WO2001013079A1

WO2001013079A1 - Method and apparatus for the resolution of beams of electromagnetic radiation

Info

Publication number: WO2001013079A1
Application number: PCT/AU2000/000990
Authority: WO
Inventors: Kiang Wei Kho; Martin Russell Harris; Gavan Edmund Rosman
Original assignee: Swinburne University
Priority date: 1999-08-18
Filing date: 2000-08-18
Publication date: 2001-02-22

Abstract

The invention provides method and apparatus for the resolution of beams of electromagnetic radiation. In one aspect, there is provided a method for resolving overlapping emission spectra, involving: providing a plurality of polarised incident beams (14) of electromagnetic radiation for irradiating a sample (36), each of the beams having a different polarisation; irradiating the sample with the beams; receiving respective return emission spectra from the sample; and resolving (38, 40) the emission spectra according to their respective polarisations. In another aspect, there is provided a method of spatially resolving a beam of electromagnetic radiation according to polarisation, involving: passing the beam through a polarising beam splitter to form first and second orthogonally polarised components; and altering the plane of polarisation of at least the second component and translating the path of at least one of the components, so that the components are polarised in substantially the same plane, are substantially parallel and are spatially displaced.

Description

METHOD AND APPARATUS FOR THE RESOLUTION OF BEAMS OF ELECTROMAGNETIC RADIATION

The present invention relates to a method and apparatus for the spatial and polarization resolution of electromagnetic radiation, typically laser beams, for use in, for example, materials analysis.

Existing apparatuses and techniques for collecting emission spectra from samples are employed widely, including - for example - in the use of confocal microscopy in which a biological sample may be double-labelled in order to gain knowledge of the relative spatial distribution of two internal biological structures . However, spectral leakage between channels resulting from the overlapping of emission spectra can reduce image contrast. In addition, the requirement to have adequate separation of fluorescence spectra in the conventional techniques causes one channel to give less detailed structural information, owing to very long emission wavelength. These problems have been seen, for example, in double-labelling observations of a MDCK cell using FITC and Rhodamine. This spectral overlap is often referred to as bleed-through or, sometimes, fluorescein spectral tail.

Further, in various existing devices, such as laser scanning confocal microscopes, Raman spectrometers and two photon scanning spot microscopes, there is a growing need for rapid acquisition of optical spectra at low light levels, to be used in applications such as process control and biological diagnostics. In addition, attention to the state of polarization is often required. Existing geometries are, however, frequently or the spectra of the two polarization components are not available in the same plane .

It is an object of the present invention, therefore, to provide a method and apparatus that at least reduces some of these disadvantages .

SUMMARY OF THE INVENTION The present invention provides, therefore, a method for resolving overlapping emission spectra, involving: providing a plurality of polarized incident beams of electromagnetic radiation for irradiating a sample, each of said beams having a different polarization; irradiating said sample with said beams; receiving respective return, emission spectra from said sample; and resolving said emission spectra according to their respective polarizations.

It should be noted, however, that the term emission spectra is used herein to refer to electromagnetic radiation emitted by the sample, which through excitation of the sample, fluorescing by the sample, reflection from the sample or otherwise. Further, the emission spectra may comprise very narrow spectra that might generally be described as beams .

The plurality of polarized incident beams may have a single beam path. The plurality of polarized incident beams may comprise a plurality of components of a beam. The plurality of polarized incident beams may be from a single source. The source may comprise a source of unpolarized beams or beam components combined with polarizing means.

Preferably said source is a laser source and said plurality of components comprise different excitations of said laser source. Preferably said laser source is an argon laser. Preferably said plurality of components comprise the 488 nm and 514 nm excitations of argon.

Preferably said incident beams have different wavelengths, for exciting respective different states in said sample.

Preferably said method includes resolving said emission spectra by means of a polarizing beam splitter. Preferably said polarizing beam splitter includes a dichroic mirror.

Preferably said plurality of incident beams comprises two orthogonally polarized incident beams.

Preferably said method includes providing said different polarizations .

Preferably said method includes polarizing or altering the polarization of at least one of said plurality of incident beams by means of polarizing means. Preferably said polarizing means includes a half wave plate.

Preferably said sample comprises one or more labels for labelling a biological sample.

The present invention also provides an apparatus for separating overlapping emission spectra, having: a source of a plurality of polarized incident beams of electromagnetic radiation for irradiating a sample, each of said beams having a different polarization; means for receiving receiving respective return, emission spectra from said sample; and means for resolving said emission spectra according to their respective polarizations.

The plurality of polarized incident beams may comprise a plurality of components of a beam, more preferably from a single source.

The source may comprise a plurality of light sources.

Preferably said apparatus includes a polarizing beam splitter for resolving said emission spectra. Preferably said polarizing beam splitter includes a dichroic mirror.

Preferably said apparatus includes polarizing means for polarizing or altering the polarization of at least one of said plurality of incident beams. Preferably said polarizing means includes a half wave plate.

The present invention further provides a method of spatially resolving a beam of electromagnetic radiation according to polarization, involving: passing said beam through a polarizing beam splitter to form first and second orthogonally polarized components; and altering the plane of polarization of at least said second component and translating the path of at least one of said components, so that said components are polarized in substantially the same plane, are substantially parallel and are spatially displaced.

Preferably said altering the plane of polarization of at least said second component comprises changing at least said second component from s-polarization to a p-state.

Preferably said polarizing beam splitter comprises a compound prism. Preferably said altering said plane of polarization of at least said second component includes internally reflecting said second component within a prism. Preferably said translating the path of at least one of said components includes internally reflecting said at least one of said components within a prism. More preferably said altering said plane of polarization of at least said second component and said translating the path of at least one of said components includes internally reflecting said second component within a prism.

Preferably said passing said beam through a polarizing beam splitter, said altering the plane of polarization of at least said second component and said translating the path of at least one of said components is by means of a single prism that includes at least one twisted periscope.

In one embodiment, said altering the plane of polarization of at least said second component is by means of an achromatic half-wave plate that changes an input s- polarization to a p-state.

Preferably said method includes directing said substantially parallel and spatially displaced components into a dispersing means. Preferably said dispersing means comprises at least one prism (and may comprise a train of prisms), and said method includes directing said substantially parallel and spatially displaced components against said at least one prism at substantially the Brewster angle.

Preferably said dispersing means comprises one or more diffracting elements operating in transmission or reflecting mode. The present invention also provides a method of spectrographically resolving a beam of electromagnetic radiation, including the method of spatially resolving said beam of electromagnetic radiation according to polarization described above.

The present invention also provides an apparatus for spatially resolving a beam of electromagnetic radiation according to polarization, involving: a polarizing beam splitter for forming first and second orthogonally polarized components of said beam; means for altering the plane of polarization of at least said second component; and means for translating the path of at least one of said components; wherein said components exit said apparatus polarized in substantially the same plane, substantially parallel and spatially displaced.

Preferably said polarizing beam splitter comprises a compound prism. Preferably said means for altering said plane of polarization of at least said second component comprises a prism for internally reflecting said second component. Preferably said means for translating the path comprises a prism arranged to internally reflect said at least one of said components . More preferably said means for altering said plane of polarization and said means for translating the path comprise a single prism.

Preferably said apparatus includes a prism including at least one twisted periscope, said prism constituting said polarizing beam splitter, said means for altering the plane of polarization of at least said second component and said means for translating the path.

In one embodiment said means for altering the plane of polarization of at least said second component comprises a half wave plate.

Preferably said apparatus includes a dispersing means for dispersing said substantially parallel and spatially displaced components. Preferably said dispersing means comprises at least one prism for receiving said substantially parallel and spatially displaced components at substantially the Brewster angle.

The present invention still further provides a spectrograph, including the apparatus for spatially resolving said beam of electromagnetic radiation according to polarization as described above.

The present invention still further provides a Raman spectrophotometer including the apparatus for spatially resolving said beam of electromagnetic radiation according to polarization as described above.

The present invention still further provides a laser scanning confocal microscope (LSCM) including the apparatus for spatially resolving said beam of electromagnetic radiation according to polarization as described above.

The present invention still further provides a two photon scanning spot laser microscope including the apparatus for spatially resolving said beam of electromagnetic radiation according to polarization as described above.

The present invention still further provides a volume holographic spectral dispersion system including the apparatus for spatially resolving said beam of electromagnetic radiation according to polarization as described above.

BRIEF DESCRIPTION OF THE DRAWING In order that the present invention may be more fully ascertained, preferred embodiments will now be described, by way of example, with reference to the accompanying drawing, in which: Figure 1 is a schematic view of an apparatus for separating overlapping emission spectra in a double labeling experiment according to a first embodiment of the present invention;

Figure 2 is the transmission curve of the dichroic mirror of the apparatus of figure 1;

Figure 3 is a schematic of a first polarizing beam splitter configuration for use with the apparatus of figure 1;

Figure 4 is a schematic of a second polarizing beam splitter configuration for use with the apparatus of figure 1;

Figure 5 is a schematic of a third polarizing beam splitter configuration for use with the apparatus of figure 1; Figure 6 is a schematic of a fourth polarizing beam splitter configuration for use with the apparatus of figure 1;

Figure 7 is a schematic view of a polarizing spectrograph according to a second embodiment of the present invention;

Figure 8 is a schematic view of a polarization insensitive version of the spectrograph of figure 6; and

Figure 9 is a schematic view of a polarizing spectrograph according to a third embodiment of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS An apparatus for separating overlapping emission spectra in a double labeling experiment according to a first embodiment of the present invention is shown generally at 10 in figure 1. The apparatus 10 includes an Argon or Argon/Krypton laser 12 for generating a beam 14 of multiple laser lines (in this example comprising components of excitation energies 488 nm and 514 nm respectively) , filter set 16, polarizing means 18, double dichroic mirror 20, beam scanning system 22, microscope objective 24, polarizing beam splitter cube 26 and first and second photomultiplier tubes 28 and 30.

In use, laser lines 14 are isolated by means of filter set 16 and then orthogonally polarized by means of polarizing means 16 (described in further detail below) . The now polarized lines 32a,b are reflected by dichroic mirror 20, scanned by means of scanning system 22, then focussed by microscope objective 24 to a diffraction-limited spot 34 in a sample 36 that has been double-labelled (such as with Fluorescein and Rhodamine) . Fluorescence from that focus spot 34 is polarized as defined by the polarization of the excitation wavelengths, and - after returning through dichroic mirror 20 - is readily separated into two channels 38 and 40 by mean of using polarizing beam splitter cube 26. These channels may then be detected with photomultiplier tubes 28 and 30.

Figure 2 illustrates the transmission curve 42 of the dichroic mirror 20. The notch band 44 and 46 correspond to the excitation wavelengths. Fluorescence spectra 48 and

50, corresponding to the two different labelling dyes used, will be transmitted, but fluorescence spectrum 48 will be partially reflected. It should be noted that the bandwidth of the notch band 44 and 46 is narrow, so that fluorescence energies can be maximally transmitted.

One possible configuration for polarizing the beam 14 is shown in figure 3. In this configuration, the multi-line Argon ion laser 12 emits a TEMoo beam containing two vertically polarized components 52a,b of wavelengths 488 nm and 514 nm respectively. A long pass dichroic mirror 54 reflects the 488 nm component 52a, but passes the 514 nm component 52b to a half-wave plate 54, which rotates the polarization of the 514 nm component by 90°. The beam paths 52a,b are then recombined into one beam path 56 using two mirrors 58,60 and a long-pass dichroic mirror 62. The recombined beam 56 may then be employed as shown in figure 1, in any suitable apparatus (such a LSCM, a flow cytometer or a gene chip reader) .

Alternatively, referring to figure 4, a TEMoo beam containing two vertically polarized components 52a,b of wavelength 488nm and 514nm respectively from a multi-line Argon ion laser 12 are separated by a dispersing prism 64. The two components 52a,b are then made parallel by a further prism 66. A half-wave plate 68 is placed in the path of the 514 nm component 52b, to rotate its polarization by 90°. The two components 52a,b are then recombined into one beam path 68 by mans of two prisms 70 and 72.

A further possible configuration is shown in figure 5. As above, a multi-line Argon ion laser 12 emits a TEMoo beam containing vertically polarized components 52a,b of wavelengths 488 nm and 514 nm respectively. A dual- wavelength-plate 74 (at 45° to the beam 52a,b) rotates the polarization of the 514 nm component 52b by 90°, but the

488 nm component 52a by 180°. There is no beam separation, thus eliminating image misregistration.

A further polarizing beam splitter configuration is shown in figure 6. The multi-line Argon ion laser 12 emits a

TEMoo beam containing vertically polarized components 52a,b of excitation wavelengths 488 nm and 514 nm respectively, which are separated out by a dispersing prism 76. Both separated beam paths 52a,b are then made parallel by a second prism 78. A half-wave plate 80 (at 45°) rotates the polarization of the 488 nm component 52a by 90°, leading to orthogonally polarized beams 82a,b (488 nm component 82a polarized perpendicular to the plane of figure 6 as depicted, 514 nm component 82b in the plane of figure 6) . Two prisms 84,86 recombine the two components 82a,b into a single beam path 88, which is then directed to the sample measuring apparatus. Returning, induced fluorescence follows the same beam path 88, but in the opposite direction. A mirror 90, which reflects only the fluorescence, is positioned between prisms 78 and 82 and directs the returned fluorescence 92 towards two holographic (narrow band) notch filters 94,96. These notch filters 94,96 further exclude the excitation wavelengths. The returned fluorescence 92, after passing through filters 94,96, is sent to a polarizing beam splitter 98, which resolves the returned fluorescence 92 into orthogonally polarized components 100a,b, which are focussed by lenses 102,104 respectively onto respective photomultiplier tubes 106,108.

Thus, with the apparatus 10, it is not necessary to use spectrally well separated fluorescence dyes; as the image improvement is achieved with the use of two orthogonally polarized excitation wavelengths, two fluorescent dyes having similar emission spectrum can still be used, provided that the absorption peaks are different. This is possible because rotational diffusion coefficient of biological samples is often very slow. When appropriate fluorescence dyes are chosen, their emission polarization can be made parallel to their corresponding excitation polarization. This suggests that it may be possible, with the apparatus 10 of figure 1, to achieve almost equal resolution for both channels. It is considered that this apparatus could be used for as a gene chip reader using the Affymetrix patented gene chip. It would also be possible to apply this technique in i munoassay analysis as well as flow cytometry.

Figure 7 is a schematic view of a polarizing spectrograph 110 according to a second embodiment of the present invention. Spectrograph 110 includes a collimating lens 112, a broadband polarizing beam splitter 114, a twisted periscope 116 formed as or attached to the (side) s face of the beam splitter 114, a horizontal periscope 118 formed as or attached to the (front) p face of the beam splitter 114, and a dispersing prism 120

A light beam 122 (delivered to the spectrograph 110 by, for example, a fibre or slit) is collimated by lens 122. The collimated beam 124 is available for pre-filtering before entering the polarizing beam splitter 114, where the collimated beam 124 is split into p component 126a (horizontal in this figure) and ε component 126b (initially vertical in this figure) respectively, at vertical diagonal plane 115 of beam splitter 114. The twisted periscope 116, formed as or attached to the ε face of the beam splitter 114, converts the polarization of the 8 component 126b to a p state and elevates that component, while horizontal periscope 118, formed as or attached to the (front) p face of beam splitter 114, translates the p component 126a laterally to be directly under component 126b as it leaves the twisted periscope 116. The combined action of the periscopes 116,118 results in two parallel but vertically displaced beams 126a, b of p polarization (126b above 126a in the figure) . These are directed against the dispersing prism 120 under Brewster or near-Brewster angle conditions, so that little if any energy is lost due to reflection. After passing through the dispersing prism 120, the beams 126a, b can be focused by separate lenses 128,130 to suitable detectors (such as CCD or PMT detectors) 132a,b so as to form separate spectra for each polarization p and ε which were present at the input .

In this embodiment, the height of the front face of the polarizing beam splitter 114, the extent to which twisted periscope 116 projects above the beam splitter 114 and the height of the dispersing prism 120 are respectively hι=25 mm, h₂=25 mm and h₃=50 mm.

Referring to figure 8, by using a sufficiently large diameter focussing lens 134 after the dispersing prism 120, the two beams 126a, b can be re-combined to fall on the same photodetector 136 and thereby provide a polarization- insensitive spectrograph.

In another embodiment, shown in figure 9, a similar outcome to that of the spectrograph 110 of figure 7 can be achieved by a spectrograph 140. Unlike spectrograph 110, however, spectrograph 140 has a single periscope 142 formed as or attached to the ε face of a beam splitter 144. Spectrograph 140 also includes an achromatic half-wave plate 146 in the optical path after the periscope 142.

Consequently, a beam 148 enters the beam splitter 144 and is split into p and 8 components 150a,b respectively, at diagonal vertical plane 145. The p component 150a exits the beam splitter 144 through the p face of beam splitter 144 essentially undeflected. The 8 component 150b is reflected by periscope 142, which it exits with its polarization preserved, but parallel to and spatially displaced from p component 150a. However, s component 150b then traverses achromatic half-wave plate 146, which changes the s-polarization of component 150b to a p state, so that both components 150a, b are now in the same polarization state but spatially displaced. They then enter dispersive prism 152 as described above, are focussed by one or more lenses (lens 154 in the figure) and detected by detector or detectors 156.

Thus, spectrographs 110 and 140 need use only a single dispersive means (prism, grating or otherwise), as the two exit beams are co-planar and parallel at the stage where they approach the face of the dispersive means, so that there is efficient use of the, for example, prism volume. An SF11 60° prism at minimum deviation is sufficiently close to the Brewster condition to give a power loss of only 0.6% for either input polarization from 488 nm to beyond one micron, or the limit imposed by the beam splitter.

These characteristics have the advantages, therefore, that:

• Higher throughput than conventional prism and grating devices is possible;

• One can control the polarizations presented to detectors, which can be quantified with separate photodetector channels (or recombined into one single photodetector) ; • Collimating lenses can be closer to the dispersing element than for reflective grating instruments;

• Prism volume is efficiently used by two vertically displaced beams;

• Prism chain can be extended to increase dispersion without discrimination of the two polarizations;

• Geometry allows direct insertion of a prefilter to reject laser pump light in the collimated beam;

• More equal and increased throughput for each polarization than conventional volume holograph transmission configurations; and

• Compared to scanning grating systems, the spectrographs can be operated in a non-scanning multichannel mode.

Modifications within the spirit and scope of the invention may readily be effected by persons skilled in the art. It is to be understood, therefore, that this invention is not limited to the particular embodiments described by way of example hereinabove.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS :

1. A method for resolving overlapping emission spectra, involving: providing a plurality of polarized incident beams of electromagnetic radiation for irradiating a sample, each of said beams having a different polarization; irradiating said sample with said beams; receiving respective return, emission spectra from said sample; and resolving said emission spectra according to their respective polarizations.

2. A method as claimed in claim 1, wherein said plurality of polarized incident beams have a single beam path.

3. A method as claimed in claim 1, wherein said plurality of polarized incident beams comprise a plurality of components of a beam.

4. A method as claimed in any one of the preceding claims, wherein said plurality of incident beams are from a single source .

5. A method as claimed in claim 4, wherein said source is a laser source and said plurality of components comprise different excitations of said laser source.

6. A method as claimed in claim 5, wherein said laser source is an argon laser.

7. A method as claimed in claim 6, wherein said plurality of components comprise the 488 nm and 514 nm excitations of argon.

8. A method as claimed in any one of the preceding claims, wherein said incident beams have different wavelengths, for exciting respective different states in said sample.

9. A method as claimed in any one of the preceding claims, wherein said method includes resolving said emission spectra by means of a polarizing beam splitter.

10. A method as claimed in claim 9, wherein said polarizing beam splitter includes a dichroic mirror.

11. A method as claimed in any one of the preceding claims, wherein said plurality of incident beams comprises two orthogonally polarized incident beams.

12. A method as claimed in any one of the preceding claims, wherein said method includes providing said different polarizations.

13. A method as claimed in any one of the preceding claims, wherein said method includes polarizing or altering the polarization of at least one of said plurality of incident beams by means of polarizing means.

14. A method as claimed in claim 13, wherein said polarizing means includes a half wave plate.

15. A method as claimed in any one of the preceding claims, wherein said sample comprises one or more labels for labelling a biological sample.

16. An apparatus for separating overlapping emission spectra, having: a source of a plurality of polarized incident beams of electromagnetic radiation for irradiating a sample, each of said beams having a different polarization; means for receiving receiving respective return, emission spectra from said sample; and means for resolving said emission spectra according to their respective polarizations.

17. An apparatus as claimed in claim 16, wherein said plurality of polarized incident beams comprises a plurality of components of a beam.

18. An apparatus as claimed in either claim 16 or 17, wherein said source includes a plurality of light sources.

19. An apparatus as claimed in any one of claims 16 to 18, wherein said source includes a laser source and said plurality of components comprise different excitations of said laser source.

20. An apparatus as claimed in claim 19, wherein said laser source is an argon laser.

21. An apparatus as claimed in claim 20, wherein said plurality of components comprise the 488 nm and 514 nm excitations of argon.

22. An apparatus as claimed in any one of claims 16 to 21, wherein said incident beams have different wavelengths, for exciting respective different states in said sample.

23. An apparatus as claimed in any one of claims 16 to 18, including a polarizing beam splitter for resolving said emission spectra.

24. An apparatus as claimed in claim 23, wherein said polarizing beam splitter includes a dichroic mirror.

25. An apparatus as claimed in any one of claims 16 to 24, wherein said plurality of incident beams comprises two orthogonally polarized incident beams.

26. An apparatus as claimed in any one of claims 16 to 25, wherein said apparatus includes polarizing means for polarizing or altering the polarization of at least one of said plurality of incident beams and thereby providing said different polarizations.

27. An apparatus as claimed in claim 26, wherein said polarizing means includes a half wave plate.

28. An apparatus as claimed in any one of claims 16 to 27, wherein said sample comprises one or more labels for labelling a biological sample.

29. A method of spatially resolving a beam of electromagnetic radiation according to polarization, involving: passing said beam through a polarizing beam splitter to form first and second orthogonally polarized components; and altering the plane of polarization of at least said second component and translating the path of at least one of said components, so that said components are polarized in substantially the same plane, are substantially parallel and are spatially displaced.

30. A method as claimed in claim 29, wherein said altering the plane of polarization of at least said second component comprises changing at least said second component from s- polarization to a p-state.

31. A method as claimed in either claim 29 or 30, wherein said polarizing beam splitter comprises a compound prism.

32. A method as claimed in any one of claims 29 to 31, wherein said altering said plane of polarization of at least said second component includes internally reflecting said second component within a prism.

33. A method as claimed in any one of claims 29 to 32, wherein said translating the path of at least one of said components includes internally reflecting said at least one of said components within a prism.

34. A method as claimed in any one of claims 29 to 31, wherein said altering said plane of polarization of at least said second component and said translating the path of at least one of said components includes internally reflecting said second component within a prism.

35. A method as claimed in any one of claims 29 to 34, wherein said passing said beam through a polarizing beam splitter, said altering the plane of polarization of at least said second component and said translating the path of at least one of said components is by means of a single prism that includes at least one twisted periscope.

36. A method as claimed in any one of claims 29 to 34, wherein said altering the plane of polarization of at least said second component is by means of an achromatic half- wave plate that changes an input s-polarization to a p- state.

37. A method as claimed in any one of claims 29 to 36, including directing said substantially parallel and spatially displaced components into a dispersing means.

38. A method as claimed in claim 37, wherein said dispersing means comprises at least one prism, and said method includes directing said substantially parallel and spatially displaced components against said at least one prism at substantially the Brewster angle.

39. A method as claimed in either claim 37 or 38, wherein said dispersing means comprises one or more diffracting elements operating in transmission or reflecting mode.

40. A method of spectrographically resolving a beam of electromagnetic radiation, including the method of spatially resolving said beam of electromagnetic radiation according to polarization as claimed in any one of claims 29 to 39.

41. An apparatus for spatially resolving a beam of electromagnetic radiation according to polarization, involving: a polarizing beam splitter for forming first and second orthogonally polarized components of said beam; means for altering the plane of polarization of at least said second component; and means for translating the path of at least one of said components; wherein said components exit said apparatus polarized in substantially the same plane, substantially parallel and spatially displaced.

42. An apparatus as claimed in claim 41, wherein said polarizing beam splitter comprises a compound prism.

43. An apparatus as claimed in either claim 41 or 42, wherein said means for altering said plane of polarization of at least said second component comprises a prism for internally reflecting said second component.

44. An apparatus as claimed in any one of claims 41 to 43, wherein said means for translating the path comprises a prism arranged to internally reflect said at least one of said components.

45. An apparatus as claimed in any one of claims 41 to 44, wherein said means for altering said plane of polarization and said means for translating the path comprise a single prism.

46. An apparatus as claimed in any one of claims 41 to 45, including a prism including at least one twisted periscope, said prism constituting said polarizing beam splitter, said means for altering the plane of polarization of at least said second component and said means for translating the path.

46. An apparatus as claimed in any one of claims 41 to 44, wherein said means for altering the plane of polarization of at least said second component comprises a half wave plate.

47. An apparatus as claimed in any one of claims 41 to 46, including a dispersing means for dispersing said substantially parallel and spatially displaced components.

48. An apparatus as claimed in claim 47, wherein said dispersing means comprises at least one prism for receiving said substantially parallel and spatially displaced components at substantially the Brewster angle.

49. A spectrograph, including the apparatus for spatially resolving said beam of electromagnetic radiation according to polarization as claimed in any one of claims 41 to 48.

50. A Raman spectrophotometer, including the apparatus for spatially resolving said beam of electromagnetic radiation according to polarization as claimed in any one of claims 41 to 48.

51. A laser scanning confocal microscope, including the apparatus for spatially resolving said beam of electromagnetic radiation according to polarization as claimed in any one of claims 41 to 48.

52. A two photon scanning spot laser microscope, including the apparatus for spatially resolving said beam of electromagnetic radiation according to polarization as claimed in any one of claims 41 to 48.

53. A volume holographic spectral dispersion system, including the apparatus for spatially resolving said beam of electromagnetic radiation according to polarization as claimed in any one of claims 41 to 48.