating excitation light beam, particularly when the microscope employs a coherent light source, such as a laser. A further difficulty is maintaining a stable and reproducible operating condition for a TIRF microscope over a period of time.

In general terms, the present invention provides a microscope test sample comprising: a transparent substrate, a transparent fluorescent medium interfacing with the substrate, the medium being of lower refractive index than the transparent substrate, and fluorescent entities within the medium which, when excited to fluoresce, are fluorescently distinguishable from the medium. More particularly, in a first aspect, the present invention provides a microscope test sample comprising: a transparent substrate, a transparent fluorescent medium interfacing with the substrate, the medium being of lower refractive index than the transparent substrate, and fluorescent entities within the medium at the substrate- medium interface which, when excited to fluoresce, are fluorescently distinguishable from the medium.

The transparent substrate may be, for example, of glass (e.g. sapphire doped glass) or quartz. It may take the form of a conventional microscope slide.

In use, the sample is positioned on the microscope so that the excitation beam traverses the transparent substrate to arrive at the substrate-medium interface. When the microscope is operating under TIRF conditions an EF is produced in the fluorescent medium at that interface. The EF causes a fluorescent emission from the medium and a distinguishable fluorescent emission from the fluorescent entities. The ratio of the intensities of these two emissions can be indicative of whether the microscope is providing epi or EF illumination, and also be used to evaluate the quality of the EF illumination. Further, it is possible to monitor the operating stability of the microscope over a period of time, or to ensure that different microscope systems are operating at the same level of performance .

This can be particularly useful, for example, in screening applications in which a TIRF microscope has to operate stably for significant periods of time and/or over significant numbers of samples. Regular monitoring of the intensities of the two emissions from the test sample, followed by, if necessary, adjustment of the microscope, allows a stable TIRF operating condition for the microscope to be maintained. Also, in high throughput screening applications involving parallel TIRF microscopes, the microscopes can be monitored to endure that they are all at the same operating condition.

In contrast, if the known test sample, in which the liquid carrying the freely diffusing fluorescent beads is not fluorescent, were used for such purposes, an operator would have to rely on analysis of the lengths of time which the freely diffusing beads were illuminated, or their rates of entry and into and exit from the objective focal plane. These would be unreliable indicators of operating condition.

The microscope can be adjusted to vary the incidence angle of the excitation beam at the substrate-medium interface so that the ratio of the intensity of the emission fluorescence produced by the fluorescent entities to the intensity of the emission fluorescence produced by the fluorescent medium is maximised. An advantage of having the fluorescent entities at the substrate-medium interface is then that the microscope should be at an optimal setting for TIRF microscopy when the ratio is maximised in this way.

Preferably, the fluorescent medium is homogeneously fluorescent. This helps to reduce error in measuring the ratio of the intensities of the emissions from the fluorescent medium and the fluorescent entities. However, a further advantage of a homogeneously fluorescent medium is that it allows the operator to determine if the excitation light beam is providing the flat illumination which is important for quantitative image analysis.

More particularly, coherent light sources, such as lasers, are commonly used for TIRF microscopy. However, the illumination provided by a coherent light source can contain undesirable interference patterns if, for example, there is misalignment of the beam path, or dust or dirt in the beam path (e.g. in the immersion oil which is typically used to optically couple the objective to the sample) . Such interference patterns will show up in the background image provided by the homogeneously fluorescent medium, thereby alerting the operator to their presence. If necessary, the microscope can then be adjusted to improve the flatness of the background image.

Preferably, the fluorescent entities, for a given excitation illumination intensity, produce a more intense fluorescence emission than the medium. More preferably, the fluorescent entities and fluorescent medium emit fluorescence of the same wavelength. Such an arrangement allows the entities to be easily distinguishable from the medium, and mimics a real biological sample. Alternatively, however, the fluorescent medium may have a first fluorescent wavelength and the fluorescent entities may have a second fluorescent wavelength which is different from the first fluorescent wavelength.

Preferably, the fluorescent medium is encapsulated in an airtight container on the substrate. This helps to preserve the integrity of the sample.

Typically, the fluorescent medium is a liquid. The liquid may contain, for example, a fluorescent dye which should be photo- stable, i.e. resistant to photo-bleaching. Generally, higher concentrations of dye result in higher refractive indices for the medium. Thus preferably, the dye concentration is sufficient to allow clear visualisation of the EF, but also provides a refractive index comparable to that of a biological material (around 1.33 to 1.36) . A solution of 5-10 μM fluorescein in phosphate-buffered saline may be used.

However, the fluorescent medium may be, for example, a gel or a solid.

Preferably, the fluorescent medium has a refractive index in the range of from 1.3 to 1.4, and more preferably has a refractive index in the range of from 1.33 to 1.36.

To facilitate the positioning of the fluorescent medium over the microscope objective and to facilitate production using commonly available materials, preferably the fluorescent medium extends over at least 5 mm² (and more preferably at least 25 or 100 mm²) of the surface area of the transparent substrate.

The test sample may be configured such that, when viewing the fluorescent medium through a microscope with the fluorescent medium extending across the entire field of view, the fluorescent entities occupy in the range of from 0.2 to 10% (preferably from 0.4 to 5%) of the area of the field of view. Such a proportion of the area of the field of view can allow accurate measurement of the intensity of the fluorescent emission from the entities, while also providing a sufficiently large background fluorescent emission from the medium for assessment of the flatness of the excitation illumination.

Desirably, the fluorescent entities are photo-stable, i.e. resist photo-bleaching, in order that long-term performance of the test sample can be maintained.

The fluorescent entities may have a refractive index in the range of from 1.3 to 1.4, and more preferably in the range of from 1.33 to 1.36. The fluorescent component of the fluorescent entities may comprise green, red or blue fluorophores (e.g. green, red or cyan fluorescent protein) . Indeed, as TIRF microscope set-up conditions can vary depending on the colour of the fluorophores which the microscope is to image, a second aspect of the invention provides a set of test samples of the first aspect, the respective fluorescent entities of each test sample of the set having a different fluorescent colour (for example green, red and blue) .

The fluorescent entities may be provided by fluorescent beads, e.g. carrying such fluorophores. Preferably, the fluorescent beads are less than 100 nm (more preferably less than 50 nm) in diameter so that the beads are of the order of size of, or smaller than, the extent of the EF.

The fluorescent beads may be attached to the substrate at the substrate-medium interface, e.g. by molecular linkages, covalent bonds etc. to the surface of the substrate. The fluorescent beads are preferably evenly dispersed over the substrate surface .

Alternatively, however, the fluorescent entities may be provided by a patterned fluorescent layer (such as a fluorescent grid or spots) on the surface of the substrate. Such a layer can be formed, for example, by microlithography or other micro- patterning technique. An advantage of a fluorescent layer over fluorescent beads is that the layer can more easily be configured to regularly sample the illumination field. An additional advantage of the patterned layer is that it can have a thickness significantly less than the extent of the EF, making it a more accurate indicator of the transition from epi to EF illumination. The test sample may be used for setting up or maintaining an operating condition of a TIRF microscope.

A further aspect of the invention provides a method of calibrating a TIRF microscope comprising the steps of: (a) positioning the test sample of the first aspect on the microscope such that an excitation beam of the microscope traverses the transparent substrate to produce an evanescent field in the fluorescent medium at the substrate-medium interface, and (b) measuring the relative intensities of the fluorescent emissions produced by the fluorescent entities and the fluorescent medium.

The method may comprise the further step of calculating the ratio of the intensity of the fluorescent emission produced by the fluorescent entities to the intensity of fluorescent emission produced by the fluorescent medium.

Another aspect of the invention provides a method of performing TIRF microscopy comprising performing at intervals the calibration of the previous aspect, and, when necessary, adjusting the TIRF microscope to maintain the relative intensities of the fluorescent emissions produced by the fluorescent entities and the fluorescent medium.

This aspect is, therefore, particularly applicable to screening operations using TIRF microscopy.

A further aspect of the invention provides a method of adjusting a TIRF microscope comprising the steps of:

(a) positioning the test sample of first aspect on the microscope such that an excitation beam of the microscope traverses the transparent substrate to arrive at the substrate- medium interface, and (b) adjusting the microscope such that the ratio of the intensity of fluorescent emission produced by the fluorescent entities to the intensity of fluorescent emission produced by the fluorescent medium is maximised.

For example, the microscope can be adjusted by varying the incidence angle of the excitation beam at said interface.

In step (a) of the above method of calibrating a TIRF microscope, performing TIRF microscopy, or adjusting a TIRF microscope, the test sample may have a fluorescent medium which is homogeneously fluorescent, an evanescent field being produced in the fluorescent medium at the substrate-medium interface. The method may then comprise the further step (c) of adjusting the microscope to improve the flatness of the fluorescent emission produced by the fluorescent medium.

Another aspect of the invention provides a kit comprising the test sample of the first aspect (or set of test samples of the second aspect) , and instructions for performing the method of any one of the previous three aspects.

(a) positioning the test sample of the first aspect on the microscope such that an excitation beam of the microscope traverses the transparent substrate to produce an evanescent field in the fluorescent medium at the substrate-medium interface, the fluorescent medium of the test sample being homogeneously fluorescent, and

(b) adjusting the microscope to improve the flatness of the fluorescent emission produced by the fluorescent medium.

Another aspect of the invention provides a kit comprising the test sample of the previous aspect (or set of test samples of the second aspect) , the fluorescent medium of the (or each) test sample being homogeneously fluorescent, and instructions for performing the method of the previous aspect.

Brief Description of the Drawings

The invention will be described by way of example with reference to the accompanying drawings, in which:

Figure Ia shows a time series of images for epi illumination of two beads, and Figure Ib shows a time series of images for EF illumination of two beads;

Figures 2a and b show respectively a schematic cross-section and a schematic top view of a first embodiment of a test sample;

Figures 3a and b show respectively a schematic cross-section and a schematic top view of a second embodiment of a test sample;

Figure 4 plots the predicted decay in EF intensity with distance from a substrate surface for six different illumination angles;

Figure 5 shows plots, at different laser powers, of the improvement in image contrast achieved by TIRF over epi illumination for varying illumination incident angles;

Figure 6a is an image of an interference pattern produced under EF illumination of a test sample, and Figure 6b is a plot of pixel intensity against position along the horizontal white line of Figure 6a.

Detailed Description

Known Test Sample

For live cell TIRF applications, the specimen features of interest generally consist of discreet structures at the cell surface containing multiple fluorophore labels, such as adhesion sites, microtubules, actin filaments, clathrin coated pits, and membrane receptors. Background, on the other hand, results from fluorophores distributed more evenly throughout the cytoplasm. A test sample should mimic the conditions of foreground and background in a typical biological specimen.

A known test sample for demonstration and evaluation of TIRF microscopy consists of sub-resolution fluorescent latex beads floating in solution. Under epi-illumination beads are illuminated throughout the sample, and individual beads drift in and out of focus through Brownian motion. Under EF illumination, beads are illuminated only when they are within the EF, i.e. within approximately 150 nm of the substrate. Beads diffusing in and out of the EF appear and disappear suddenly, and the bulk of beads disappear when the illumination angle reaches the critical angle for epi to TIRF illumination. This difference in the "behaviour" of beads is a commonly used standard for the verification of EF illumination.

Figure Ia shows a time series of images for epi illumination of two beads. The left hand bead (indicated with an arrow in the top image) is fixed in the image plane, and the intensity of its emitted fluorescence does not vary significantly with time. The right hand bead, on the other hand, is not fixed and the intensity of its emitted fluorescence gradually changes as it drifts in and out of focus. Figure Ib shows a time series of images for EF illumination of two beads. Again, the left hand bead (indicated with an arrow in the top image) is fixed in the image plane, and the intensity of its emitted fluorescence does not vary significantly with time. The right hand bead is not fixed. As it drifts in and out of the EF, relative to the unfixed bead under epi illumination, the intensity of its emitted fluorescence varies much more abruptly.

However, the known test sample has several shortcomings. A primary weakness is that evaluation of the behaviour of moving beads is subjective. Furthermore, such a sample does not mimic the features of a biological specimen (bright features and diffuse background) . In particular, the low or non-existent background signal from such a sample is very different from a typical biological sample, in which features of interest are generally bathed in a background of diffuse cytoplasmic fluorescence. Additionally, this type of sample provides no information about variations in the intensity of EF illumination across the specimen plane. Flatness of the illumination field is important for quantitative fluorescence microscopy. However, EF illumination is susceptible to inhomogeneities due to the use of laser light. In particular, the coherence of laser light can lead to the generation of interference patterns, which can result from improper alignment of the system (clipping) and from dust in the illumination pathway.

Embodiments of Test Samples According to the Present Invention

A first embodiment of a test sample according to the present invention is shown schematically in Figures 2a and b, which are respectively a cross-section and a top view of the sample.

The sample has a relatively high refractive index transparent substrate 1 which supports a volume of lower refractive index, homogenously fluorescent liquid 2. The liquid is encapsulated by a surface of the substrate and a cover 3. Beads 4, which fluoresce at the same wavelength to the liquid but with greater intensity, are attached to and evenly dispersed across the surface of the substrate. A non-encapsulated version of this embodiment can be made by replacing the cover 3 with a simple microscope cover slip.

The beads are fluorescently excited at the wavelength of the EF illumination and are sized to not extend substantially beyond the point where the EF decays to 50% of its initial intensity. In practice this means that the beads are typically less than 100 nm in diameter. For manual analysis of the emitted fluorescence from the beads, the number density of beads on the substrate surface is preferably such as to provide around 20 to 50 beads in a given image field. Suitable beads are e.g. PS- Speck™ point source beads from Molecular Probes, Fluoresbrite™ microspheres from Polysciences, Inc., or plain fluorescent polystyrene nanospheres from Corpuscular, Inc.

The liquid can be a fluorescent dye solution. The concentration of the solution used to generate a background signal should be sufficiently high to clearly visualize any illumination pattern in the EF. However, the refractive index of the background solution is influenced by the concentration of the dye, a higher concentration dye generally having a higher refractive index. Since the basis of EF illumination is a refractive index step, and biological material typically has a refractive index of around 1.33 to 1.36, the concentration of the dye used to generate a background solution preferably produces a refractive index in this range. A solution of 5-10 μM fluorescein in phosphate-buffered saline seems to satisfy these requirements.

A second embodiment of a test sample according to the present invention is shown schematically in Figures 3a and b, which are respectively a cross-section and a top view of the sample.

This sample is the same as that of the first embodiment, except that instead of the fluorescent beads, a pattern 5 of fluorescent dye is formed on the surface of the substrate. Microlithography or other micro-patterning techniques known to the skilled person can be used to adsorb a layer of fluorescent dye directly or indirectly (through linkage to a carrier molecule such as bovine serum albumin, dextran, polyethylene glycol, etc.) to the surface of a substrate. An advantage of this approach is that a regular fluorescent pattern, such as a grid or a 2D lattice of spots, which evenly covers the illumination field, can be created. Such a fluorescent pattern on the glass surface samples the illumination field more evenly than randomly positioned fluorescent beads. Also the thickness of a fluorescent layer formed on the substrate surface can be much less than the diameter of a bead, whereby the pattern can be more fully retained within the EF. Preferably, the pattern should cover less than 10% of the total image field, so that the background fluorescence of the liquid can be used to assess the flatness of the excitation illumination.

Theoretical and Practical Considerations

According to theory (Axelrod, D., (1981), Cell-substrate contacts illuminated by total internal reflection fluorescence,

J. Cell. Biol. 89, 141-5, and Thompson, N. L., Burghardt, T. P. and Axelrod, D., (1981), Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy, Biophys . J. 33, 435-54) the exponential decay in intensity of the EF from the substrate surface into the sample is given by the following expression:

I_z = I_oe^"z/d, where d = λ/4π (ni²sin²θ - n₂ ²)^1/2

I_z being the intensity at a distance z from the substrate surface, I₀ being the maximum intensity of the EF at the substrate surface according, and d being the EF depth. d itself is determined by the wavelength of the incident illumination (λ) , the refractive indicies of the substrate (ni) and fluorescent medium (n₂) , and the angle of incident (θ) of the illumination.

Thus the fluorescence intensity of foreground objects at z = 0 is determined by I₀, whereas the background intensity is determined by integration of the intensity profile of the EF.

Further, the illumination angle (θ) is the primary factor expected to influence image contrast as measured by the optical sectioning ratio (foreground intensity/background intensity) . Figure 4 plots the predicted decay in EF intensity with distance from the substrate surface for six different illumination angles. The illumination intensity profile drops more steeply with larger illumination angles, suggesting that better optical sectioning can be achieved using higher angles. The illumination intensity I₀ should have little effect on the observed optical sectioning ratio, since the intensity of both foreground and background are linearly dependent on Io . Note however that I₀ is the intensity of the EF at the substrate surface, which is not simply the intensity of the collimated beam focused on the sample.

The present inventors have observed, as expected, that the fluorescence intensity of foreground objects increases as the incident angle increases from below the critical angle for total internal reflection to that angle and as the illumination correspondingly changes from epi to EF. However, in contrast to the predictions of theory, they have also surprisingly found that further increases in the incident angle beyond the critical angle often lead to reductions in image contrast, as estimated by comparison of foreground (entities) and background (medium) fluorescence intensity. The inventors have further surprisingly found that increasing the power of illumination generally produces higher image contrast. These trends may be caused by different dependencies of I₀ and e^"z/d on the angle and power of illumination.

The Image Contrast Estimate (ICE) can be defined as the ratio of the average foreground intensity (e.g. due to fluorescent emission produced by the fluorescent entities) to the average background intensity (e.g. due to fluorescent emission produced by the fluorescent medium) . Illustrating the inventors findings, Figure 5 shows plots, at different laser powers, of the experimentally determined improvement in image contrast achieved by TIRF over epi illumination (as characterised by the TIRF optical sectioning ratio (TOSR) , which is the ratio of TIRF ICE to epi ICE) for varying illumination incident angles. The TIRF microscopy was performed using Nikon TIRF objectives on an Eclipse E2000 inverted microscope. The Nikon epi-fluorescence condensor was replaced with a custom condensor in which laser light was introduced into the illumination pathway directly from the output of a 3.5 μm optical fiber. Output was parallel to the optical axis of the microscope. The TIRF illumination angle was controlled through mechanical translation of the fiber output. Increasing distance of the output fiber from the optical axis resulted in increasing the angle of TIRF illumination. The light source was a 473 nm diode laser (Omicron) controlled by a DAC 2000 card in a PC running MetaMorph™ (Molecular Devices) . A 473/10 laser clean up filter was used together with an FT500 dichroic and a 525/50 emission filter (Chroma) . Epi-fluorescence was performed by centering the fiber output on the illumination pathway to generate a collimated laser beam which illuminated the sample at 0°. A glass hemisphere was used for measurement of the TIRF illumination angle. The hemisphere was placed in oil contact with the objective and centered using backreflections of the laser beam within the hemisphere. A ruler was erected on the microscope stage at a known distance from the objective aperture, and the height of the beam above the microscope stage was measured over a range of approximately 55 to 70 degrees.

Thus, in practice the best optical sectioning results are often achieved at incident angles close to the critical angle. It is, therefore, important for the optimal operation of TIRF microscopes to be able to set up the microscope so that it operates at such an angle. Test samples according to the present invention in which the fluorescent entities are at the surface of the substrate are particularly suited for achieving this. Such samples can be readily used to measure the average foreground intensity and the average background intensity. The Image Contrast Estimate (ICE) can then be calculated. With the test sample on the microscope, it is relatively straightforward to adjust the microscope to vary, for example, the incidence angle of the excitation beam such that the ICE is maximised.

However, in practice, there may be complex interactions occurring within a microscope, such as scattering, reflection, and mis-alignment. Therefore, a test sample, such as that provided by the present invention, which can mimic a biological sample and can be quantitatively analyzed, is useful for maximising instrument performance. Aside from varying the angle of incidence of the excitation beam, other features that can be adjusted include the position of lenses in a condensor assembly, the opening diameters of various apertures (especially the field diaphragm) , the illumination intensity, selection among different objectives, and the flatness of optical components such as mirrors . As mentioned above, flatness of TIRF illumination can be a problem for quantitative image analysis. In particular, misalignment of laser beam light paths or dirt in the paths can produce interference patterns in the EF illumination. However, using a test specimen of the present invention in which a homogenously fluorescent medium is carried by the substrate, such patterns can be readily identified so that with appropriate adjustment of the microscope the flatness of the excitation fluorescence can be improved^ By way of example, Figure 6a is an image of an interference pattern produced under EF illumination of a test sample according to the present invention. The test sample has a liquid fluorescent medium containing fluorescent beads. The bright spots in the image are caused by fluorescent emission from the beads. Figure 6b is a plot of pixel intensity against position along the horizontal white line of Figure 6a.

Using a Test Sample According to the Present Invention

The test sample may be appropriate for two phases of experimental microscopy:

1. The set up phase

When a TIRF microscope system is being optimized for use, there are a significant number of alignment issues concerning e.g. passage of the illumination beam through the microscope, clipping of apertures, flatness of mirrors, perfection/imperfection of other optical surfaces. A test sample with a homogenously fluorescent medium can be used to visualise and assist in the removal of interference effects associated with laser illumination, or any other inhomogeneities in the illumination field. By adjusting the incident angle of excitation beam to maximise the ICE, an optimal TIRF operating condition can be achieved.

Additionally, if the ICE is calculated for both TIRF and epi images acquired with the same objective, an estimation of the improvement in image contrast associated with TIRF illumination can be obtained.

If a plurality of microscopes are being operated in parallel, for example to provide a high-throughput screen, the ICEs of the different microscopes can be matched to improve uniformity of analysis across the microscopes.

2. The experimental phase

When system performance must be controlled over the course of an experimental series (which, particularly for screening applications, could extend over days, weeks, or months) it may be necessary to ensure that results obtained at different times are quantitatively comparable. This can be achieved by periodically measuring the relative intensities of the fluorescent emission produced by the fluorescent entities and the fluorescent emission produced by the fluorescent medium. Conveniently, the relative intensities can then be used to calculate the ICE. If necessary, the microscope can be adjusted to maintain a constant ICE value.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

Claims

1. A microscope test sample comprising: a transparent substrate, a transparent fluorescent medium interfacing with the substrate, the medium being of lower refractive index than the transparent substrate, and fluorescent entities within the medium at the substrate- medium interface which, when excited to fluoresce, are fluorescently distinguishable from the medium.

2. A test sample according to claim 1, wherein the fluorescent medium is homogeneously fluorescent.

3. A test sample according to claim 1 or 2, wherein, for a given excitation illumination intensity, the fluorescent entities produce a more intense fluorescence emission than the fluorescent medium.

4. A test sample according to any one of the previous claims, wherein the fluorescent medium is encapsulated in an air-tight container on the substrate.

5. A test sample according to any one of the previous claims, wherein the fluorescent medium is a liquid.

6. A test sample according to any one of the previous claims, wherein, the fluorescent medium extends over least 1 mm² of the surface area of the transparent substrate.

7. A test sample according to any one of the previous claims, wherein, the fluorescent medium has a refractive index in the range of from 1.3 to 1.4.

8. A test sample according to any one of the previous claims which is configured such that, when viewing the fluorescent medium through a microscope with the fluorescent medium extending across the entire field of view, the fluorescent entities occupy in the range of from 0.5 to 10% of the area of the field of view.

9. A test sample according to any one of the previous claims, wherein the fluorescent entities are provided by fluorescent beads .

10. A test sample according to claim 9, wherein the fluorescent beads are less than 100 nm in diameter.

11. A test sample according to claim 9 or 10, wherein the fluorescent beads are evenly dispersed over the substrate surface.

12. A test sample according to any one of claims 1 to 8, wherein the fluorescent entities are provided by a patterned fluorescent layer on the surface of the substrate.

13. Use of the test sample according to any one of the previous claims for setting up a total internal reflection fluorescence (TIRF) microscope.

14. Use of the test sample according to any one of claims 1 to 12 for maintaining an operating condition of a TIRF microscope.

15. A method of calibrating a TIRF microscope comprising the steps of:

(a) positioning the test sample of any one of claims 1 to 12 on the microscope such that an excitation beam of the microscope traverses the transparent substrate to produce an evanescent field in the fluorescent medium at the substrate- medium interface, and

(b) measuring the relative intensities of the fluorescent emissions produced by the fluorescent entities and the fluorescent medium.

16. A method of performing TIRF microscopy comprising performing at intervals the calibration of claim 15, and, when necessary, adjusting the TIRF microscope to maintain the relative intensities of the fluorescent emissions produced by the fluorescent entities and the fluorescent medium.

17. A method of adjusting a TIRF microscope comprising the steps of: (a) positioning the test sample of any one of claims 1 to 12 on the microscope such that an excitation beam of the microscope traverses the transparent substrate to arrive at the substrate-medium interface, and

(b) adjusting the microscope such that the ratio of the intensity of fluorescent emission produced by the fluorescent entities to the intensity of fluorescent emission produced by the fluorescent medium is maximised.

18. A kit comprising the test sample of any one of claims 1 to 12, and instructions for performing the method of any one of claims 15 to 17.

19. A method of adjusting a TIRF microscope comprising the steps of:

(a) positioning the test sample of claim 2, or of any one of claims 3 to 12 as dependent on claim 2, on the microscope such that an excitation beam of the microscope traverses the transparent substrate to produce an evanescent field in the fluorescent medium at the substrate-medium interface, and

20. A kit comprising the test sample of claim 2, or of any one of claims 3 to 12 as dependent on claim 2, and instructions for performing the method of claim 19.