WO2000013574A1 - Intravital fluorescence and luminescence monitoring microinstrument and method of using same - Google Patents

Abstract

This invention is a microinstrument, and method for measuring fluorescence, luminescence or other light emitted or reflected by cells, tissues, organs, tumors or other structures or substances inside or outside the body of a living or dead organism, or in cell or organ cultures. The microinstrument includes a detector (68) for detecting light emitted or reflected by cells and producing a signal indicative of light intensity, an analog to digital converter (93) for converting the signal from the detector into a digital signal indicative of light intensity, and a transmitter for transmitting the digital signal to a data processing system. The detector, analog to digital converter, and transmitter may be mounted to a base portion (74) of the device of sufficient size to be inserted within a subject's vasculature. In addition, the microinstrument may be integrated with a catheter.

Description

INTRA VITAL FLUORESCENCE AND LUMINESCENCE MONITORING MICROINSTRUMENT AND METHOD OF USING SAME

CROSS-REFERENCE TO RELATED PROVISIONAL APPLICATION

The present application claims the benefit of the earlier filing date of U.S.

Provisional Patent Application Serial No. 60/098,827, filed September 2, 1998, which is

incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to a device and method for measuring light

emitted or reflected by cells, tissues, organs, tumors or other structures or substances inside or

outside a living or dead organism, or in cell or organ cultures. In particular, the present invention relates to a microinstrument, and method of using the same, that may be inserted into a blood vessel or other body space to monitor in vivo the success of a transfection or

infection process, or the presence of any light emitting or reflecting substance.

BACKGROUND OF THE INVENTION

Transfection and infection protocols are widely used to introduce genetic

materials into cells of living organisms for research or therapeutic purposes, e^, gene therapy, or diagnostic purposes. Typically, to assess the efficacy of such procedures, reporter

genes such as Green Fluorescent Protein ("GFP"), luciferase, beta-galactosidase, and

chloramphenicol acetyltransferase, to name a few, are introduced at the same time as

therapeutic or diagnostic genes. The intensity of the light emitted or reflected by such

reporter genes provides an indication of whether or not the introduction of the therapeutic or

diagnostic genetic materials was successful. However, quantitatively detecting such expression of the reporter genes has proven to be challenging.

For example, U.S. Patent No. 5,419,323 to Kittrell, et al, which is

incorporated by reference herein in its entirety, discloses a method for irradiating tissue in vivo to detect atherosclerotic lesions in a vessel wall. The Kittrell, et al. method requires

transmitting fluorescence light emitted by the tissue through a fiber optic cable to a location

outside the body where it can be analyzed. One significant problem with systems such as this

is that substantial light attenuation occurs in the fiber optic lightguide. In addition, transporting the light outside the body results in the introduction of noise. Combined with the fact that the light emission is very small to begin with, it is apparent that such attenuation and noise seriously erode measurement sensitivity. Similar problems are encountered by systems

such as that disclosed in U.S. Patent No. 5,438,985 to Essen-Moller, which is drawn to a gastric probe for monitoring pH by detecting fluorescence in vivo and evaluating such

fluorescence using a data processing system outside the body. Thus, there is a need for a system for detecting and conditioning in vivo any light emitted or reflected by cells, tissues,

organs, tumors or other structures or substances inside or outside a living or dead organism,

or in cell or organ cultures, and particularly such a system which provides improved

measurement sensitivity.

Yet another problem with devices such as those disclosed by Kittrell, et al. and

Essen-Moller is that they are bulky, expensive to produce, and are not amenable to use as a

one-use, disposable product. Thus, there is a need for a system for detecting and conditioning

in vivo any light emitted or reflected by cells, tissues, organs, tumors or other structures or

substances inside or outside a living or dead organism, or in cell or organ cultures which is simple and inexpensive to produce, such that it is viable as a one-use product.

Accordingly, it is an object of the present invention to provide a new device for detecting in vivo any light emitted or reflected by cells, tissues, organs, tumors or other structures or substances inside or outside a living or dead organism, or in cell or organ cultures.

It is another object of the present invention to provide such a device that

comprises an integrated light detection and signal generating and conditioning device.

It is another object of the present invention to provide such a device that provides improved measurement sensitivity. It is yet another object of the present invention to provide such a device that is

simple and inexpensive to produce, such that it is viable as a one-use product. SUMMARY OF THE INVENTION

In accordance with the principles of the present invention, a device and

method are provided for measuring fluorescence, luminescence or other light emitted or reflected by cells, tissues, organs, tumors or other structures or substances inside or outside

the body of a living or dead organism, or in cell or organ cultures. The device of the present

invention includes a means for detecting light emitted or reflected by cells or tissues and

producing a signal indicative of the intensity of the light, and a means for transmitting the signal to a data processing system which may be, but need not be, a part of the device. Where

the signal is an analog signal, the device of the present invention also includes a means for

converting the analog signal into a digital signal indicative of light intensity. Each of these

means may be mounted to a base portion of the device, which is preferably sufficiently small that it may be inserted within an organism, such as a human body.

The means for detecting light may be any type of photodetection device, such as a photodiode, phototransistor, semiconductor metal diode, or array of one or more types of

such devices. Similarly, the means for converting the analog signal into a digital signal may be any type of analog-to-digital ("A/D") converter, and the means for transmitting the digital

signal to a data processing system may include any type of serial output driver.

In one embodiment of the light intensity detection device of the present invention, the photodetection device is particularly adapted to achieve dual-emission wavelength optical sensing using a single excitation light wavelength. This embodiment is

intended to be particularly useful where background cells or tissues produce fluorescence or luminescence, and the fluorescent or luminescent emission spectra of marker molecules

(whether inherently present or introduced by a clinician for therapeutic, diagnostic or other

purposes) and background differ sufficiently to readily distinguish between them. In this embodiment, the photodetection device has a first photodetector and a second photodetector. The first photodetector detects a first band of light passing through a first optical filter and

produces a first analog signal indicative of the intensity of the first band of light. The second

photodetector detects a second band of light passing through a second optical filter and

produces a second analog signal indicative of the intensity of the second band of light. The wavelengths comprising the first band of light are determined by the first optical filter, and

may include, for example, emission spectra of a marker such as GFP, luciferase or any other

marker molecule. The wavelengths comprising the second band of light are determined by

the second optical filter and may include, for example, emission spectra of background cells or tissues around the location where the first band of light was detected. The band of

excitation light used preferably has a range of wavelengths corresponding to a strong

excitation wavelength of the marker molecules being used, but may have a range of wavelengths corresponding to an area of overlap in the excitation spectra of the marker

molecules and the that of the background cells or tissues.

The signal conversion device may include a first switched capacitor bandpass filter corresponding to the first analog signal, and a second switched capacitor bandpass filter

corresponding to the second analog signal, a multiplexer that selectively receives the respective first and second analog signals from the first and second switched capacitor

bandpass filters, and an A/D converter. By this configuration, the signal conversion device produces a first digital signal indicative of the intensity of the first band of light and a second

digital signal indicative of the intensity of the second band of light.

This embodiment of the light intensity detection device of the present

invention may be utilized in many applications, such as to measure the intensity of a

fluorescent or luminescent marker at a site of interest, e_^g., in the body of a human or animal.

One way to employ the device in such an application where a fluorescing marker is used, once the marker has been placed within the site of interest, is to introduce a band of excitation

light to the site of interest, then filter the resulting emission light using the first optical filter and detect the intensity of the emission light using the first photodetector. Either the resulting

emission light or emission light resulting from a subsequent or simultaneous introduction of

the band of excitation light to the site of interest is filtered using the second optical filter, and

the intensity of that light detected using the second photodetector. The two intensity measurements may then be compared.

In another embodiment particularly adapted to measuring fluorescence, and in separating the background fluorescence of cells or tissues from that of marker molecules,

dual-wavelength excitation may be achieved by alternating the wavelength of light used to excite fluorescing marker molecules and background cells or tissues. For each of the two

excitation light wavelengths used, a single photodetection device is used to measure light

intensity. The range of wavelengths of the first excitation light band preferably are selected to correspond to a strong peak excitation wavelength of the marker being used, whereas the

range of wavelengths of the second excitation light band preferably are selected to correspond

to a strong peak emission wavelength of the background cells or tissues at the site of interest.

An optical filter positioned in front of the photodetection device is designed to pass a first band of emission light at a first range of wavelengths, and reject a second band of emission light at a second range of wavelengths. The first range of wavelengths is selected at a point

of overlap in the emission spectra of the marker molecules and background cells or tissues.

For each measurement taken corresponding to the two excitation wavelengths, the photodetection device detects the first band of light passing through the optical filter and

produces an analog signal indicative of the intensity of that light.

A signal conversion device connected to the photodetection device converts

the analog signal into a digital signal indicative of light intensity. The signal conversion

device of this embodiment may include, for example, a switched capacitor bandpass filter and

an A/D converter. Alternatively, it may comprise a trans-impedance amplifier and an A/D

converter. A signal transmission device receives the digital signal from the signal conversion device and transmits the digital signal to a data processing system. Preferably, the light

intensity detection device has a base portion to which the photodetection device, signal

conversion device, and signal transmission device are mounted, the base portion being sufficiently small that the device may be inserted within a body. More preferable still, the

components and base portion of the light intensity detection device are of such a size as to be inserted within a blood vessel of a human body.

An exemplary use of this embodiment is as a device for measuring the light

intensity of a transfected fluorescent marker such as E-GFP™ in order to determine the success of a therapeutic gene transfection procedure (e.g.. to inhibit the growth of scar tissue

after balloon angioplasty) in a human blood vessel. In this exemplary embodiment, the base portion of the light intensity detection device may be an integrated circuit chip on which the

optical filter, photodetection device and signal conversion device are integrated. The integrated circuit chip may be manufactured using a bipolar, FET, CMOS, BiCMOS or other applicable process, but in the present embodiment is manufactured using a MOSIS AMI

CMOS 1.2 μm process. The integrated circuit chip preferably has a size of about 2.2 mm x

2.2 mm or smaller, and most preferably about 0.5 mm x 2 mm or smaller. The optical filter may be a high or low pass or bandpass filter which is separate from, or attached directly or

indirectly to, the integrated circuit chip, but for purposes of this embodiment preferably is a

high wavelength-pass filter attached to a thin sheet of glass which is in turn attached to the

integrated circuit chip.

The photodetection device may comprise, for example, a photodiode,

phototransistor, or semiconductor metal diode in order to obtain a discrete intensity indication at a site of interest. The photodetection device may also comprise an array of one or more

types of such devices corresponding to an array of optical filters in order to create a map of a

site of interest. In this exemplary embodiment, the photodiode chosen is an N-diffusion/P- base junction photodiode having a width that is about 0.5 mm or smaller.

In this embodiment, the signal conversion device includes an A/D converter, which may be any type of A D converter, such as a digital-to-analog-based A/D converter, a counter-ramp converter, a tracking converter, a successive approximation converter or a flash

converter. The A D converter chosen for this embodiment is an 8-bit, dual-slope converter, preferably with a size that is about 0.45 mm x 1.95 mm or less. The A/D converter comprises an analog portion and a digital portion. The analog portion has an integrator, a comparator,

an analog multiplexer and an analog switch, and the digital portion has a control unit, an a 8-

bit counter, a serial output driver and a clock generator. The signal transmission device in

this embodiment is a serial output driver, which transmits digital data to the data processing

system.

The data processing system may include any type of data receiving, storing,

analyzing and displaying hardware or software, and preferably includes data storage and display hardware, as well as digital signal processing software which performs spectral

analysis of the digital signal and calculates the amplitude of the emitted or reflected light to

determine a value of light intensity.

In using this embodiment to measure the intensity of a marker at a site of

interest, for example, a first band of excitation light having a first range of wavelengths may

be introduced at that site and the intensity of the resulting emission measured using the device. A second band of excitation light having a second range of wavelengths may then be introduced to the site and the device used to measure the intensity of the resulting emission.

The two values may be compared by the data processing system to eliminate or reduce the noise in the signal and determine accurately the intensity of the light emission from the marker molecules.

Using any of the foregoing embodiments, one alternative method for

measuring the intensity of luminescent or fluorescent light emitted by a marker, instead of by using dual excitation light wavelengths or dual emission filters and detectors, is by simply

repositioning the light intensity detection device between measurements. In particular, the

light intensity detection device may be first positioned at a first location proximal to the marker molecules at the site of interest. Then, if a luminescing marker is used, the device is used to obtain a measurement of light intensity at that location. If a fluorescent marker is

used, a band of excitation light is introduced to the site of interest, and the device is used to measure the intensity of the resulting light emission. The device may then be repositioned to

a second location proximal to the background cells or tissues (and away from the location of

the marker molecules) at the site of interest, and the excitation light introduced for a second

time. The light intensity detection device may be used to measure the intensity of light emitted in response to the excitation light at this second location, and the two measurements

may be compared by the data processing system to determine the intensity of the marker

molecules alone.

The light intensity detection device of the present invention is ideally suited

for use in conducting research or diagnostic or therapeutic procedures using the microinstrument. For example, the microinstrument of the present invention may be used to evaluate in vivo the success of a gene transfection or infection procedure. In such an

application, marker molecules are placed at a site of interest in a body cavity, blood vessel or

other part of a body. The microinstrument of the present invention may be inserted into the body and transported to the site of interest, e^g., by mounting the device to the distal portion

of a balloon-type catheter, or to another suitable device. The microinstrument may then be used to measure the intensity of the light emitted by the marker molecules and the

background cells or tissues at the site of interest.

The light intensity measuring microinstrument of the present invention therefore overcomes one of the most significant shortcomings of prior systems by its ability to detect in situ the presence of specific markers in cells of human blood vessels, tissues,

tumors, organs, body cavities, as well as cell culture containers, for example, where competing background fluorescence is present, and to configure that data in situ as a digital signal instead of piping the light detected to a location outside the body or region of interest.

In addition, the attributes of the dual-wavelength embodiment of the present invention

overcome the limitations of single wavelength detection methods by enabling quantitative

analysis of relative degrees of transfection using low light fluorescence methods.

The foregoing and other features, objects and advantages of the present

invention will be apparent from the following detailed description, taken in connection with

the accompanying figures, the scope of the invention being set forth in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic illustration of the microinstrument of the present invention.

FIG. 2 is a schematic illustration of another embodiment of the microinstrument of the present invention.

FIG. 3 is a schematic illustration of yet another embodiment of the microinstrument of the present invention.

FIG. 4 is a schematic illustration of a particular embodiment of the microinstrument of FIG. 3.

FIG. 5a is a circuit diagram of the microinstrument of FIG. 4.

FIG. 5b is a layout diagram of the microinstrument of FIG. 4.

FIG. 6 is a graph illustrating the transmission of an optical filter as a function of wavelength of light.

FIG. 7 is a cross-sectional diagram of a photodiode which may be used in the

microinstrument of the present invention.

FIG. 8 is a circuit diagram of an operational amplifier which may be used in

the microinstrument of the present invention.

FIG. 9 is a circuit diagram of an A/D converter which may be used in the

microinstrument of the present invention.

FIG. 10 is a graph illustrating the integrator output waveform of the A/D

converter of FIG. 9.

FIG. 11 is a circuit diagram of an analog switch which may be used in the A/D

converter of FIG. 9. FIG. 12 is a circuit diagram of a multiplexer which may be used in the A/D converter of FIG. 9.

FIG. 13a is a circuit diagram of a finite state machine which may be used in the analog-to digital converter of FIG. 9.

FIG. 13b is a schematic illustration of the operation of the finite state machine of FIG. 13a.

FIG. 14 is a circuit diagram of the combination logic circuit of the A/D converter of FIG. 9.

FIG. 15 is a circuit diagram of an 8-bit counter which may be used in the A/D converter of FIG. 9.

FIG. 16a is a circuit diagram of a serial output driver which may be used in the microinstrument of the present invention.

FIG. 16b is a circuit diagram of a multiplexer which may be used in the serial

output driver of FIG. 16a.

FIG. 16c is a circuit diagram of a 4-bit counter which may be used in the serial

output driver of FIG. 16a.

FIG. 17 is a circuit diagram of a clock generator which may be used in the

microinstrument of the present invention.

FIG. 18 is a schematic illustration of an embodiment of the present invention

in which one embodiment of the microinstrument of the present invention has been integrated

into a balloon catheter.

FIG. 19 is a graph of the response curves of five different diodes which may be used in connection with the microinstrument of the present invention.

FIG. 20 is a graph of the quantum efficiency curves of the five diodes of FIG. 19.

DETAILED DESCRIPTION OF THE INVENTION

The device of the present invention comprises a microinstrument 10 which

may be used for measuring light emitted or reflected by cells or tissues. Microinstrument 10 comprises a means for detecting light being emitted or reflected by cells or tissues and

producing a signal indicative of the intensity of the detected light, and means for transmitting the signal to a data processing system. The signal produced by the light detecting means may be analog or digital, and where it is analog, microinstrument 10 will also comprise means for

converting the analog signal into a digital signal. For purposes of convenience only, and not intending to limit the breadth of the present invention, the various embodiments of the present

invention described herein will be discussed as including such a signal conversion means. Once produced, and after being converted to digital if applicable, the signal is then analyzed

by the data processing system to produce a result useful to the user (e.g.. diagnostician,

clinician, research scientist, etc.). Each of the light detection means, signal conversion means

(if used) and signal transmission means of microinstrument 10 is integrated into

microinstrument 10 such that microinstrument 10 is of a sufficiently small size to be inserted

into a cell culture, body cavity or, most preferably, a vein or artery.

As schematically illustrated in FIG. 1, microinstrument 10 comprises a base

portion 11, such as a microchip, to which the light detection means, signal conversion means

and signal transmission means are mounted. The light detection means of microinstrument 10 may be any photodetection device 12 known in the art for detecting fluorescence,

luminescence or other light 13, and that is capable of being implemented on a micro scale.

Similarly, the signal conversion means may be any signal conversion device 16 capable of receiving data from photodetection device 12 and converting it to a digital signal, and the

signal transmission means may be any signal transmission device 18 that is capable of

transmitting the resulting digital signal to a data processing system 19, and that is capable of being implemented on a micro-scale. Although shown in the embodiment of FIG. 1 as being

located apart from base portion 11, data processing system 19, or any part of that system,

alternatively may be attached or integral to base portion 11.

FIG. 2 schematically illustrates one embodiment of the present invention, in which microinstrument 10 is particularly adapted to achieve dual-emission wavelength optical sensing using a single excitation light wavelength and two optical filters having

different wavelength-pass characteristics. This embodiment is intended to be useful

particularly where non-fluorescent or luminescent markers are used, or when the background

cells or tissues emit sufficient amounts of fluorescence or luminescence to provide a reliable means for discrimination between the marker and background. In this embodiment, the light

detection means comprises first and second optical filters 20 and 22 selected to narrow the range of light wavelengths to which respective first and second photodetectors 24 and 26 are

exposed. As will be appreciated, first and second optical filters 20 and 22 may be any type of

filter adequate to the purpose, such as high or low-pass filters or bandpass filters amendable

to use in an integrated circuit environment. First optical filter 20 and first photodetector 24

correspond to a first signal conversion means including a first switched capacitor bandpass filter and gain 28. Second optical filter 22 and second photodetector 26 correspond to a

second signal conversion means including a second switched capacitor bandpass filter and gain 32. Both first and second signal conversion means (if used) include multiplexer 36,

rectifier and gain 37, and A/D converter 38. The signal transmission means of

microinstrument 10 of FIG. 2 includes a serial output or port driver 40, which may be part of

A/D converter 38. Control logic circuit 42 controls the circuit in a conventional manner.

In this embodiment, fluorescing, luminescing or other marker molecules may

be deposited in an organism or cell culture, as is known in the art, at a specified transfection or infection location. Where a non-luminescing marker is used, a modulated light source is

used to introduce excitation light of a particular wavelength band to the marker molecules and surrounding cells or tissues. The excitation light wavelength preferably is selected to have a

range of wavelengths corresponding to a strong excitation wavelength of the marker molecules being used, and where the emission spectra of the marker molecules and

background cells or tissues are sufficiently distinct. For example, where E-GFP™

(manufactured by Clonetech of Palo Alto, California) is used for the marker and the background tissues at the site of interest are vascular wall tissues in a human blood vessel, a wavelength band including a wavelength between about 450 nm and about 488 nm is suitable

for the excitation light wavelength. Accordingly, the excitation light causes emission from

the marker molecules at a first wavelength, and from the background cells or tissues at a

second wavelength. First optical filter 20 is designed to pass light at the first wavelength, permitting first photodetector 24 to sense the marker molecules' emission. Photodetector 24

may comprise any type of photodetection device such as, for example, a photodiode, phototransistor, semiconductor metal diode or an array of one or more types of such devices

corresponding to optical filter 20. Photodetector 24 transmits a respective analog current, which is then converted to a first voltage signal and amplified by first switched capacitor

bandpass filter and gain 28. Similarly, second optical filter 22 is designed to pass light at the

second wavelength, permitting second photodetector 26, which also may be a photodiode, to sense the marker molecules' emission. Photodetector 26 transmits a respective analog

current, which is then converted to a second voltage signal and amplified by second switched capacitor bandpass filter and gain 32. Multiplexer 36 selectively and rapidly channels the

first and second voltage signals, permitting the measurement of light intensities quasi-

simultaneously. After multiplexer 36, first and second voltage signals are amplified by rectifier and gain 37, then are converted into digital voltage signals by A/D converter 38. The resulting digital signals 41 are transmitted to a data processing system 44 by serial port driver 40, where they are stored. Once the first and second digital voltage signals have been

recorded by data processing system 44, data processing system 44 may compare them and display a corresponding output (e.g.. by visual display, electronic printer, or other appropriate

device).

Another embodiment of the present invention is schematically illustrated in

FIG. 3, in which microinstrument 10 is particularly adapted to achieve dual- wavelength

fluorescence excitation by alternating the wavelength of light used to excite fluorescing

marker molecules and background cells or tissues. In contrast to the embodiment of FIG. 2,

this embodiment is intended to be useful particularly where excitation spectra of marker

molecules and of background fluorescence are sufficiently different to permit detection of one or both components using two excitation wavelengths. In this embodiment, the light

detection means comprises a photodetector 46 and an optical filter 48 selected to narrow the

range of light to which photodetector 46 is exposed, the signal conversion means includes

switched capacitor bandpass filter and gain 50, rectifier and gain 52, and A/D converter 54, and the signal transmission means includes serial port driver 56, which may be a separate unit or part of A/D converter 54. Control logic circuit 58 controls the circuit in a conventional

manner. Marker molecules are deposited as is known in the art at a specified transfection or infection location. The marker molecules and surrounding cells or tissues are exposed to a

first wavelength of light which preferably corresponds to a strong excitation wavelength of

the marker molecules, and to as weak an excitation wavelength of the background cells or tissues as possible. As in each of the embodiments of the present invention presented here, the excitation light may be modulated at 100-300 Hz or any other frequency that is required

or desirable. The resulting first emission wavelength passes through optical filter 48, which is designed to pass a certain emission wavelength or range of such wavelengths and to reject

the wavelength of the excitation light used, and is sensed by photodetector 46, which may be

a photodiode. Photodetector 46 transmits a respective analog current, which is then converted to a voltage signal and amplified by switched capacitor bandpass filter and gain 50,

which ensures that only the signal close to the modulating frequency can be amplified and go

to rectifier and gain 52. Thus, most of the dark current of photodetector 46 which is near

direct current ("DC") is eliminated, and all other out-of-band noise is rejected. Following the

rectifier and gain 52, the positive part of the signal is integrated and digitized by A/D

converter 54. The resulting digital signal 59 is transmitted to a data processing system 62 by serial port driver 60, where it is stored.

Before, after or simultaneously with the introduction of the first wavelength of

light, the marker molecules and surrounding cells or tissues are exposed to a second

wavelength of light preferably corresponding to a strong excitation wavelength of the background cells or tissues, and to as weak an excitation wavelength as possible of the marker molecules. Optical filter 48 and photodetector 46 are selected to pass and detect light

at a overlapping emission wavelength of the marker molecules and background cells or

tissues. Accordingly, the resulting second emission wavelength is sensed by photodetector 46

and processed by microinstrument 10 as previously described in connection with the first emission wavelength. Once the first and second emission wavelengths have been recorded by data processing system 62, data processing system 62 may compare them and provide a corresponding output (e_^g., by visual display, electronic printer, or other appropriate device).

As will be appreciated by those of ordinary skill in the art, the embodiments of FIGS. 2 and 3 may be combined to further improve the reliability of marker identification

where both excitation and emission spectra of marker molecules and background cells or

tissues differ to a usable extent. In addition, where it is desired to produce a broader image or

map of an organ, body cavity or other sample of tissue or cells, the embodiment of

microinstrument 10 of FIG. 2 may be modified to utilize a plurality (perhaps thousands) of

sets of first and second optical filters 20 and 22 and first and second photodetectors 24 and

26, and the embodiment of microinstrument 10 of FIG. 3 may similarly be modified to utilize

an array of optical filters 48 and photo detectors 46. In contrast to conventional devices,

microinstrument 10 is totally self-contained, capable of being very small in size and highly portable and reliable, virtually noise immune, stable, and inexpensive to manufacture (it is

estimated that the particular embodiment disclosed in reference to FIG. 4 may be manufactured for less than $1 apiece).

The embodiments of FIGS. 2-3 address situations in which background fluorescence or luminescence is relevant. It will be appreciated, however, that in the simplest

case, where background fluorescence or luminescence is not appreciable or otherwise of concern, luminescence or fluorescence may be detected by an embodiment of the microinstrument of the present invention using a single wavelength and a single signal

channel. The embodiment of FIG. 3 is particularly amenable to such an application, wherein,

instead of switching the wavelength of the excitation light between light intensity measurements, a single excitation light band such as that used in connection with the embodiment of FIG. 2 may be used and the position of the microinstrument changed between

measurements. For example, the position of the microinstrument during the first measurement may be proximal to the location of the marker molecules at the site of interest,

and the position of the microinstrument during the second measurement would then be

proximal to the background cells or tissues at that site.

Whether used in a dual or single-wavelength application, the microinstrument

10 of FIG. 3 may alternatively be implemented as illustrated in FIG. 4, in which the switched

capacitor and bandpass filter is implemented by circuitry including a trans-impedance

amplifier. In FIG. 4, microinstrument 10 includes a base portion 64 to which is attached light

detection means including at least one optical filter 66 and a corresponding photodiode 68,

and signal conversion means including a trans-impedance amplifier 69, a voltage amplifier 70 and an A/D converter 72. In this embodiment, microinstrument 10 is particularly designed

for measuring light 71 emitted from E-GFP™ (S65T-GFP with a strong emission peak

wavelength at about 514 nm) at a site of interest in the vascular wall tissue of a human blood vessel (one way to determine the emission wavelength of this background tissue is to measure

intensity at a peak E-GFP™ emission wavelength, then back off from that peak to, e^, 540

nm and obtaining another measurement). It will be appreciated, however, that the design of microinstrument 10 of FIG. 4 is readily modifiable for use in connection with any GFP

mutant, or any other fluorescence, luminescence, or other light source.

Base portion 11 (FIG. 1) comprises an integrated circuit ("IC") chip 74 (FIG.

4) that is generally preferably small enough to be used safely in a catheter-based system for

use in blood vessels, or in other in vivo applications. For use in the former type of application, a size for IC chip 74 of about 0.5 mm to about 2 mm by about 0.5 mm is desirable. The IC chip 74 of the present embodiment, as shown in FIG. 5b, has dimensions of about 2.2 mm x 2.2 mm. To help IC chip 74 perform the function of a multiple wavelength

fluorescence excitation measurement device useful for measuring the intensity of E-GFP™, which has a peak excitation wavelength of about 396 nm and a peak emission wavelength of

about 514 nm, at a site on the vascular wall of a human blood vessel, optical filter 66

comprises a multi-layer thin film interference long-wave-pass filter that is cemented to IC

chip 74 and designed to transmit nearly 100% of the light being emitted or reflected by the

cells or tissues under observation at a wavelength of 510 nm (i.e., greater than 95%

transmission for wavelengths greater than 460 nm), while blocking excitation light (indeed,

all wavelengths of light) below about 420 nm. The excitation light is modulated sinusoidally at 250 Hz, resulting in similar modulation of the fluorescence light of the E-GFP™.

Photodiode 68 detects the fluorescence light transmits a 250 Hz sinusoidal signal which is

proportional or otherwise related to the fluorescence light intensity. The photocurrent signal is then converted into a voltage signal by trans-impedance amplifier 69, and the voltage signal is further amplified by voltage amplifier 70. The A/D converter 72 integrates the voltage

signal from voltage amplifier 70 preferably until the maximum signal strength available from

the chosen photodiode has been achieved (about 25.6 μs if no deviations are made form the particular embodiment described here), and converts the integrated voltage to digital data. The digital data 75 is then transmitted serially by a serial data port driver 60 (FIG. 9), which

may be part of A/D converter 72, to a data processing system 76 (which may be external to or attached to or integral with IC chip 74) to be analyzed by a digital signal processing program,

as is well-known in the art. The digital signal processing program extracts the amplitude of the sinusoidal signal, which provides a quantification of light intensity. For example, where microinstrument 10 is used in gene therapy applications, the amplitude of the sinusoidal

signal provides a direct indication of the extent of gene expression.

One embodiment of IC chip 74 of FIG. 4 and its components is illustrated by

the circuit diagram of FIG. 5a, in which R, = 5 kΩ, R₂ = 100 Ω, R₃ = 10 kΩ, R₄ = 500 Ω, R_f =

100 kΩ and C = 78 pF. This embodiment of IC chip 74 is also illustrated in the layout

diagram of FIG. 5b. The IC chip 74 of FIGS. 5a and 5b was produced through a standard

MOSIS AMI CMOS 1.2 μm process, although it will be appreciated that another

microelectronics process could be used, such as a bipolar, FET or BiCMOS process, or

another CMOS process. As is known in the art, optical filter 66 may be made as a discrete unit apart from IC chip 74, or may be deposited directly on the surface of IC chip 74 or on any optically transparent media that may then be attached to IC chip 74 using cement, epoxy

or another adhesive. In present embodiment, it is constructed on glass 0.1 mm thick and cemented on IC chip 74. Once the filter is formed on the glass, the glass may be cut to size and cemented onto IC chip 74. Direct deposition of the optical filter on the surface of IC chip

74 is believed to be advantageous for mass production.

In the present embodiment, as can be appreciated from FIG. 6, optical filter 66 of FIGS. 4 and 5 is a high- wavelength-pass interference filter designed to reject wavelengths

of light below about 420 nm and to transmit wavelengths of about 510 nm. In IC chip 74 of

the present embodiment, the ratio of rejection to transmittance is approximately 10,000.

Photodiode 68 detects light passing through optical filter 66 and generates a photocurrent. In the particular embodiment of FIGS. 4 and 5, wherein photodiode 68 is a single photodiode, the photocurrent generated is approximately proportional to light intensity.

As will be understood by persons having ordinary skill in the art, photodiode 68 of IC chip 74 should be designed for optimal performance considering the particular design of IC chip 74

and the characteristics of the light to be measured. The number of photodiodes to be used in IC chip 74 also necessarily depends upon the application being considered. Given the design

of IC chip 74 of FIGS. 4 and 5, the selection of photodiode 68 of this embodiment is based on

testing results obtained with five different pn junctions. Of these, photodiode 68 preferably

comprises the N-diffusion/P-base junction, since this was observed to have the greatest

response in the region of interest. A cross-sectional diagram of photodiode 68 is provided in

FIG. 7. Although the size of photodiode 68 preferably is small enough to fit in a vein or artery along with IC chip 74 (e_^g., a width of 0.5 mm or less), in this embodiment it is about 0.25 mm x 0.25 mm.

Trans-impedance amplifier 69 receives a current signal from photodiode 68 and converts the current signal into a voltage signal. The voltage signal is then amplified by voltage amplifier 70. As illustrated in FIGS. 5a and 5b, trans-impedance amplifier 69 and

voltage amplifier 70 each comprise an amplifier 78. Amplifier 78 may be any amplifier device, such as an operational amplifier, voltage amplifier, current amplifier, trans-impedance

amplifier, charge-pump amplifier, or any other class of amplifier. In the present embodiment,

however, amplifier 78 is an operational amplifier including a bias circuit, an input stage, and an output stage.

As can be seen in the circuit diagram of FIG. 8, the bias circuit of operational amplifier 78 includes MOSFET Ml 3, Ml 2, Ml 1 and integrated poly-resistor R_b. The integrated poly-resistor R_b is connected between the node BIAS and the ground to set the

operating currents of devices through current mirrors M13-M12 and M13-M11. For example,

setting R_b = 14.1 kΩ sets the operating current of M12 to 100 μA and that of Ml 1 to 150 μA.

If there are several operational amplifiers in IC chip 74, the bias resistors comprising R_b can be combined to save die area. In IC chip 74 of FIGS. 4 and 5, two bias resistors of 7.05 kΩ

are used instead of four 14.1 kΩ resistors. One of them is for the operational amplifiers in

trans-impedance amplifier 69 and voltage amplifier 70, and the other is for the integrator and

comparator in A/D converter 72. In single power supply applications, R_b can only be

connected from node BIAS to power supply V_ss, or Ground equivalently. However, in dual-

power supply applications such as IC chip 74 of the present embodiment, the other end of resistor R_b can be connected either to power supply V_ss or Ground. The latter case results in a small bias resistor value, which is favorable for an on-chip bias-resistor.

The input stage of operational amplifier 78 consists of source-coupled pair M1-M2, current mirror M3-M4 as active load, and bias current mirror M12-M13. The operating current of Ml 2 is set to 100 μA, such that the currents in Ml, M2, M3 and M4 are

all 50 μA. Under this bias condition, the gate to source voltage V_gs of transistor M3 is about 1.4V, which will make the DC voltage at node N5 about -3.6V.

The output stage from M5 to Ml 1 is a push-pull, inverting amplifier working in the class AB mode. The DC voltages at nodes N4 and N5 are both about -3.6 V. By the

level shifter circuit comprising M5, M7, M8, M9 and Ml 1, the DC voltage at node N7 is

about 3.1 V. This arrangement results in both output NMOS and PMOS transistors working at the optimal operating points, and the quiescent current of the output stage is small. Thus, the power conversion efficiency of the output stage is improved significantly compared with

the case in which the node voltages of N5 and N7 have the same DC voltage. The bias

current of Ml 1 is about 150 μA, which is divided into the bias currents of M5 and M7. When the voltage of N5 becomes positive, the current of M5 is increased and that of M7 is

decreased. The decreased M7 current is current-mirrored to that of M8, increasing the

voltage of node N7. The careful sizing of M5, M7, M8 and M9 results in the AC voltage at

node N5 being almost the same as that at node N7.

When there is no load resistor, using small-signal analysis, the voltage gain of

the output stage is A2 = (g_m6 + g_ml0) / (g_o6 + g_ol0) = 440, where g_m6 and g_ml0 are the trans-

conductance of M6 and Ml 0, respectively, and g_o6 and g_ol0 are the output conductance of M6 and Ml 0, respectively. Combining this with the input stage, the total voltage gain of the

operational amplifier is about 120 K, or 110 dB. The highest output voltage where the

operational amplifier is still linear is V (N7) + V_Tp = 3.95 V, and the lowest voltage is V(N5)

- ^vτ_n ⁼ -4-3 V. Out of this region, either M6 or M10 will be in the triode region.

The output resistance of the output stage is fairly high with a value R₀= r_o6/

r_ol0 = 110 kΩ, where r_o6 and r_ol0 are the output resistances of M6 and M10, respectively. In

some applications, such as driving small load resistors or large capacitors where small output resistance is required, it may be desirable to place a low output resistance configuration

source follower may after this inverting amplifier stage.

When there is a load resistor R_L connected between V_out and Ground, the bias conditions make the output current I_L = 0 A, and the output voltage V_out = 0 V when no AC

signal is applied on node N5. If the voltage at node N5 is perturbed in the positive direction, the current of M6 is increased and that of Ml 0 is decreased. Accordingly, the load current is sunk through M6 and the maximum value is 910 μA, which is the DC current of M6. On the

other hand, when the voltage is perturbed in the negative direction, M10 will source the load

current with the maximum source current 910 μA, which is the quiescent current of M10. The linear output voltage swing will be from - I_LR_L to I_LR_L if this region is smaller than the

region from - 4.3 V to 3.95 V. Using a small signal model, the gain of the output stage at the

center of the voltage swing is (g_m6 + g_ml0)R_L, which is much smaller than the case without the

load resistor. The 4.88 pF capacitor C and 1.5 kΩ resistor R_c are for compensation to

eliminate oscillation when there is a feedback in the operational amplifier configuration.

All the large transistors of operational amplifier 78, including Ml, M2, M6 and M10, are composed of several smaller transistors in parallel to reduce the parasitic

resistance of polysilicon gate. Transistors Ml and M2 are laid out in the common centroid

fashion to minimize the effect of process variations. N+ and P+ guard rings are used widely

to provide good contacts from n-well to power source V_dd and from p-substrate to power

source V_ss, as well as to prevent cross talk from substrate to the signal and controllers. The compensation capacitor is separated into Cl and C2 to facilitate the tailoring of compensation for different feedback factors. For example, the rightmost capacitor may easily be removed

when operational amplifier 78 is used as a comparator in A/D converter 72, as discussed below.

A/D converter 72 of IC chip 74 may be any type of A D converter. For

example, it may be a digital-to-analog-based A/D converter, a counter-ramp converter, a tracking converter, a successive approximation converter or a flash converter. In the

embodiment of FIGS. 4 and 5, an 8-bit, dual-slope A/D converter was chosen for A/D converter 72 because of its small die area (less than 0.45 mm x 1.95 mm in the embodiment

of FIG. 8), simplicity, high precision and relatively low-cost. A schematic representation of A/D converter 72 of FIGS 4 and 5 is shown in FIG. 9. The A/D converter 72 comprises an

analog portion 91 and a digital portion 93. The analog portion includes an integrator 61, a

comparator 63, an analog multiplexer 73 and an analog switch 88. The digital portion of A/D

converter 72 includes a control unit 80, an 8-bit counter 82, a serial port driver 60 and an

internal clock generator 77 with a frequency of 10 MHz. As illustrated by the waveform of

the integrator output shown in FIG. 10, the A/D converter 72 works three successive stages:

signal integration SI, reference integration RI, and data transmission and reset DT. In the SI stage, control unit 80 connects the input signal V, to the integrator input and activates counter 82. The integrator integrates input signal V, for 256 clock cycles and the voltage output V₀ decreases as shown in FIG. 10, with a slope proportional to the

input signal until counter 82 overflows. The voltage change of the integrator output during

this stage is ΔV₀ = (V, / RC) x 256T_clk, where V, is the input voltage, T_clk is the period of the clock, and R and C are the integrator resistor 99 and the integrator capacitor 86, respectively.

In the RI stage, control unit 80 connects the reference voltage V_r to the integrator input and again starts counter 82. Because at the end of the previous stage counter 82 stops at zero after overflowing, counter 82 does not need to be reset. The polarity of V_r is

opposite to the polarity of V_j5 so the output of the integrator increases with a fixed slope until

the integrator output voltage V₀ crosses over zero, and as a result the comparator output becomes active and control unit 80 stops counter 82 at count N. The voltage change of the

integrator output during this second stage is ΔV₀ = (V_r / RC) x N x T_clk. This voltage change should be equal to that of the first stage, thus N = 256 (V, / V_r). The digital word or count N

is proportional to the input voltage V, and is the output of A/D converter 72.

In the DT stage, control unit 80 sends a transmission enable signal 84 to the

serial port driver 60, and closes switch 88 to discharge all residue charges in the integrator

capacitor 86. The transmission process takes the 16 clock cycles counted by an internal 4-bit

counter 114 in serial port driver 60. In the first two clock cycles high bits are sent as start

bits, followed by 8 bits of data 83 sent within 8 clock cycles, then 5 bits of high bits as stop

bits. Finally, the output line is pulled down to the low value as the idle state until the

beginning of next data transmission and reset stage, and the transmission-finish signal 89 is asserted. The 8-bit counter is then reset and the system moves on to another analog to digital

conversion. Evident from this description is the fact that the conversion accuracy of dual-

slope A/D converter 72 is independent of R, C and T elk. As long as these quantities remain stable over the conversion period, they affect the two integration stages equally so that long-

term drifts are automatically eliminated. The A/D converter 72 also offers an excellent integral linearity and resolution as well as virtual zero differential non-linearity. Resolution,

within the limits of integrator and comparator performance, depends on the counter length,

which can be improved conveniently. The differential non-linearity is virtually zero since the integrator output is a continuous monotonic function. In addition, since A/D converter 72

integrates the input signal for a fixed time, it provides excellent noise rejection, and the sample-and-hold circuits are not necessary.

In the analog portion of A/D converter 72, the integrator RC time constant is set to 25 μs, which is close to the time interval of the first stage. Integrator capacitor 86 is a 100 pF double-poly capacitor and integrator resistor 99 is a 250 kΩ first layer polysilicon

resistor. The comparator is a less compensated operational amplifier used in the open-loop

mode. The comparator has a slew-rate 100 V/μs and a response time 50 μs, which causes an

A/D converter offset error 'Λ LSB. The analog switch 88 of the analog portion of A/D

converter 72, illustrated in FIG. 11 comprises a transmission gate consisting of PMOS Ml

and NMOS M3 and an inverter to generate the opposite polarity control signals for the gates

of Ml and M3. The analog multiplexer 73 of the analog portion of A/D converter 72 is

illustrated in the schematic of FIG. 12. It consists of three analog switches 90, 92 and 94 and

a decoder circuit to generate control signals for switches 90, 92 and 94. In the DT stage, when the output of the finite state machine q,q₀ = 00, switch 90 is on and the V_zero is

connected to the output. In the SI and RI stages, when q,q₀ = 01 or 11, respectively, V, or V_ref are connected to the output, respectively.

In the digital portion of A/D converter 72, control unit 80 is used to monitor and control all parts of A/D converter 72. Control unit 80 includes a finite state machine 96

and a combinational logic circuit 98. Schematic illustrations of finite state machine 96 and combinational logic circuit 98 are shown in FIGS. 13 and 14, respectively. The two D-flip-

flops 100 and 102 in finite state machine 96 of FIG. 13a have four states, as illustrated in FIG. 13b. The three states q,q₀ = 01, 11, 00 correspond to the three A/D conversion stages (Le,, SI,

RI and DT stages). The state q,q₀ = 10 is unused, and finite state machine 96 can go to state

q,q₀ = 00 from it if state machine 96 happens to be in it during power-on. When the system is powered on, finite state machine 96 will begin with state q,q₀ = 00 and the data transmitted in

this DT stage should be discarded.

Referring to FIGS. 13a, 13b and 14, finite state machine 96 toggles state when

one of the following conditions is met: a. During the RI stage, the comparator output zero-pass is asserted or the

counter overflows (see counter overflow 79, FIG. 9). The latter means that input signal is

too large and is out of range. b. During the SI stage, the counter overflows.

c. During the DT, the transmission-finish signal 89 is active.

d. When finite state machine 96 falls into the unused q,q₀ = 10 stage

unexpectedly. In FIG. 14, D-flip-flop 100 in the zero-pass path is used to synchronize the zero-pass signal 101, which is from the comparator in FIG. 13a, and is an asynchronous

signal. The counter-enable signal 85 is used to gate 8-bit counter 82 and is asserted when q₀ = 1 (Le., in the SI and RI) stage. The trans-enable signal 84 is used for serial output driver 60

to enable its clock 81 and is asserted only in the q,q₀ = 00, (Le., the DT) stage. The counter

reset signal 87 is used to reset the 8-bit counter when the data transmission is completed.

The 8-bit counter 82 of the digital portion of A/D converter 72 is shown

schematically in FIG. 15. Generally, counter 82 should be designed to minimize die area. In the present embodiment, 8-bit counter 82, like many asynchronous counters, includes eight

one-bit counters cascaded with each of the one-bit counter's clock controlled by the previous counter output. The first 1-bit counter 104 toggles every clock. The second counter 106 toggles once every two clocks when the output of first counter 104 is zero (negative output).

In the same way, the third counter 108 toggles once every four clocks, fourth counter 110 toggles once every eight clocks, and so on. All the 1 -bit counters toggle at the same time.

The toggling time is one gate delay after the clock rising edge if it is assumed that the gates

controlling the clock (inverter for the first 1-bit counter 104, NOR for the other seven) have

the same propagation delays - this is actually a synchronous counter. When the results of all

the eight 1-bit counters are zero, the overflow signal is asserted.

Serial port or output driver 60 is used to transmit parallel digital bits in a serial

fashion. As schematically illustrated in FIG. 16a, it includes a 16-1 multiplexer 112, 4-bit

counter 114, and other circuits. Figure 16c is a circuit diagram for 4-bit counter 114. The 4-

bit counter 114 consists of four one-bit counters 107, 109, 111 and 113, and is very similar to 8-bit counter 82.

For easy understanding, a simplified version of 16-1 multiplexer 112 is provided in FIG. 16b. When s₃s₂s,s₀ = 000 in serial output driver 60, the output is equal to

the first input d₀; when s₃s₂s,s₀ = 0001, the output is equal to the second input d,; and so on.

When the DT stage begins, trans-enable signal 84 becomes low and the OR gate is open to the clock signal 81. The 4-bit counter 114 begins with s₃s₂s,s₀ = 0000 and increases by 1 at each rising edge of clock. When s₃s₂s,s₀ = 1111, trans-finish signal 89 is asserted and at the

next rising clock edge control unit 80 (FIG. 9) disables trans-enable signal 84. The OR gate

115 is then closed and the counter stops at s₃s₂s,s₀ = 0000. The D flip-flop latch 119 at the output of multiplexer 73 at falling edge of the clock eliminates the races caused by the combinational circuit transition. While 4-bit counter 114 counts from s₃s₂s,s₀ = 0000 to

s₃s₂s,s₀ = 1111 and to s₃s₂s,s₀ = 0000 again, serial output driver 60 sends 2 bits high as start bits, then 8 bits data from the most significant bit to least significant bit, then 5 bits high as stop bits. Then the output line is frozen at low until the next data transmission stage begins.

The clock generator 77 of the digital portion of A/D converter 72,

schematically illustrated in FIG. 17, is based on a 5-stage ring oscillator. The five capacitors

118, 120, 122, 124 and 126 at the output of each stage are used to slow down and adjust the

oscillation frequency to about 10 MHz. In the embodiment of FIG. 17, each of these

capacitors has a value of 2.5pF. The two inverters 128 and 130 after the ring oscillator 117

make the rising and falling edge steep. All the inverters in FIG. 17 have PMOS transistors

with W/L = 7.2/1.2 and NMOS transistors W/L = 2.4/1.2.

As discussed previously, a data processing system 76 is used to receive, store and analyze the digital data transmitted serially by serial data port 60. In the present

embodiment, data processing system 76 is located externally of IC chip 74. The data processing system 76 includes digital signal processing software and computer storage and

display hardware. It preferably also includes on-line, real-time analysis functions. If

necessary, off-line analysis software may be included. The software should be able to complete spectral analysis for the sampled data from microinstrument 10 and measure the

amplitude of the modulating signal to determine light intensity.

The microinstrument and method of the present invention is useful for

measuring any light emitted or reflected by (or transmitted through), cells, tissues, organs, tumors or other structures (e.g., gall stones) or substances inside or outside a living or dead organism, or in cell or organ cultures. For example, microinstrument 10 may be incorporated into a catheter for intravascular, extravascular or endoscopic applications. One such

application is to monitor the gene transfection ameliorating restenosis after balloon

angioplasty. Balloon angioplasty procedures are commonly used to relieve blockage in blood

vessels that will result in critical medical conditions. As illustrated in FIG. 18, during the angioplasty procedure a catheter-based balloon 132 typically is inflated using a balloon pump

133 and used to remove the blockage at vessel wall 134. As frequently occurs, the subsequent formation of scar tissue at vessel wall 134 can render the procedure ineffective.

Therapeutic genes whose products reduce the scarring may be directly introduced into the

cells of the blockage site during the ballooning procedure. This delivery occurs via a gene

transfection or viral infection process, or the like. Although the quantitative success of the

transfection has not heretofore been amenable of monitoring m vivo, the present invention permits such monitoring.

In particular, microinstrument 10 of the present invention may be mounted at the distal end of angioplasty catheter 136 (e.g., in or around the balloon 132) and used to transport microinstrument 10 to a site S of interest in a blood vessel 138 (or another vessel or

body cavity of an organism) where balloon angioplasty is to be applied. Following the

transfection of the therapeutic genes into the region of interest in blood vessel 138 a reporter gene such as GFP (whether wild-type GFP or any mutant GFP), luciferase or any other reporter or marker gene may be transfected from a source 144 into such region.

Microinstrument 10 may then be used to monitor the intensity of the reporter gene's

fluorescence or luminescence, thereby indicating the success of the therapeutic transfection

procedure. For example, if fluorescence is used, the light source 140 used to excite the fluorescence may be introduced to site S with optical fiber 142 by the way of catheter 136, or

it may be produced by microinstrument 10 (e.g.. IC chip 74). Microinstrument 10 may then

measure the emitted fluorescent light (Stokes shifted) or the light produced by luminescence

and send the digital results to data processing system 76, which is connected to microinstrument 10 by wires 143, fiber optic, telemetry or any other data transmission means.

Data processing system 76 may integrate the measurement result as long as necessary to

produce appropriate sensitivity and signal to noise ratio. Data processing system 76 may also

analyze and display the data for easy interpretation by the doctor.

In the embodiment of FIG. 18, microinstrument 10 preferably is of a similar

size to the microinstrument embodiment described in connection with FIGS. 4-17 herein, or

smaller. Such a device is capable of very inexpensive manufacture. Given these characteristics, in addition to making possible for the first time the in vivo measurement and conditioning of light intensity data, the microinstrument of the present invention may be made as a disposable unit.

Example

The components of a microinstrument were assembled in accordance with the embodiment of the present invention illustrated in FIGS. 4-17, and tested as follows. An ORIEL 7340 white light source was used to emit a wide wavelength span light including the

region of interest between about 400 nm and 1100 nm. Light from the light source was passed though an ORIEL 77700, a grating monochromator which utilizes grating interference phenomenon to disperse light with different wavelengths at different angles. The optical

filter was used to reject harmonic wavelengths. For example, the angle at which the first

maximum of 800 nm lies is also the second maximum of 400 nm, and the optical filter was used to reject the 400 nm wavelength light while accepting the 800 nm wavelength light. The monochromic light was then passed to the photodiode, and a photocurrent was generated

which was proportional to light intensity. The photocurrent was converted to a voltage by the

trans-impedance amplifier. After further amplification by the voltage amplifier, the voltage signal was converted into digital data by the A/D converter and sent to the external data

processing system by the serial port driver. Commercial LAB VIEW™ software, distributed

by National Instruments Corporation of Austin, Texas, was used to perform the measurement

operation continuously from 400 nm to 1100 nm.

The monochromator irradiance curve was obtained using a known reference

photodiode, according to the following equation: Irid (W/cm²) = ( A)) / ((R_ref(A/W))(A_ref(cm²)))

where R,._ef and A_ref are the response and area of the reference photodiode, and I _ref(A) is the

photo-current. Measurements were then taken from five different, similarly sized (about 200

μm x 200 μm) photodiodes. Using the above monochromator irradiance, the response R( A/W) and quantum efficiency QE of the five diodes were obtained using the following equation:

R(A/W) = (1(A)) / ((Irid(A/cm²))(A(cm²)))

QE = (1.24R(A/W)) / (λ(um))

where 1(A) is the current generated by one of the five photodiodes, and A is the area of that photodiode.

The response and quantum efficiencies of the five photodiodes are shown in FIGS. 19 and 20, respectively, in which curve A corresponds to an N-diffusion P-base diode,

curve B corresponds to a P-diffusion N-well diode, curve C corresponds to a P-base/N-well diode, curve D corresponds to a N-well/P-substrate diode, and curve E corresponds to a N-

diffusion P-substrate diode. From FIG. 19, photodiode A can be seen to have the greatest response. Actually, this photodiode consists of two junction in parallel, N-diffusion/P-base

junction and P-base/N-well junction, which is the reason for the greatest responsivity. As seen in FIG. 20, the maximum quantum efficiency in the region of interest is only about 25%

for the photodiodes.

Responsivity measurements were also taken for various sizes of photodiode A

(76 μm x 84 μm, 153 μm x 165 μm, and 247 μm x 236 μm), from which it was determined

that the smallest photodiode has the greatest responsivity, although the largest photodiode exhibits the greatest degree of simplicity and the largest output current.

It is believed that the many advantages of the present invention will now be

apparent to those of ordinary skill in the art. It will also be apparent that a number of variations and modifications, several of which have been specifically mentioned and many of which have not but will be appreciated by those of ordinary skill in the pertinent art, may be

made thereto without departing from its spirit and scope. Accordingly, the foregoing description is to be construed as illustrative only, rather than limiting. The present invention is limited only by the scope of the following claims.

Claims

What is claimed is:

1. A device for measuring light intensity, said device comprising:

means for detecting light emitted or reflected by cells or tissues, said light detecting means producing a signal indicative of light intensity;

means for transmitting said signal to a data processing system, said signal

transmission means being operatively connected to said light detection means; and a base portion, said means for detecting light and said means for transmitting said

signal to a data processing system being mounted to said base portion, and said base portion being sufficiently small that said device may be inserted within a body.

2. The device of claim 1 , wherein said signal indicative of light intensity comprises an analog signal and said device further comprises means for converting said analog signal into a

digital signal indicative of light intensity, said signal conversion means being operatively connected to said light detection means and said signal transmission means, and further being

mounted to said base portion.

3. The device of claim 2, wherein: said means for detecting light emitted or reflected by cells or tissues comprises a

photodiode; said means for converting said analog signal into a digital signal indicative of light

intensity comprises an analog-to-digital converter; and said means for transmitting said digital signal to a data processing system comprises a serial output driver.

4. A device for measuring light intensity, said device comprising:

a photodetection device that detects light emitted or reflected by cells or tissues, said photodetection device producing a signal indicative of light intensity; a signal transmission device that transmits said signal to a data processing system,

said signal transmission device being operatively connected to said photodetection device; and

a base portion, said photodetection device and said signal transmission device being

mounted to said base portion, and said base portion being sufficiently small that said device may be inserted within a body.

5. The device of claim 4, wherein said signal indicative of light intensity comprises an analog signal and said device further comprises a signal conversion device that converts said

analog signal into a digital signal indicative of light intensity, said signal conversion device

being operatively connected to said photodetection device and said signal transmission

device, and further being mounted to said base portion.

6. The device of claim 5, further comprising a first optical filter and a second optical

filter, and wherein said photodetection device comprises a first photodetector that detects a

first band of light passing through said first optical filter and produces a first analog signal

indicative of the intensity of said first band of light, and a second photodetector that detects a

second band of light passing through said second optical filter and produces a second analog signal indicative of the intensity of said first band of light.

7. The device of claim 6, wherein said signal conversion device comprises a first switched capacitor bandpass filter corresponding to said first analog signal and a second

switched capacitor bandpass filter corresponding to said second analog signal, a multiplexer that selectively receives said respective first and second analog signals from said first and

second switched capacitor bandpass filters, and an analog-to-digital converter, said signal

conversion device producing a first digital signal indicative of the intensity of said first band of light and a second digital signal indicative of the intensity of said second band of light.

8. The device of claim 7, further comprising a data processing system mounted to said

base portion.

9. The device of claim 8, wherein said data processing system comprises digital signal

processing software which performs spectral analysis of said digital signal and calculates the

amplitude of said light emitted or reflected by cells or tissues to determine a value of the

intensity of said band of light.

10. The device of claim 8, wherein said data processing system comprises data storage

and display hardware.

11. The device of claim 5, wherein said first band of light comprises a strong emission wavelength of a marker molecule and said second band of light comprises emission spectra of background cells or tissues, wherein the intensity of said first band of light may be

determined by comparing said first digital signal and said second digital signal.

12. The device of claim 5, further comprising an optical filter positioned in front of said photodetection device, and wherein:

said optical filter is configured to pass a first band of light and reject a second band of light;

said photodetection device comprises a photodetector that detects said first band of light passing through said optical filter;

said signal conversion device comprises a switched capacitor bandpass filter and an analog-to-digital converter.

13. The device of claim 5, further comprising an optical filter positioned in front of said

photodetection device, and wherein: said optical filter is configured to pass a first band of light and reject a second band of

light; said photodetection device comprises a photodetector that detects said first band of

light passing through said optical filter; and said signal conversion device comprises a trans-impedance amplifier and an analog-

to-digital converter.

14. The device of claim 13 , wherein said first band of light is selected such that said device may measure the light intensity of fluorescent marker molecules expressed by gene transfection or infection.

15. The device of claim 14, wherein said first band of light is selected such that said device may measure the light intensity of fluorescent marker molecules expressed by gene

transfection or infection in a blood vessel of a human body.

16. The device of claim 13, further comprising an integrated circuit chip on which said

optical filter, said photodetection device and said signal conversion device are integrated.

17. The device of claim 16, wherein said integrated circuit chip is manufactured using a

bipolar process.

18. The device of claim 16, wherein said integrated circuit chip is manufactured using a

BiCMOS process.

19. The device of claim 16, wherein said integrated circuit chip is manufactured using a

FET process.

20. The device of claim 16, wherein said integrated circuit chip is manufactured using a

CMOS process.

21. The device of claim 20, wherein said integrated circuit chip is manufactured using a MOSIS AMI CMOS 1.2 ╬╝m process.

22. The device of claim 16, wherein said integrated circuit chip has a size of about 2.2 mm x 2.2 mm or smaller.

23. The device of claim 22, wherein said size of said integrated circuit chip is about 0.5 mm x 2 mm or smaller.

24. The device of claim 16, wherein said optical filter is separate from said integrated circuit chip.

25. The device of claim 16, wherein said optical filter is attached to said integrated circuit chip.

26. The device of claim 25, wherein said optical filter comprises a high wavelength-pass

filter.

27. The device of claim 26, wherein said optical filter is attached to glass which is

attached to said integrated circuit chip.

28. The device of claim 16, wherein said photodetection device comprises a

phototransistor.

29. The device of claim 16, wherein said photodetection device comprises a semiconductor metal diode.

30. The device of claim 16, wherein said photodetection device comprises a photodiode.

31. The device of claim 30, wherein said photodetection device comprises an array of photodiodes.

32. The device of claim 30, wherein said photodiode comprises an N-diffusion/P-base junction photodiode.

33. The device of claim 32, wherein said photodiode has a width that is about 0.5 mm or smaller.

34. The device of claim 16, wherein said signal conversion device further comprises a

current amplifier operatively connected between said trans-impedance amplifier and said

analog-to-digital converter.

35. The device of claim 16, wherein said signal conversion device further comprises a

charge-pump amplifier operatively connected between said trans-impedance amplifier and

said analog-to-digital converter.

36. The device of claim 16, wherein said signal conversion device further comprises a

voltage amplifier operatively connected between said trans-impedance amplifier and said analog-to-digital converter.

37. The device of claim 36, wherein said trans-impedance amplifier and said voltage amplifier each comprise an operational amplifier.

38. The device of claim 37, wherein said analog-to-digital converter is an 8-bit, dual-slope analog-to-digital converter with a size that is about 0.45 mm x 1.95 mm or less.

39. The device of claim 38, wherein said analog-to-digital converter comprises an analog portion and a digital portion, said analog portion comprising an integrator, a comparator, an analog multiplexer and an analog switch, and said digital portion comprising a control unit,

an 8-bit counter, a serial output driver and a control circuit.

40. A system for measuring light intensity in a body in vivo, said system comprising:

a catheter having a proximal portion and a distal portion; and a microinstrument for measuring light intensity mounted to said distal portion of said

catheter; said microinstrument comprising:

a photodetection device that detects light emitted or reflected by cells or

tissues, said photodetection device producing a signal indicative of light intensity; and a signal transmission device that transmits said signal to a data processing system, said signal transmission device being operatively connected to said photodetection;

wherein said microinstrument is of a sufficiently small size that said microinstrument may be inserted within a body along with said catheter.

41. The system of claim 40, wherein said signal indicative of light intensity comprises an analog signal, and said microinstrument further comprises a signal conversion device that

converts said analog signal into a digital signal indicative of light intensity, said signal conversion device being operatively connected to said photodetection device and said signal transmission device.

42. The system of claim 41, further comprising a data processing system that receives said digital signal from said signal transmission device of said microinstrument.

43. The system of claim 42, wherein said catheter is an angioplasty catheter comprising a

balloon portion, said microinstrument being mounted to said balloon portion of said

angioplasty catheter.

44. A method for evaluating in vivo the success of a transfection or infection procedure,

said method comprising:

providing a data processing system; providing a light intensity detection device that is sufficiently small to be inserted

within a body, and that comprises a photodetection device that detects light emitted or reflected by cells or tissues and produces a signal indicative of light intensity and a signal transmission device that transmits said signal indicative of light intensity to said data processing system;

positioning said light intensity detection device at a site of interest, said site having a plurality of marker molecules and background cells or tissue;

using said light intensity detection device to measure the intensity of light emitted or reflected by the marker molecules and the background cells or tissues at said site of interest; and

using said data processing system to determine a value for the intensity of light emitted or reflected by the marker molecules and the background cells or tissues at said site of interest.

45. The method of claim 44, wherein said step of positioning said light intensity detection device at a site of interest comprises inserting said light intensity detection device into a human body and transporting said light intensity detection device to said site of interest in

said human body.

46. The method of claim 44, wherein said step of positioning said light intensity detection device at a site of interest comprises inserting said light intensity detection device into a cell

culture.

47. The method of claim 46, wherein said step of using said light intensity detection

device to measure the intensity of light emitted or reflected by the marker molecules and the background cells or tissues at said site of interest further comprises detecting the intensity of

luminescent light emitted from the marker molecules using said light intensity detection device.

48. The method of claim 46, wherein said light intensity detection device comprises a first

optical filter corresponding to a first photodetector and a second optical filter corresponding to a second photodetector, and said step of using said light intensity detection device to measure the intensity of light emitted or reflected by the marker molecules and the background cells or tissues at said site of interest further comprises: introducing a band of excitation light to said site of interest, resulting in emission light being emitted from said site of interest; filtering said emission light using said first optical filter, and detecting the intensity of

said emission light using said first photodetector; and filtering said emission light using said second optical filter, and detecting the intensity

of said excitation light using said second photodetector.

49. The method of claim 48, wherein said step of introducing a band of excitation light to

said site of interest comprises: introducing said band of excitation light to said site of interest for a first time,

resulting in a first emission light being emitted from said site of interest; and

introducing said band of excitation light to said site of interest for a second time,

resulting in a second emission light being emitted from said site of interest; and

wherein said step of filtering said emission light using said first optical filter comprises filtering said first emission light, and said step of filtering said emission light using said second optical filter comprises filtering said second emission light.

50. The method of claim 48, wherein said band of excitation light has a range of wavelengths.

51. The method of claim 46, wherein said step of using said light intensity detection device to measure the intensity of light emitted or reflected by the marker molecules and the background cells or tissues at said site of interest further comprises: introducing a first band of excitation light to said site of interest; using said light intensity detection device to measure the intensity of light emitted at said site of interest in response to said introduction of said first band of excitation light; introducing a second band of excitation light to said site of interest; and using said light intensity detection device to measure the intensity of light emitted at said site of interest in response to said introduction of said second band of excitation light.

52. The method of claim 51 , wherein said first band of excitation light has a first range of wavelengths, said first range of wavelengths including a strong excitation peak wavelength of

either the marker molecules or the background cells or tissues, and said second band of

excitation light has a second range of wavelengths, said second range of wavelengths

including a strong excitation peak wavelength of the other of the marker molecules or the

background cells or tissues.

53. The method of claim 46, wherein said step of using said light intensity detection device to measure the intensity of light emitted or reflected by the marker molecules and the background cells or tissues at said site of interest further comprises:

positioning said light intensity detection device at a first location proximal to said marker molecules at said site of interest;

introducing a band of excitation light to said site of interest; using said light intensity detection device to measure the intensity of light emitted in response to said excitation light at said first location proximal to said marker molecules; repositioning said light intensity detection device to a second location proximal to said background cells or tissues at said site of interest; introducing said band of excitation light to said site of interest; and using said light intensity detection device to measure the intensity of light emitted in response to said excitation light at said second location proximal to said background cells or

tissues.

54. The method of claim 46, further comprising, following said step of using said data processing system to determine a value for the intensity of said light emitted or reflected by

the marker molecules, displaying said intensity value.