WO1996037761A1

WO1996037761A1 - Multi-channel acquisition using integrating sphere

Info

Publication number: WO1996037761A1
Application number: PCT/US1996/006808
Authority: WO
Inventors: David L. Tomei
Original assignee: Lxr Biotechnology Inc.
Priority date: 1995-05-26
Filing date: 1996-05-13
Publication date: 1996-11-28
Also published as: EP0775296A1; JPH10503597A; CA2198779A1; AU5744896A

Abstract

The present invention is a novel apparatus that employs an integrating sphere (62) as a source of diffused light for multiple PMTs (64). The PMTs (64) are coupled to the integrating sphere (62) to receive diffused light from the integrating sphere (62). An optional faceplate (66) may be employed at the input port of the integrating sphere (62) in order to select certain characteristics of the light according to the characteristics of the faceplate (66).

Description

MϋLTI-CHANNEL ACQUISITION USING INTEGRATING SPHERE

FIELD OF THE INVENTION The present invention relates in general to a method and apparatus for the multi-channel acquisition of image data and, in particular, to the multi-channel acquisition of image data using an integrating sphere.

BACKGROUND OF THE INVENTION

In the field of image processing, there is a need to acquire image data from multiple channels simultaneously. For example, a double antibody labeling protocol generally requires two channels of information. However, a single channel provides only a single, monochromatic image at a given time. To obtain information provided by any other wavelength, another image needs to be acquired. This will not only double the acquisition time for a single channel system, but sequential image acquisition cannot resolve the time dependencies that are sometimes present when imaging a sample having dynamic aspects. For many protocols, these correlation problems cannot be resolved. Thus, imaging different wavelength simultaneously is one way to avoid these problems.

Simultaneous imaging in multiple colors may be accomplished by detecting each color separately, where each color image provides distinct information. The information content from the different color images depend on the interactions of the sample with the excitation wavelengths. An example is multiple fluorescent labeling. Each fluorescent label has a different absorption and emission wavelength. Attachment of such label to antibodies for preferential labeling of specific sites will yield multiple information simultaneously in a single image.

In the art, it is known to use beam splitters to simultaneously acquire multi-channel data. Figure 1 depicts a typical beam splitting imaging system. Sample 10 is illuminated with a focused beam 12 of light at an area of interest on the sample. Forward light (i.e. light that either passes through the sample unaltered or light that is scattered forward and through the sample after interaction with particles in the sample) is transmitted to a beam splitter - depicted in Figure 1 as dielectric mirror 14.

The simplest function of a beam splitter is to divide the beam amplitude into two separate beams. Beam splitter are generally one of two types - plate or cube splitters. Plate splitters (such as dielectric mirror 14) are more efficient than cube splitters but are exposed to the environment making them less reliable. Plate splitters are 1 mm crown glass plates with a surface coated with a thin dielectric film. Cube splitters are a pair of identical right angle prisms glued together on their hypotenuse faces. The hypotenuse faces are coated with the dielectric film.

In both types of splitters, the primary design of wavelength bandwidth and splitter ratio is determined by this dielectric film. The ability to obtain equal apportionment is wavelength dependent. Beam splitters have a wider effective band (i.e. covering the visible spectrum) and have higher absorption, thus lowering the signal seen at the PMTs by that amount. This reduction in efficiency is detrimental to the signal-to-noise ratio (S/N) of the image. Returning to Figure 1, mirror 14 splits the input beam into two constituents - beams 16 and 18 respectively. Beam 16 is reflected to another mirror 20 which in turn redirects the beam to a filter 22 and a first photomultiplier tube (PMT) 24 for subsequent detection. Beam 18 passes through the dielectric mirror 14 and onto a filter 26 and a second PMT 28 for detection.

Once these two beams have been detected by PMTs 24 and 28, the data is acquired from these PMTs. The PMTs are typically biased to be in the linear region. The output of the PMTs is proportional to the intensity of incidental light. The filters in front of the PMTs help to extract desired information according to the characteristics of the filters. For example, if the filters are simple color filters, the PMTs will detect color information according to the color of the filters.

One of the drawbacks with acquiring multi¬ channel image data with beam splitters is that it is difficult to maintain uniformity in a multi-channel beam splitter with the number of channels greater than two. The main reason is that uniformity of the PMT signals depends on the lens or diffuser system that couples light into the particular PMT. These independent lens or diffuser systems for each PMT add additional cost to the overall system. Additionally, the alignment of multiple PMTs gets more difficult as the number of channels increases.

Additionally, the intensities of the two beams after splitting are determined by the physical characteristics of the splitter. The splitter can be obtained in different output ratios. The exact ratio value needs to be known in order to extract quantitative information from the acquired images. ithout such information, a multicolor image display, for example, will give a false color tone due to the misrepresentation of the color intensities. A standard procedure to balance the beam splitter is to place a polarizing plate prior to the beam splitter. This plate will linearly polarize the incident beam. Used in conjunction with a polarizing broad beam splitter, the half wave plate can be rotated until a 50/50 ratio is obtained. Placing a polarizing plate in the optical path will reduce the incidence intensity, thus reducing the S/N.

If three colors are needed, the beam splitting arrangement gets more complicated. One possible arrangement is using a 33/67 ratio beam splitter first, followed by a 50/50 beam splitter for the 67% split beam. Calibration would need to be done for all three beams and PMTs. For more colors, the arrangements can get considerably more complicated; and alignment of such arrangement gets considerably more difficult. Thus, there is a need for to acquire multi¬ channel image data such that the problems of uniformity and calibration are avoided. There is also a need for a method to achieve multi-channel image data acquisition without the additional expense of using independent optical systems for each PMT.

SUMMARY OF THE INVENTION Other features and advantages of the present invention will be apparent from the following description of the preferred embodiments, and from the claims.

The present invention is a novel apparatus that employs a integrating sphere as a source of diffused light for multiple PMTs. The PMTs are coupled to the integrating sphere to receive diffused light from the integrating sphere. An optional faceplate may be employed at the input port of the integrating sphere in order to select certain characteristics of the light according to the characteristics of the faceplate.

One advantage of the present invention is that the integrating sphere homogenizes the incident beam and produces a uniform output shared equally among multiple PMTs.

Another advantage is that, with use of an integrating sphere, optical path length calibration and intensities calibration are not needed.

Yet another advantage is cost. The present invention requires only one optical path for the light to reach the various PMTs. Additionally, because PMTs require being physically housed in a light tight environment, the integrating sphere of the present invention provides a single light tight environment for the various PMTs.

For a full understanding of the present invention, reference should now be made to the following detailed description of the preferred embodiments of the invention and to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts one method for acquiring multi-channel image data by use of a conventional beam splitter.

Figure 2 is an high level block diagram of the laser imaging system that utilizes the apparatus integrating sphere. Figure 3 is a cut-away view of a multi-channel imaging system using an integrating sphere.

DETAILED DESCRIPTION OF THE INVENTION As mentioned above, the need for acquiring multi-channel image data arises in a number of imaging applications and systems. However, prior art imaging systems do not provide multi-channel data that avoid the problems specified above. One system, typical in the art, is described in U.S. Patent 5,037,207 issued to Tomei et al. on August 6, 1991 entitled "LASER IMAGING SYSTEM", which is hereby incorporated by reference. Figure 2 is a block diagram of the conventional laser imaging system 30 as shown in Tomei et al. A primary laser 32 provides a beam 34 to a beam expander 36 composed of an objective lens and a spatial filter. Beam 34 exits the beam expander 36 as collimated beam 38. A three dimensional beam position controller 40 receives collimated beam 38. The beam controller 40 includes an imaging lens and galvanometrically driven mirrors to provide control of the spot focus on sample target 42.

Forward light is captured by the detector assembly 44. Detector assembly 44 comprises an optical fiber faceplate 46, diffusion elements 48 and a photomultiplier tube 50. The image signal produced by tube 50 is subsequently sent to a support computer system 52 which further processes the image signal for display on a high resolution monitor 54 or for storage in an image storage unit 56 for later playback. Further details concerning the overall construction and operation of the laser imaging system are provided in the above- incorporated, Tomei et al. patent.

A single channel imaging system, built on the design shown in Figure 2, could be re-designed for multi¬ channel data acquisition. By simply replacing detector assembly 44 in Figure 2 with the assembly shown in Figure 3, a multi-channel imaging system is easily designed and built. Referring now to Figure 3, a cut-away view of multi-channel imaging system 60 using an integrating sphere having multiple output ports is given. Integrating spheres with a single port are generally known in the art and are made available through Melles Griot or other optical companies. Imaging system 60 of the present invention, however, comprises integrating sphere 62 that has a plurality of PMTs 64. PMTs 64 are coupled (e.g. by a threaded screw arrangement or any other light-tight coupling means known in the art) to integrating sphere 62 with the detection face of the PMTs inside the sphere. The coupling is such that it provides a light tight coupling to integrating sphere 62. It will be appreciated that any type of light measuring device that can be light-tightly coupled to the body of the integrating sphere will suffice for the purposes of the present invention; including, but not limited to: spectrometers, coupled-charge devices, or the like. At the top of integrating sphere 62 is a light input port comprising a fiberoptic faceplate 66. Depending on the design of fiberoptic faceplate 66, the faceplate can simply diffuse the incident light or it can exclude scatter above or below a scattering angle. Faceplate 66 acts as a pre-filter and only one is sufficient on the input end of the integrating sphere.

Integrating sphere 62 provides a uniform output of light from the non-uniform source input. As seen in Figure 3, the incident light 68 impinges upon the sample target 70 and the forward scattered light 72 from that interaction is transmitted into the integrating sphere through faceplate 66. That light enters integrating sphere 62 and reflects generally off the bottom of the sphere. An integrating sphere typically uses a non- specular, high reflectance coating 74 on the interior surface. As a result, the light rays are reflected to a very high degree. A typical light ray may reflect off the interior surface a great number of times before it finds its way into the input of a PMT. At the input port of the PMT, optional filters 76 may be employed to discriminate the input light by wavelength or some other characteristic selected by a suitable filter. The use of these filters enables the acquisition of multiple channel data from various wavelengths or other characteristics. It will be appreciated that other optical elements could be used to provide suitable wavelength selection, such as prisms, diffraction gratings and the like. There has thus been shown and described a novel apparatus that uses an integrating sphere with multiple PMTs that allow for multi-channel light analysis that may analyze each channel according to some independent characteristic. As stated above, many changes, modifications, variations and other uses and applications of the subject invention will, however, become apparent to those skilled in the art after considering this specification and accompanying drawings which disclose preferred embodiments thereof. All such changes, modifications, variations and other uses and applications which do not depart from the spirit and scope of the invention are deemed to be covered by the invention which is limited only by the claims which follow.

Claims

IN THE CLAIMS :

1. In a light imaging system comprising a light source, a means of focusing said light source on a sample target; en apparatus for the multi-channel collection of uniform light output derived from a non- uniform light input transmitted by said sample target, said apparatus comprising: an integrating sphere, said sphere having a light input port and a plurality of output ports; a plurality of light measuring devices, such that at least one light measuring device is coupled to one of said plurality of output ports.

2. The apparatus as recited in claim 1 wherein said light measuring device is a photomultiplier tube.

3. The apparatus as recited in claim 1 wherein said coupling of the plurality of light measuring devices is light-tight.

4. The apparatus as recited in claim 1 wherein said coupling is a threaded screw arrangement.