GB2251701A - UV Microscope illuminator - Google Patents

Abstract

A microscope objective tube 12 includes a lateral input port 13 for UV light and adjacent the port a beam splitter e.g. a UV-reflective mirror 15 necessary to reflect the incoming UV light towards the objective, and subsequently to reflect away the returning UV light (if any) whilst allowing the visible fluorescence-generated light to pass through and into the microscope barrel 18 and onward to the ocular. The tube has means 16, 17 at its ends for mounting an objective lens 22, 23 and to be mounted on the microscope barrel 18. A UV barrier filter 19 is shown. <IMAGE>

Description

Microscopes This invention relates to microscopes, and concerns in particular objectives suitable for use with microscopes operating with ultraviolet light.

A conventional microscope has a stage on which is placed the object - the specimen - to be viewed, and a combination of lenses with which to view that object, the combination including a magnifying object lens (or "objective") nearest the object to be viewed, and a magnifying eyepiece lens (or "ocular") to which the user places his or her eye to see the magnified object, there being a barrel separating the two and spacing them at approximately the correct distance for the ocular to be able to focus on the real (primary) image formed by the objective. The whole combination of lenses and barrel is moved to and from the object to get it in focus.

Light from the object is magnified by the object lens, and then passed along the barrel for further magnification by the eyepiece lens, where it leaves the microscope and enters the user's eye.

In order to make sufficiently bright the image of the object, it is conventional to provide a light source illuminating the object - the source may be a mirror, to catch light from some distant light such as a window, or it may be a light bulb adjacent, but part of, the microscope. Moreover, it is common to view the object not by reflected light - light shining down onto the object and being reflected back into the objective - but by transmitted light - light originating beneath the object, and being shone through it and thence to the objective. In either case it is common to stain the object with a coloured dye, to bring out detail otherwise "invisible".

Conventional optical microscopes employ visible light, but there are many occasions when it is more useful to view the object with ultraviolet light UV light - because numerous objects glow, or fluoresce, under UV, and in this way reveal greater or different detail about their constitution. Typically, parts of an object illuminated with UV (in the 300 to 400 nanometre wavelength range) will glow white, or blue, or green or even yellow (visible light, in the 400 to 700 nanometre wavelength range), and this generated glow can impart useful information. In some cases, indeed, it is common to stain the object with a fluorescent dye - that is, a dye which fluoresces (usually yellow or green, but red and orange, and other colours, are possible) under UV light - in order to accentuate detail.One such dye is eosin, which absorbs both blue C480 nm) and UV C400 nm) light and emits green (540 nm) light. This technique is commonly employed in immunodiagnosis, where stains, counterstains and colour suppressants may be used to indicate whether a particular antibody-antigen complex is present in the specimen.

An ordinary light microscope can be used as a fluorescent microscope (provided that the lenses present in the eyepieces, objective and condensors are not themselves fluorescent). The light source should be rich in blue and UV light, and mercury and xenon arcs are commonly used, as well Cmore recently) as certain types of high intensity quartz halogen lamp. However, since the emitted fluorescent light is weak it has to be conserved carefully inwits passage through the microscope's lens and filter systems. Moreover, UV light can be intensely damaging to the retina of the human eye, and it is obviously necessary when utilising it to ensure that any that travels from the specimen toward the objective and thence toward the eyepiece be removed, by some form of filtering, before it reaches the eyepiece and the human eye beyond.One technique proposed for dealing with both these problems is that of viewing the object by epi-fluorescence (that is, surface fluorescence); the object is illuminated from above (rather than from below) by UV light, and the fluorescence-generated visible light (plus any reflected UV light) is led back up, to the eyepiece, via a "filter" device where only the harmless fluorescencegenerated light is permitted to pass onwards.It is common to supply the UV light from an external off-axis source, feeding it into the microscope above the objective and then reflecting it down towards the objective using a suitably-positioned dichroic (or "beam-splitting") mirror - that is, a mirror which reflects one band of spectral energy while transmitting all others - which also acts as a "filter" for the returning mixture of fluorescence-generated visible light and reflected UV light. As will be appreciated, not only does epi-fluorescence illumination reduce the likelihood of user eye-damage, but it also eliminates the need for the weak fluorescence-generated light to pass through the glass of the substage condensors and the glass of the microscope slide on which the object is mounted, and so does much to strengthen the fluorescent signal seen by the observer.

A typical form of microscope operating under epi-fluorescence conditions is one wherein the barrel is especially built, or modified, to have a port allowing UV light to be shone thereinto via an excitation filter (chosen to transmit UV and block visible light), there then being placed within the barrel adjacent the port a dichroic mirror so positioned and angled as to cause the UV light to be directed down the barrel to and out of the objective so that it falls on and illuminates the specimen, the resultant fluorescence, and some reflected UV light, being gathered by the objective and directed back up the barrel to the mirror. The dichroic mirror now acts as a filter, reflecting the UV component to one side, out of the barrel, and allowing the rest - the visible light from the fluorescing specimen - to pass on along to the ocular (via a UV barrier filter), and thence to the eye.Such a microscope is very satisfactory, save from one viewpoint, namely that of cost. The modifications necessary to make the barrel, or to convert a conventional (visible light microscope) barrel into one suitable for UV light epi-illumination, can be expensive and time-consuming. The present invention seeks to overcome this problem by the entirely novel approach of providing the UV input not to the barrel but instead to the objective.

For the most part, a microscope objective is a very short focal length lens (or lens pair) carried at the tip of a short tube that mounts, co-axially, commonly by a screw thread, onto the bottom of the barrel. A microscope may have a number of objectives, of different magnifying power, and it is conventional to mount these on a rotatable platform under the barrel, each objective being rotated into position aligned with the barrel as it is required.The present invention stems from the realisation that the conventional objective may be replaced by one wherein the short tube on its eyepiece side is designed both to include the lateral input port for the UV light and to contain adjacent the port the UV-reflective mirror necessary to reflect the incoming UV light towards the objective, and subsequently to reflect away the returning UV light (if any) whilst allowing the visible fluorescence-generated light to pass through and into the microscope barrel and onward to the ocular. In this way a perfectly conventional visible light microscope may be used for fluorescence microscopy, without any need in any way expensively to "convert" the microscope, simply by providing it with a relatively inexpensive special objective.

In one aspect, therefore, the invention provides an objective for a microscope to be used for viewing specimens by ultraviolet-generated epi-fluorescence, the objective including: an objective lens system; and an objective tube, having co-axially mounted at one end the objective lens system, and adapted to be mounted at the other end to the objective end of the microscope barrel, which tube has a lateral port, via which supplied UV light can enter the tube, and contains within itself a beam splitter, reflective for UV light but transmissive for the expected fluorescence, mounted adjacent the port to reflect towards the objective UV light entering the tube via the port but transmit fluorescence light entering via the objective lens system.

The invention concerns an objective for a fluorescence microscope using UV light epi-illumination.

The microscope itself may - apart from the objective be any conventional visible-light optical microscope, and needs no further comment here.

The objective of the invention includes an objective lens system. This system may, indeed, in essence be the objective lens system of a conventional microscope, and so needs no further discussion here save perhaps to say that the lens system is conveniently mounted into the tube exactly as it would be in a conventional microscope, and will normally have a magnification power in the range 40X to 60x. In addition is is worth observing that the brightness of an image, as seen by the eye, is proportional to NA2/M2, where NA is the Numerical Aperture of the objective lens and M is the total magnification of objective and eyepiece. It will be apparent that the light collected by the lens increases as the square of the numerical aperture but decreases as the square of the magnification.To obtain the brightest fluorescent image it is therefore best to use objectives with the highest numerical aperture and the lowest possible magnification (commensurate with the need to magnify the object, of course). Moreover, since some light is invariably lost as it traverses optical surfaces, the simplest lenses, with the fewest components, are usually the best.

The objective lens system is mounted into one end of the objective tube; it is this tube that is the heart of the present invention. The tube is a short one, preferably of a length sufficient to incorporate the port and the beam splitter and provide parfocality thus, the whole is about 1.75in (4.3cm) long. It can be made of any suitable strong rigid material, such as machined brass, and be of any appropriate cross-section, such as circular. It can be attachable to the microscope barrel in any convenient way, and is indeed preferably screw-mounted onto the end of the microscope barrel (or onto the rotatable nosepiece) in place of the ordinary objective.

The objective tube has firstly a lateral port via which light from a UV source can be shone into the tube, and secondly a UV beam splitter that reflects towaras the objective UV light entering via the port while transmitting to the barrel (and on to the ocular) visible light entering via the objective lens system from the fluorescing specimen. The lateral port is in principal merely an aperture into which UV light can be shone, but advantageously it incorporates a narrow band excitation filter - that is, a filter that passes the UV component of the light received from whatever source is providing UV light and blocks the visible light component.A typical excitation filter is an FITC 4a filter - that is, a filter that specifically enhances that UV component of the input light that is "tuned" to the frequency of one of the more common of the fluorescent dyes used in fluorescence microscope, namely fluoroscein isothiocyanate. Such a filter is commercially available from Balzers High Vacuum Ltd of Milton Keynes, England.

The source itself may be any convenient UV source one such, for example, is that source the subject of McArthur British Fatent Application No: 90/20,288.8 (P1178), which is in essence a light box containing a low voltage quartz halogen bulb and a "filter" directing solely, or mostly, UV light out through a suitable aperture in the box. Such a source, like many others, is best operatively connected to the present invention's objective's tube's port by an optical fibre light guide (preferably one optimised for UV/blue light), and so in addition the port is most preferably equipped with mounting means (such as an appropriate screw thread, or push fit aperture) whereby the UV source may operatively be connected to the port.

Within the objective tube is a beam splitter that separates UV from visible light. With respect to the UV light entering via the port - it enters at rightangles to the tube's optical axis, as in a standard epi-fluorescence arrangement - the beam splitter is positioned across the optical axis adjacent the port so as to reflect the UV component along the optical axis toward the objective lens system (the other light components, if any, are transmitted, and absorbed by the facing tube wall). In operation this UV light shines through the objective lens system onto the specimen, causing fluorescence, and both the generated, visible "glow" light and any reflected UV light is gathered by the objective lens system and directed back to the beam splitter.Here again the UV component is reflected off-axis (sideways, to the port) and the visible component is transmitted . . . but now it is the former component that is disposed of (harmlessly, away from the user's eye) and the latter that is allowed to proceed onwards into the microscope barrel and so to the ocular.

A typical (and preferred) beam splitter is a suitable dichroic mirror, for example one that reflects UV light in the 300 to 400 nanometre band while transmitting the rest (and particularly that in the visible 400 to 700 nanometre band), such as that DC Blue mirror sold by Bailers (loc cit). It is positioned adjacent the port, and at 45' to both the port and the tube optical axis.

Just as the objective tube employs an excitation filter to select the input UV light, so it very preferably includes a barrier filter to absorb any UV component in the light exiting the tube towards the microscope barrel, for 'despite the UV-reflective effect of the beam splitter some UV may pass through rather than be reflected. This barrier filter, which is advantageously mounted across the tube optical axis at the exit (barrel) end of the tube, is matched to the expected fluorescence, and normally is conveniently a DT Yellow filter, available from Balzers (loc cit), and needs no further comment here.

It will be appreciated that the excitation filter, the beam splitter and the barrier filter are, with the specimen itself, components of a "tuned'5 system, and so need to be selected with care, to match one another.

Thus, where sequential specimens are, for instance, to be stained with differently-fluorescing dyes, it will be desirable either to change the filters within the set or, and preferably (given the relative inexpensiveness of the invention's objective), to provide a second, or even a third, objective to match the new stain's properties.

In summary, the objective of the invention, by combining ail the essential elements of a standard fluorescence system into one single objective by providing the objective tube with its integral filter system, has made the technique of fluorescence microscopy available, at a very low comparative cost (around a fifth or a sixth of the conventional cost), to almost any user of a conventional microscope system.

This is achieved by exploiting the near universal RMS thread-type fitting for all major microscope objective systems. The invention's objective is simply screwed into an empty port on the standard microscope nosepiece, an external UV light source is connected, and fluorescence microscopy is instantly available.

An embodiment of the invention is now described, though by way of illustration only, with reference to the accompanying diagrammatic Drawings in which: Figure 1 shows a cutaway perspective view of an objective of the invention; and Figure 2 shows a diagrammatic axial cross-section through an objective like that of Figure 1, mounted directly on a microscope barrel and viewing a specimen.

The objective of the invention (generally 10) is based very simply on a combination of a conventional microscope objective lens system (11) and the type of filter arrangement usually found within the body of a standard fluorescence microscope. These have been combined to form a unit providing fluorescence capabilities for even the simplest of microscopes possessing a standard RMS-tppe nosepiece.

This unit comprises a tubular objective body (12) having a lateral port (13) leading via a UV excitation filter (14) to a 45 cross-axial dichroic mirror beam splitter (15; not shown per se in Figure 1). The body 12 has suitable threads Cas 16, 17) at either end to allow the objective lens system 11 to be screwed thereon, and to allow the body to be screwed to the microscope barrel (18; in Figure 2 only), and at the barrel end there is a UV barrier filter C19).

In operation, UV light (.20) enters the unit from a remote source (not shown) which produces a UV/far bluerich light, via the short tubular entry port 13, which incorporates the UV excitation filter 14. This filter provides excitation in the most suitable waveband, and also does not permit a significant level of other wavebands to be transmitted, which facilitates subsequent filtering further on in the system.

The resultant light passes on to the beam splitter 15 (dichroic-blue type), where it is reflected by go. down into the body of the objective. The splitter is mounted on a brass wedge (21) which is accurately machined to preserve the angle to within acceptable tolerances.

The objective elements (shown diagrammatically as 22, 23) are contained within the separate objective unit 11. They form a simple achromat objective lens system (the optical system may be kept basic, to keep the total price of the system as low as possible while retaining operational integrity). The system may be designed to keep to the usual working distance for a standard objective of the chosen power to ensure compatibility with a wide variety of microscope systems.

The objective lens system unit 11 is of the dry type, to avoid the inherent inconvenience of immersiontype objectives when rapidly changing between magnifications This also facilitates rough focussing.

The light 20 then passes to the specimen (30), where any fluorescent material is excited. The visible (blue/green) "glow" component generated (31) is then transmitted back through the lens system to the beam splitter 15 which it passes through unhindered, most of any specimen-reflected UV light (32) being reflected by the beam-splitter off towards port 13.

A simple DT Yellow filter C19) acts as a barrier for any residual UV light.

Finally, the visible light 31, in the blue/green region of the spectrum, is then carried through the conventional optical system (not shown) of the microscope to the eyepiece Cnot shown).

For most popular types of microscope, the objective is designed to be parfocal with the existing objectives on the nosepiece, thus allowing trouble-free change-over of the objectives during use, and low-power quickscanning of the specimen.

A large number of the diagnostic kits available at present utilise FITC Cfluoroscein isothiocyanate) as the fluorochrome marker. The invention's objective is therefore usually supplied with a filter set suitable for the excitation of this dye. However, if different dyes are used, and thus different excitation wavelengths are required, then the FITC set can easily be replaced by a set specific for the fluorochrome employed.

Claims (11)

1. An objective tube for a microscope to be used for viewing specimens by ultraviolet-generated epi-fluorescence, the objective tube including at one end means for co-axially mounting thereon an objective lens system, and being adapted to be mounted at the other end to the objective end of the microscope barrel, which tube has a lateral port, via which supplied UV light can enter the tube, and contains within itself a beam splitter, reflective for UV light but transmissive for the expected fluorescence, mounted adjacent the port to reflect towards the objective lens system end UV light entering the tube via the port but transmit fluorescence light entering via the objective lens system end.

2. An objective tube as claimed in Claim 1, wherein the objective lens system is to be mounted thereon by screw-mounting means.

3. An objective tube as claimed in either of the preceding Claims, which is a short one, of a length sufficient both to incorporate the port and the beam splitter and to provide parfocality.

4. An objective tube as claimed in any of the preceding Claims, which is directly screw-mountable onto the end of the microscope barrel.

5. An objective tube as claimed in any of the preceding Claims, wherein the lateral port is an aperture into which UV light can be shone, and incorporates a suitable narrow band excitation filter.

6. An objective tube as claimed in any of the preceding Claims, wherein the lateral port is equipped with mounting means whereby the UV source may operatively be connected to the port.

7. An objective tube as claimed in any of the preceding Claims, wherein the beam splitter is a suitable dichroic mirror, positioned adjacent the port, and at 450 to both the port and the tube optical axis.

8. An objective tube as claimed in any of the preceding Claims, which includes a suitable barrier filter to absorb any UV component in the light exiting the tube towards the microscope barrel, this barrier filter being mounted across the tube optical axis at the exit (barrel) end of the tube.

9. An objective tube for a microscope as claimed in any of the preceding Claims and substantially as described hereinbefore.

10. An objective lens assembly for a microscope to be used for viewing specimens by ultraviolet-generated epi-fluorescence, the assembly incorporating an objective lens system mounted on an objective tube as claimed in any of the preceding Claims.

11. A microscope to be used for viewing specimens by ultraviolet-generated epi-fluorescence, which microscope incorporates an objective tube as claimed in any of Claims 1 to 9.