Fluorescence receiving apparatus and fluorescence receiving method

Patent metadata

Field	Value
Publication number	US-9740166-B2
Application number	US-201414772570-A
Country	US
Kind code	B2
Filing date	Mar 4, 2014
Priority date	Mar 6, 2013
Publication date	Aug 22, 2017
Grant date	Aug 22, 2017

Abstract

A fluorescence receiving apparatus comprises an excitation light source, a spatial light modulator of a phase modulation type for phase-modulating excitation light to obtain phase-modulated light, a focusing optical system configured to focus the phase-modulated light to a specimen, a specimen stage for supporting the specimen, a fluorescence receiver for receiving fluorescence generated by focus of the phase-modulated light to the specimen, a control unit for displaying a first CGH on the spatial light modulator, and a correction unit for correcting the first CGH. The correction unit comprises a receiver-specific sensitivity information storage preliminarily acquiring and storing sensitivity information per reception position specific to the fluorescence receiver, and a second hologram generator for correcting the first CGH, based on intensities of the fluorescence and the sensitivity information, to generate a second CGH. The control unit displays the second CGH on the spatial light modulator.

First claim

The invention claimed is:

1. An apparatus for receiving fluorescence generated from a specimen by focus of phase-modulated excitation light, comprising:
light source configured to output an excitation light;
a spatial light modulator of a phase modulation type configured to input the excitation light, modulate phase of the excitation light and output the phase-modulated light;
a focusing optical system configured to input the phase-modulated light and focus the phase-modulated light to the specimen;
a stage configured to support the specimen;
a light detector configured to detect fluorescence generated by the phase-modulated light, via the focusing optical system;
a controller configured to control the spatial light modulator so as to display a first hologram to modulate a phase of the excitation light at each of a plurality of two-dimensionally arrayed pixels, thereby focusing the phase-modulated light at a plurality of focusing positions of the specimen by the focusing optical system; and
a correction unit configured to correct the first hologram based on intensities of the fluorescence at the focusing positions and sensitivity information per reception position specific to the light detector, to generate a second hologram and,
wherein the controller controls the spatial light modulator so as to display the second hologram.

2. The apparatus according to claim 1 , wherein the correction by the correction unit is performed for each of the plurality of focusing positions.

3. The apparatus according to claim 1 , wherein the light source outputs a short pulsed laser capable of inducing multiphoton excitation.

4. The apparatus according to claim 1 , wherein the light detector is a multi-anode type photomultiplier tube.

5. The apparatus according to claim 1 , wherein the specimen is comprised of a homogeneous fluorescent material without location-dependent variation in a ratio of intensities of the fluorescence generated from the specimen to the phase-modulated light focused to the specimen.

6. The apparatus according to claim 1 , wherein the correction by the correction unit is performed for each of scanned layers at predetermined intervals along an optical-axis direction of an objective lens included in the focusing optical system.

7. The apparatus according to claim 6 , wherein scanning for each of the scanned layers is performed in such a manner that the spatial light modulator controls the focusing positions in the optical-axis direction of the objective lens.

8. A method for receiving fluorescence generated from a specimen mounted on a stage by focus of phase-modulated excitation light, comprising:
outputting an excitation light;
modulating phase of the excitation light by a spatial light modulator of a phase modulation type and outputtings phase-modulated light;
focusing the phase-modulated light to the specimen by a focusing optical system;
detecting fluorescence generated by the phase-modulated light, via the focusing optical system, using a light detector;
controlling the spatial light modulator so as to display a first hologram to modulate a phase of the excitation light at each of a plurality of two-dimensionally arrayed pixels, thereby focusing the phase-modulated light at a plurality of focusing positions of the specimen by the focusing optical system;
correcting the first hologram based on intensities of the fluorescence at the focusing positions and sensitivity information per reception position specific to the light detector, to generate a second hologram;
controlling the spatial light modulator so as to display the second hologram.

Assignees

• Hamamatsu Photonics Kk

Inventors

• Matsumoto Naoya

Classifications

• G03H2001/005

  in microscopy, e.g. digital holographic microscope [DHM] · CPC title

• G01N21/6458 Primary

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• G03H1/2294

  Addressing the hologram to an active spatial light modulator · CPC title

• G02B21/0076

  arrangements using fluorescence or luminescence · CPC title

• G03H2001/0825

  Numerical processing in hologram space, e.g. combination of the CGH [computer generated hologram] with a numerical optical element · CPC title