US20170031148A1

US20170031148A1 - LED Microscope

Info

Publication number: US20170031148A1
Application number: US15/162,497
Authority: US
Inventors: Pablo Albertos Sanchez
Original assignee: Streambio LLC
Current assignee: Streambio LLC
Priority date: 2015-07-31
Filing date: 2016-05-23
Publication date: 2017-02-02
Also published as: ES2599056A1; ES2599056B2; EP3124952A1; US20170030887A1; US10345288B2

Abstract

Disclosed is an LED fluorescence microscope without a need for light filters or dichroic mirrors and a method for using the same. The light source for this microscope is made of one or more LEDs directly illuminating the specimen 306 being observed without the use of any light filters or dichroic mirrors.

Description

RELATED APPLICATIONS

This application is related under 35 USC 120 to the patent application entitled, Magnetophoresis System for Separation of Biological Particles, Ser. No. 15/135,415, filed on Apr. 21, 2016, which in turn was related under 35 USC 119 to Spanish provisional application, serial no. P201500574, filed on Jul. 31, 2015, both which are incorporated herein by reference.

BACKGROUND

The invention relates to the field of microscopes specially adapted to specific functions and, in particular, to CPC class G02B (Optical Elements Systems or Apparatus) and/or G02F (Devices or Arrangements, Optical Operation).
A known fluorescence microscope described in U.S. Pat. No. 3,860,813 involves passing light from the light source through an excitation filter to obtain an excitation wavelength. This wavelength is then reflected by a dichroic mirror through the objective lens to the specimen. The fluorescence band is then collected through the objective lens and passes through the dichroic mirror with minimal light of the excitation band. The light passing through the dichroic mirror is then passed through a barrier (absorbing) filter which stops the remaining light from the exciting band from traveling to the eyepiece. This leaves just the fluorescence light to be observed.
Disadvantageously, this set up requires the use of filters and a dichroic mirror specific to the specimen being studied. Thus in order to observe a diverse range of specimens a variety of filters and dichroic mirrors must be purchased. Further, these filters and mirrors require the use of special materials and significantly increase the cost of the microscope.
A known fluorescence stereomicroscope described in U.S. Pat. No. 6,040,940 involves passing light from the light source through an excitation filter to obtain an excitation wavelength. This excitation light is then shown directly on the specimen instead of being reflected off of a dichroic mirror and passed through the objective lens. The light fluoresced from the specimen is then collected through the objective lens where it is directed toward each eye. Before reaching each eye, the light collected is and passed through a barrier (absorbing) filter which absorbs the light from the excitation band, preventing it from being observed. This leaves just the fluorescence light to be observed by each eye.
Disadvantageously, this set up still requires purchasing costly filters. Further, it still requires purchasing a variety of costly filters to observe a diverse range of specimens.

BRIEF SUMMARY

In the illustrated embodiments of this invention, I have made a fluorescence microscope which can be used without the need for filters or dichroic mirrors. The light source is provided by a light emitting diode (LED). The LED shines directly on the specimen and is not passed through the objective lens first.
The LED is located adjacent to the objective lens with the objective lens and the LED opposing the specimen such that the excitation light is not transmitted through the specimen, but instead illuminates the surface of the specimen.
After the light from the specimen passes through the objective lens, the image of the specimen is directly observed, the light is not first passed through a filter. In a preferred embodiment the light is collected by a camera. One skilled in the art can readily select a camera which is suited to microscopy.
In a preferred embodiment the LED is not coupled with a filter, but instead excites the specimen with the full wavelength band of light it emits. In a more preferred embodiment, multiple LEDs are present and able to excite the specimen. In this preferred embodiment, the LEDs each emit light at different wavelengths and the LEDs can be controlled such that only a chosen excitation band is directed at the specimen at once.
In a preferred embodiment the image of the specimen is collected by a camera attached to a computer such that the image can be analyzed to obtain useful information.
In a preferred embodiment the angle of the incident, excitation, light with respect to the optical axis is minimized by selecting an objective lens with a longer working distance.
In some embodiments, a florescence microscope assembly for observing a specimen may include an objective lens assembly and/or a light source.
In some embodiments, the light source is a light emitting diode (LED), the light source with the objective lens assembly opposes the specimen, the light source is located off the optical axis of the objective lens assembly, and the light source illuminates the specimen when the specimen is located on the optical axis of the objective lens assembly.
In some embodiments, the objective lens assembly has a working distance of at least 2 mm.
In some embodiments, the objective lens assembly may include an amplification device with an amplification power of 40 and a working distance of 3.5 t0 3.7 mm.
In some embodiments, such a florescence microscope assembly may further include two anti-reflective transparent sheets spaced apart wherein they contain the specimen.
In some embodiments, the anti-reflective transparent sheets are formed of fused silica.
In some embodiments, such a florescence microscope assembly may further include a camera located on the optical axis of the objective lens assembly and optically coupled thereto.
In some embodiments, such a florescence microscope assembly may further include a computer.
In some embodiments, the camera is attached to the computer for data processing and analysis.
In some embodiments, the light emitting diode may further include an LED array of a group of LEDs having differing frequencies.
In some embodiments, such a florescence microscope assembly may further include a microcontroller coupled to the LED array.
In some embodiments, the LED array is controlled by a microcontroller.
In some embodiments, such a florescence microscope assembly may further include a light path without any light filters or dichroic mirrors.
In some embodiments, a method for performing florescence microscopy with an objective lens assembly may include exciting a specimen located with narrow band of light having a specific peak on an optical axis of a lens assembly using a light source, fluorescing light from the specimen to the objective lens in response to the excitation by the light source, passing light fluoresced from the specimen through the objective lens assembly, and/or observing the light passed through the objective lens.
In some embodiments, the light source is a light emitting diode (LED), the light source is off the optical axis, the light source and lens assembly oppose the specimen, and the light source illuminates the specimen when the specimen is located on the optical axis.
In some embodiments, where light fluoresced from the specimen is passed through the objective lens assembly, the method may include passing that light to the objective lens assembly across a working distance of at least 2 mm.
In some embodiments, where light fluoresced from the specimen is passed through the objective lens assembly, the method may include passing the light through an objective lens assembly having an amplification power of 40 and a working distance of 3.5-3.7 mm.
In some embodiments, such a method may further include placing the specimen between anti-reflective transparent sheets.
In some embodiments, where the specimen is placed between anti-reflective transparent sheets, the method may include placing the specimen on anti-reflective transparent sheets formed of fused silica.
In some embodiments, where the light passed through the objective lens assembly is observed, the method may include detecting the light passed through the objective lens assembly with a camera.
In some embodiments, the camera is connected to a computer for data processing and analysis.
In some embodiments, such a method may further include exciting the specimen again with a narrow band of light having a new and different specific peak, fluorescing light from the specimen to the objective lens in response to the new and different excitation wavelength, passing this new light light fluoresced from the specimen through the objective lens assembly, and/or observing this new light passed through the objective lens.
In some embodiments, a fluorescence microscope light source may include an LED, an LED mount, and an LED receptacle.
In some embodiments, the light emitting diode (LED) may include a light emitting diode circuit, a lens to focus emission of LED light, and/or an LED housing to hold the light emitting diode circuit and lens.
In some embodiments, the LED mount may include a tube and/or a flange attached to the tube at each side of the gap.
In some embodiments, the tube has a gap along its length, the internal diameter of the tube is sized to fit over a microscope objective, and the length of the tube is approximately the length of the microscope objective.
In some embodiments, an LED receptacle is attached to the tube.
In some embodiments, the LED receptacle is specially sized to correspond to the LED housing.
In some embodiments, the LED housing is held in the LED receptacle.
In some embodiments, the receptacle is adjustable so that the angle with respect to the axis of the tube can be adjusted such that the area of focus of the light coming from the LED is changed.
While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 USC 112, are not to be construed as necessarily limited in any way by the construction of “means” or “steps” limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 USC 112 are to be accorded full statutory equivalents under 35 USC 112. The disclosure can be better visualized by turning now to the following drawings wherein like elements are referenced by like numerals.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

To easily identify the discussion of any particular element or act, the most significant digit or digits in a reference number refer to the figure number in which that element is first introduced.

FIG. 1 illustrates the side and bottom of the subject matter in accordance with one embodiment of the invention.

FIG. 2 illustrates the side and bottom of FIG. 1 without the LED array.

FIG. 3 illustrates the front of the embodiment illustrated in FIG. 1 and also includes a specimen placed between anti-reflective transparent sheets and a computer.

FIG. 4 illustrates the light path of the fluorescence microscope in accordance with one embodiment. It shows a simplified cross-section of FIG. 3.

FIG. 5 contains a flowchart which illustrates the steps of a method for fluorescence microscopy in accordance with one embodiment of this invention.

FIG. 6 illustrates the fluorescence microscopy light source 102 of FIG. 1 without the remaining embodiment of FIG. 1 present as one embodiment of the invention.

DETAILED DESCRIPTION

FIG. 1 illustrates one embodiment of the invention. The light source 102 is attached to the fluorescence microscope assembly 100. The light source 102 holds one or more LEDs 110 which excites the fluorescence specimen. In this embodiment multiple LEDs 110 are used. The light source 102 is attached to the microscope via the objective lens assembly 104. The LED mount tube 112 is located coaxially around the objective lens assembly 104. LED mount tube 112 is a tube with a gap along its length. The LED mount tube 112 includes flanges 114 with holes 118 defined through them at each side of the gap. These flanges 114 can be forced apart or together to increase or decrease the size of the gap and thus the internal diameter of the LED mount tube 112, respectively. The light source 102 is seated on the outer surface of the objective lens assembly 104 by forcing the flanges 114 together with a load. In a preferred embodiment the load is applied by running a fastener through the holes 118 and the fastener down.
The objective lens assembly 104 is the device in the microscope which is the primary magnifier of the specimen. The objective lens assembly 104 has an input where light from the unmagnified specimen enters the objective lens assembly 104. After being magnified by the objective lens assembly, the light exits the output of the objective lens assembly into the microscope tube 108.
The microscope tube 108 is a round tube connected on one end to the objective lens assembly 104 and on the other end to the camera 106. It serves as a path for the light exiting the objective lens assembly 104 to travel through on its way to the camera 106 without interference from other, non-magnified, light.
The camera 106 is attached to the microscope tube 108 and collects the light which comes from the objective lens assembly 104. This serves to provide a user with a magnified image of the specimen.
The microscope mount 116 is composed of two half-circles capable of being attached to each other such that they create a circle having an internal diameter equivalent to the external diameter of the microscope tube 108. Mount 116 is then attached around the microscope tube 108. Radially attached to the half circles is the end of a rod. At the other end, the rod is attached to the center of a disk with holes for fasteners. This disk is can be attached to a stabilizing device. In this way the fluorescence microscope assembly 100 can be mounted to a stable surface and be effectively used.
FIG. 2 illustrates an embodiment of the invention. This illustration provides a view of the fluorescence microscope assembly 100 illustrated in FIG. 1. This is portrayed from the same viewpoint; however, in FIG. 2 the light source 102 from FIG. 1 is not illustrated. FIG. 2 better illustrates that in this embodiment the microscope tube 108 is coupled on one end the objective lens assembly 104 and on the other end to the camera 106. This coupling must be optically light-tight. Being coupled optically light-tight means that no light is able to leak through the coupling. Thus, the only light capable of entering the microscope tube 108 is that which passes through the objective lens assembly 104. This ensures that the camera 106 is optically coupled only to the objective lens assembly 104.
FIG. 3 illustrates a preferred embodiment of the invention in use. Here LEDs 110 provide light 304 to specimen 306. This light excites the specimen 306 as is standard in fluorescence microscopy. In response to this excitation light, the specimen 306 releases some fluorescence 308 as the excitation decays to a lower energy state. Some of this fluoresced light then magnified by the objective lens assembly 104 and passes into the microscope tube 108. From there it is collected as photographic data by the camera 106. This photographic data is passed to the computer 312 which provides for data analysis.
In a preferred embodiment the distance between the specimen 306 and the objective lens assembly 104 is at least 2 mm. This signifies that the light fluoresced from the specimen 308 travels at least 2 mm before it enters into the objective lens assembly 104. This is accomplished by employing an objective lens assembly 104 with a working distance of at least 2 mm. The working distance of an objective lens assembly is readily determined and understood by those skilled in the art.
In a preferred embodiment, more than one LED 110 makes up the light source 102. In a more preferred embodiment, these LEDs 110 each have a unique wavelength emission band.
The specimen 306 can be anything capable of being observed by fluorescence microscopy. When being observed, it is located on the optical axis of the objective lens assembly between two anti-reflective transparent sheets 310. In a preferred embodiment, the anti-reflective transparent sheets 310 are made of fused silica. The anti-reflective transparent sheets 310 can take several forms. For example, in one embodiment the sheets 310 are in the form of a microscope slide and cover slip. In another embodiment they form two sides of a flow cell. In any form, these transparent sheets 310 serve to contain the specimen for observation.
The computer 312 and camera 106 are capable of creating a data connection to transfer the photographic data collected by the camera 106 to the computer 312 for viewing or data analysis by a user.
The microscope mount 116 provides a mounting mechanism such that the fluorescence microscope assembly 100 can be stabilized for use. In a preferred embodiment, the specimen 306 and the anti-reflective transparent sheets 310 are mounted on an apparatus which can be moved along the optical axis to provide the user control over how far the specimen 306 is from the objective lens assembly 104. In a preferred embodiment, the microscope mount 116 is mounted on an apparatus which can be moved along the optical axis to provide user control over how far the specimen 306 is from the objective lens assembly 104.
FIG. 4 is a diagram of the light path of one embodiment of this invention in use. The light path is shown over a simplified cross section of the embodiment illustrated in FIG. 3. Light is first emitted by one or more LEDs 110 and directed at the specimen 306. In this embodiment the specimen is assumed to be suited to florescence microscopy. The excitation light 304 excites the specimen 306 causing it to fluoresce some light 308. The light coming from the specimen is then magnified by passing through the objective lens assembly 104. In this embodiment it is illustrated as a single convex lens. This magnified light 402 then is directed up the microscope tube 108 to the camera 106. A magnified image of the specimen 306 is then formed at the focal point of the camera 106. The point at which an image of the specimen 306 forms on focal point of the camera 106, a magnified image of the specimen 306 can be collected by the camera 106 for viewing or data analysis.
No dichroic mirrors or light filters are present in the light path of this embodiment of the invention.
FIG. 5 is a flowchart illustrating the steps for performing fluorescence microscopy in accordance with one embodiment of this invention. In a preferred embodiment, this illustrated method is performed on the fluorescence microscope assembly 100 illustrated in FIG. 3.
The first step 502 in this embodiment is to place the specimen 306 to be observed between anti-reflective transparent sheets 310. In a preferred embodiment the specimen 306 is known to be suited to the specific type of fluorescence microscopy which will be performed in the following steps of this embodiment of the invention. In other embodiments, it is unknown whether the specimen 306 will be suited to this type of fluorescence microscopy. In one embodiment the transparent glass sheets 310 between which the specimen 306 is placed are a microscope slide and coverslip. In a preferred embodiment, the transparent glass sheets 310 make up opposing sides of a flow cell. In a preferred embodiment regardless of the type of anti-reflective transparent sheets 310, the specimen 306 is placed between anti-reflective transparent sheets 310 made of fused silica.
The second step 504 in this embodiment is the step of exciting the specimen 306 with light from the fluorescence microscope's light source 102. In a preferred embodiment of this method the excitation radiation comes from an LED 110. This LED 110 is preferably located off of the optical axis of the objective lens assembly 104 and does not pass through the objective lens assembly 104 before exciting the specimen 306. Instead, the LED 110 and objective lens assembly 104 opposing the specimen 306. In such an embodiment they are located on the same side of the specimen 306 and are not arranged in such a way that the light from the light source 102 would have to pass through the specimen 306 before reaching the objective lens assembly 104.
The light is passed directly from the LED 110 to the specimen 306. As such, the light is not first passed through a light filter or dichroic mirror; similarly, the light is not reflected off of a dichroic mirror. In some embodiments, the light is passed through a focusing lens placed in front of the LED 110 to concentrate the light from the LED 110 on the specimen 306 before exciting the specimen 306; however, the lens transmits the full band of light emitted from the LED 110 and does not function as a wavelength filter. The light excites the specimen 306 as is standard in fluorescence microscopy and understood by those skilled in the art. In preferred embodiments the specimen 306 is located on the optical axis of the objective lens assembly 104 for ideal results.
In the third step 506 in this embodiment light is fluoresced from the specimen 306. The light fluoresced from the specimen 306 depends on the properties of specific specimen 306 being used in the method.
In the fourth step 508 in this embodiment the light fluoresced from the specimen 306 then travels up to and is passed through the objective lens assembly 104. The light must travel the distance from the specimen 306 to the objective lens assembly 104; when the specimen 306 is in focus this distance traversed is the working distance of the objective lens. The working distance of an objective lens assembly 104 is a property which can be readily understood and ascertained by a practitioner skilled in the art of fluorescence microscopy. In a preferred embodiment the light fluoresced from the specimen 306 when the specimen 306 is in focus must travel through a working distance of at least 2 mm before passing through the objective lens assembly 104. In another embodiment of the invention the light fluoresced from the specimen 306 when the specimen 306 is in focus must travel through a working distance of about 3.5-3.7 mm before passing through an objective lens assembly 104 with a magnification power of 40.
In the fifth step 510 in this embodiment the light fluoresced from the specimen 306 continues through the microscope to a camera 106 where it is detected. In a preferred embodiment the camera 106 has a connection with a computer 312 capable of transferring data to the computer 312.
In a preferred embodiment, further steps are performed in this method. These further steps include first collecting the light detected by the camera 106 as photographic data and then passing the data through a data connection to the computer 312. In a preferred embodiment the data is then analyzed on the computer 312 by a user or automated program loaded on the computer 312.
In a preferred embodiment the embodiment illustrated in FIG. 5 is performed with a light source 102 having multiple LEDs 110 with different wavelength emission bands. In that case, the preferred embodiment contains additional steps. The method illustrated in FIG. 5 is repeated with a different LED 110 having a different wavelength band as the light source 102 from the excitation step 504 through the rest of the method. In a preferred embodiment the method is performed in an automated fashion by controlling the LEDs 110 with a microcontroller 312.
In a preferred embodiment the embodiment illustrated in FIG. 5 is repeated from the excitation step 504 where the entire specimen 306 was not observable by the camera 106. Before repeating the illustrated steps, either the specimen 306 or the objective lens assembly 104 is moved into a new position where the specimen 306 has not yet been observed. In a preferred embodiment, the new positions may be preselected and the movements of either the specimen 306 or the objective lens assembly 104 automated.
In an embodiment, several images of the specimen 306 are obtained at the same place after being excited with different wavelength bands from different LEDs. In this embodiment either the specimen 306 or the objective lens assembly 104 is moved and several images of a new area of the specimen 306 are obtained. This may be repeated several times to obtain many photographic images of the fluorescence of the specimen 306 from different excitations at many different places across the specimen 306.
FIG. 6 illustrates a fluorescence microscopy light source 102 as one embodiment of the invention. This is used as the light source in the embodiments illustrated in FIG. 1 and FIG. 3. In this embodiment light source 102 contains multiple LEDs 110. Each LED 110 contains a light emitting diode circuit protected inside an LED housing 610. The light emitting diode circuit emits the light in a narrow band of visible radiation. The light emitted is then passed through a lens 612 to allow the light to be focused onto a specimen 306. In this illustration the light emitting diode circuit is not visible as it is covered up by the housing 610 and lens 612, but the terminals of the circuit 614 are shown. Light emitting diode circuits are well know in the prior art.
The LEDs 110 are positioned within the LED receptacle 602. This contains individual receptacles sized to that of the LEDs 110 being used such that the LED 110 fits snugly into the receptacle 602 and is held in place by friction. Alternatively, the LED 110 is held firmly in the receptacle 602 with glue. In a preferred embodiment, the LED 110 is not held snugly into the receptacle 602, but instead can move easily. In this preferred embodiment the LED housing 610 has pins coming out of each side which fit into holes in each side of the receptacle 602. This allows the LED 110 to pivot within the receptacle 610 such that the angle relative to the optical axis is changeable. Alternatively, the LED 110 is held by adjustable arms within which the LED 110 can pivot. The arms can be attached to the LED mount tube 112; the arms also able to pivot, with respect to the axis of the LED mount tube 112. The light source 102 is then, therefore, able to hold the LED 110 at an angle desired by the user.
The LED receptacles 602 are connected to the LED mount tube 112. This is a tube with a gap 616 along its length. The LED mount tube 112 includes flanges 114 with holes 118 defined through them at each side of the gap 616. These flanges 114 can be forced apart or together to increase or decrease the size of the gap 616 and thus the internal diameter of the LED mount tube 112, respectively. In this embodiment the LED mount tube 112 is sized such that when the flanges 114 are forced apart, the LED mount tube 112 will fit easily over the objective lens assembly 104 of a fluorescence microscope, but when forced together it will be held firmly in place around the objective lens assembly 104. In a preferred embodiment the light source 102 can be held in place over an objective lens assembly 104 by applying a load to the flanges 114 forcing them closer together.
In a preferred embodiment, application of this load can be accomplished by running a fastener through the flanges 114 via holes 118, forcing the flanges 114 toward each other. In another embodiment this can be accomplished with a clamp providing the load across the flanges 114. In another embodiment this can be accomplished by a restorative force of LED mount tube 112. In the embodiment which uses a restorative force the LED mount tube 112 can be sized so that when no force is applied, the internal diameter of the tube is smaller than the external diameter of the objective lens assembly 104 around which the light source 102 will be placed. Thus, to place the light source 102 over the objective lens assembly 104, the flanges 114 must be forced away from each other. When the tube 112 is made of a hard but flexible material which returns to its initial shape after a force is applied to it, the internal forces of the tube 112 will apply a load to the flanges 114 forcing them toward each other.
In a preferred embodiment each LED 110 is connected to a microcontroller 606 via leads 608 connected to the terminals 614 of the light emitting diode circuit. In another preferred embodiment each LED 110 is connected to a computer via the leads 608 connected to the terminals 614 of the light emitting diode circuit. In this way, an individual LED 110 can be turned off or turned on in a controlled or automated fashion.
In a preferred embodiment this fluorescence microscopy light source 102 can be used to convert most standard microscopes to a fluorescence microscope with little to no other modifications. In a preferred embodiment a standard microscope having an objective lens assembly 104 with a working distance of at least 2 mm is converted into a fluorescence microscope by placing the fluorescence microscopy light source 102 illustrated in FIG. 6 around the objective lens assembly 104 of the microscope. In a more preferred embodiment a standard reflective microscope having an objective lens assembly 104 with a working distance of at least 2 mm and an opaque stage is converted into a fluorescence microscope by placing the fluorescence microscopy light source 102 illustrated in FIG. 6 around the objective lens assembly 104 of the reflective microscope. To reduce the autofluorescence of a standard, reflective microscope converted to a fluorescence microscope, fused silica can be exchanged for the standard glass used in the microscope.
Many alterations and modifications may be made by those having ordinary skill in the art without departing from the spirit and scope of the embodiments. Therefore, it must be understood that the illustrated embodiment has been set forth only for the purposes of example and that it should not be taken as limiting the embodiments as defined by the following embodiments and its various embodiments.
Therefore, it must be understood that the illustrated embodiment has been set forth only for the purposes of example and that it should not be taken as limiting the embodiments as defined by the following claims. For example, notwithstanding the fact that the elements of a claim are set forth below in a certain combination, it must be expressly understood that the embodiments includes other combinations of fewer, more or different elements, which are disclosed in above even when not initially claimed in such combinations. A teaching that two elements are combined in a claimed combination is further to be understood as also allowing for a claimed combination in which the two elements are not combined with each other, but may be used alone or combined in other combinations. The excision of any disclosed element of the embodiments is explicitly contemplated as within the scope of the embodiments.
The words used in this specification to describe the various embodiments are to be understood not only in the sense of their commonly defined meanings, but to include by special definition in this specification structure, material or acts beyond the scope of the commonly defined meanings. Thus if an element can be understood in the context of this specification as including more than one meaning, then its use in a claim must be understood as being generic to all possible meanings supported by the specification and by the word itself.
The definitions of the words or elements of the following claims are, therefore, defined in this specification to include not only the combination of elements which are literally set forth, but all equivalent structure, material or acts for performing substantially the same function in substantially the same way to obtain substantially the same result. In this sense it is therefore contemplated that an equivalent substitution of two or more elements may be made for any one of the elements in the claims below or that a single element may be substituted for two or more elements in a claim. Although elements may be described above as acting in certain combinations and even initially claimed as such, it is to be expressly understood that one or more elements from a claimed combination can in some cases be excised from the combination and that the claimed combination may be directed to a subcombination or variation of a subcombination.
Insubstantial changes from the claimed subject matter as viewed by a person with ordinary skill in the art, now known or later devised, are expressly contemplated as being equivalently within the scope of the claims. Therefore, obvious substitutions now or later known to one with ordinary skill in the art are defined to be within the scope of the defined elements.
The claims are thus to be understood to include what is specifically illustrated and described above, what is conceptionally equivalent, what can be obviously substituted and also what essentially incorporates the essential idea of the embodiments.

Claims

What is claimed is:

1. A florescence microscope assembly for observing a specimen comprising:

an objective lens assembly having an optical axis; and

a light source, wherein the light source is a light emitting diode (LED), the light source with the objective lens assembly opposing the specimen, the light source being positioned off the optical axis of the objective lens assembly, and the light source illuminating the specimen when the specimen is located on the optical axis of the objective lens assembly.

2. The florescence microscope assembly of claim 1, wherein the objective lens assembly has a working distance of at least 2 mm.

3. The florescence microscope assembly of claim 1 wherein the objective lens assembly comprises

an amplification device with an amplification power of 40 and a working distance of 3.5 t0 3.7 mm.

4. The florescence microscope assembly of claim 1, further comprising two anti-reflective transparent sheets spaced apart whereby they contain the specimen.

5. The florescence microscope assembly of claim 4, wherein the anti-reflective transparent sheets are formed of fused silica.

6. The florescence microscope assembly of claim 1, further comprising

a camera located on the optical axis of the objective lens assembly and optically coupled thereto.

7. The florescence microscope assembly of claim 6, further comprising

a computer, wherein the camera is attached to the computer for data processing and analysis.

8. The florescence microscope assembly of claim 1, wherein the light emitting diode further comprises

an LED array of a plurality of LEDs having differing frequencies.

9. The florescence microscope assembly of claim 8 further comprising

a microcontroller coupled to the LED array, wherein the LED array is controlled by the microcontroller.

10. The florescence microscope assembly of claim 1, further comprising

a light path without any light filters or dichroic mirrors.

11. A method for performing florescence microscopy with an objective lens assembly comprising:

exciting a specimen located with narrow band of light having a specific peak within an optical axis of a lens assembly using a light source, wherein the light source is a light emitting diode (LED); the light source is off the optical axis; the light source and objective lens assembly oppose the specimen; and the light source illuminates the specimen when the specimen is located on the optical axis;

fluorescing light from the specimen to the objective lens in response to the excitation by the light source;

passing light fluoresced from the specimen through the objective lens assembly; and

observing the light passed through the objective lens.

12. The method of claim 11, where passing light fluoresced from the specimen through the objective lens assembly comprises passing the light to the objective lens assembly across a working distance of at least 2 mm.

13. The method of claim 11, where passing light fluoresced from the specimen through the objective lens assembly comprises passing the light through an objective lens assembly having an amplification power of 40 and a working distance of 3.5-3.7 mm.

14. The method of claim 11, further comprising

placing the specimen between anti-reflective transparent sheets.

15. The method of claim 14, where placing the specimen between anti-reflective transparent sheets comprises placing the specimen on anti-reflective transparent sheets formed of fused silica.

16. The method of claim 11, where observing the light passed through the objective lens assembly comprises detecting the light passed through the objective lens assembly with a camera wherein the camera is connected to a computer for data processing and analysis.

17. The method of claim 11, further comprising:

exciting the specimen again with a narrow band of light having a new and different specific peak;

fluorescing light from the specimen to the objective lens in response to the new and different excitation wavelength;

passing this new light light fluoresced from the specimen through the objective lens assembly; and

observing this new light passed through the objective lens.

18. A fluorescence microscope light source comprising

a light emitting diode (LED) comprising:

a light emitting diode circuit; and

an LED housing wherein the LED housing holds the light emitting diode circuit;

an LED mount comprising:

an LED mount tube wherein the tube has a gap along its length, the internal diameter of the tube is sized to fit over a microscope objective, and the length of the tube is approximately the length of the microscope objective; and

a flange attached to the tube at each side of the gap; and

an LED receptacle attached to the tube specially sized to correspond to the LED housing wherein the LED housing is held in the LED receptacle.

19. The fluorescence microscope light source of claim 18, the LED further comprising

a lens wherein the lens is held in front of the light emitting diode circuit by the LED housing to focus emission of LED light from the light emitting diode circuit.

20. The fluorescence microscope light source of claim 18 wherein the LED receptacle is adjustable such that the angle with respect to the axis of the tube can be adjusted such that the area of focus of the light produced by the light emitting diode circuit is changed.