Imaging fluorometer for time resolved fluorescence

• US 20030160151A1
• Filed: 09/30/2002
• Published: 08/28/2003
• Est. Priority Date: 02/28/2001
• Status: Active Grant

• Current Assignee: GE Healthcare Niagara, Inc. (General Electric Company)
• Original Assignee: Amersham Biosciences Niagara, Inc. (General Electric Company)
• Inventors: Yekta, Ahmad, Masoumi, Zahra, Ramm, Peter, Zarate, Carlos, Donders, Paul
• Granted Patent: US 6,806,455 B2
• Time in Patent Office: Days
• US Class Current: 250/208.1
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 2201/0648 Shutters

Abstract

The present invention relates to an apparatus and method for imaging time resolved fluorescence in biochemical and medical samples. In a primary aspect, the device includes a lens of large aperture, a flash lamp in the illumination path, a fast-acting solid state shutter or a gated detector in the emission path, a device for delivering homogenous monochromatic illumination to a plurality of wells distributed within a microwell plate, a digital camera of high quantum efficiency, and a computer under computer control, the lamp is pulsed at short intervals. The fast-acting emission shutter or gated detector operates to limit exposure of the camera to a period some microseconds after the extinction of each lamp pulse, during which only delayed fluorescence is transmitted to the camera. The invention achieves simultaneous time resolved imaging of a plurality of samples, with high sensitivity and high throughput.

Claims

1. An imaging system for time resolved fluorometry of chemical, biological and medical samples provided on a containing medium where they are to be subjected to illumination, causing them to emit light, the imaging system comprising:

an illumination source which can be modulated from substantially full intensity at the samples to less than approximately 1% of full intensity in a time of less than approximately 100 microseconds;

an illumination system providing illumination from the source to an area in the vicinity of the samples which can be larger than a microscope field of view;

a lens to collect light emitted from samples contained within the illuminated area;

an image formation device located such that light from the samples, passing through the lens, impinges on the device and the device produces a representation of an image of the sample emissions;

a shutter placed between the samples and the image formation device, comprising a structure which can be made opaque (off) or transparent (on), with an on-off period shorter than approximately 100 microseconds and with a contrast ratio between on and off in excess of approximately 100;

1; and

a control device for setting the on-off times of the illumination source and the open/close times of the shutter.

2. The system of claim 1, wherein the shutter is a ferroelectric crystal device configured as a polarizer that changes the rotation of polarization of light passing through it depending on an applied electric field.

3. The system of claim 2, further comprising a polarizing analyzer inserted in the emission path between the samples and the imaging formation device.

4. The system of any one of claims 1, 2 or 3, wherein the shutter is disposed within the lens, in a position to yield the best possible contrast ratio.

5. The system of claim 4, wherein the lens has optics so constructed and the shutter is so positioned in the optical path of the emitted light that the incidence angles of the light with respect to the normal onto the shutter do not exceed 20°

.

6. The system of any preceding claim, wherein the containing medium is composed of a plurality of wells arranged within a plate and the lens is constructed to detect the entire contents of wells without parallax error.

7. The illumination system of any preceding claim, wherein the illumination source is a flash lamp.

8. The system of any preceding claim, wherein the control device is constructed to operate the shutter and the illumination source through multiple on-off cycles, the image formation device accumulating the light impinging thereon during plural cycles.

9. The imaging system of any preceding claim, wherein emitted light exhibits a decay in intensity and the shutter is gated on and off more than once during the decay following an illumination of the samples, so as to acquire images at different times during the decay process.

10. The system of any preceding claim wherein the illumination system is constructed to guide light from the source into the lens and out of the lens towards the samples.

11. The system of any preceding claim wherein the illumination system comprises a beam splitter between the lens and the samples constructed to reflect light from the source towards the samples and to transmit light emitted from the samples towards the lens.

12. The system of any preceding claim further comprising a correction processor storing a leakage image of prompt fluorescence with the shutter closed and subtracting this stored leakage image from a subsequent delayed fluorescence image, to produce a corrected delayed fluorescence image.