Microscopy system and microscopy method for quantifying a fluorescence

Patent metadata

Field	Value
Publication number	US-11079587-B2
Application number	US-201815910225-A
Country	US
Kind code	B2
Filing date	Mar 2, 2018
Priority date	Mar 2, 2017
Publication date	Aug 3, 2021
Grant date	Aug 3, 2021

Abstract

A microscopy method for quantifying a fluorescence of protoporphyrin IX includes: imaging an object region onto a first detector field and a second detector field, wherein a first optical filter and a second optical filter, respectively, are arranged in the beam paths between the object region and the detector fields, the first optical filter and second optical filter respectively having a wavelength-dependent transmission characteristic; exciting at least a first fluorescence of protoporphyrin IX and a second fluorescence; recording first images and second images; and determining a spatially dependent fluorescence intensity of the first fluorescence in the object region by virtue of determining values representing a fluorescence intensity at locations in the object region, wherein the values are determined on the basis of the radiation intensities of the two detector fields detected in a spatially dependent manner and the spatially dependent wavelength-dependent detection efficiencies of the two detector fields.

First claim

The invention claimed is:

1. A microscopy method for quantifying a fluorescence of protoporphyrin IX, the method comprising:
imaging an object region onto a first detector field having a multiplicity of first pixels, wherein at least one first optical filter with a first wavelength-dependent transmission characteristic is arranged in a first beam path between the object region and each one of the pixels of the first detector field;
imaging the object region onto a second detector field having a multiplicity of second pixels, wherein at least one second optical filter with a second wavelength-dependent transmission characteristic that differs from the first wavelength-dependent transmission characteristic is arranged in a second beam path between the object region and each one of the pixels of the second detector field;
exciting at least a first fluorescence and a second fluorescence in the object region, wherein the first fluorescence is the fluorescence of protoporphyrin IX;
recording a first image of the imaged object region using the first detector field;
recording a second image of the imaged object region using the second detector field; and
determining a spatially dependent fluorescence intensity of the first fluorescence in the object region by determining in each case a fluorescence intensity value for a plurality of first pixels or a plurality of groups of first pixels in the first detector field, the fluorescence intensity value representing a fluorescence intensity at a location in the object region imaged onto the respective first pixel or the respective group of first pixels,
wherein the fluorescence intensity value is determined from a radiation intensity detected by the respective first pixel or group of first pixels of the first detector field, a wavelength-dependent detection efficiency of the respective first pixel or group of first pixels, a radiation intensity detected by a second pixel or group of second pixels of the second detector field on which the location in the object region is imaged, a wavelength-dependent detection efficiency of the second pixel or group of second pixels, a fluorescence spectrum of the first fluorescence, and a fluorescence spectrum of the second fluorescence,
wherein the respectively determined fluorescence intensity value is determinable according to the following formula:

C
F

⁡

(

x
,
y

)

=

U

1
⁢
A

·

S
2

⁡

(

k
,
l

)

-

U

2
⁢
A

·

S
1

⁡

(

i
,
j

)

U

2
⁢
F

·

U

1
⁢
A

-

U

2
⁢
A

·

U

1
⁢
F

,

and
wherein:
CF(x,y) is the fluorescence intensity value representing a fluorescence intensity of protoporphyrin IX at a location (x,y) in the object plane,
S1(i,j) represents a radiation intensity detected by the first pixel (i,j) or group (i,j) of first pixels of the first detector field, wherein the location (x,y) is mapped onto the first pixel (i,j) or group (i,j) of first pixels by way of the first beam path,
S2(k,l) represents the radiation intensity detected by the second pixel (k,l) or group (k,l) of second pixels of the second detector field, wherein the location (x,y) is mapped onto the second pixel (k,l) or group (k,l) of second pixels by way of the second beam path, and
U1F, U1A, U2F, and U2A are variables which depend on wavelength-dependent detection efficiencies of the first and second pixels or first and second group of pixels.

2. The microscopy method according to claim 1 , further comprising:
determining values for at least some of the variables U1F, U1A, U2F, and U2A in a reference measurement at a location in the object region in which a concentration of protoporphyrin IX is substantially zero.

3. The microscopy method according to claim 1 , further comprising:
determining values of at least some of the variables U1F, U1A, U2F, and U2A by calculating an associated equation selected from the group consisting of:

U

1
⁢
F

=

∫

λ
⁢

⁢
min

λ
⁢

⁢
max

⁢

S
F

Assignees

• Zeiss Carl Meditec Ag

Inventors

• Regensburger Alois
• Hauger Christoph
• Kohlhammer Susanne
• Essig Jonathan

Classifications

• G01N2021/6421

  Measuring at two or more wavelengths · CPC title

• G01N2021/6471

  Special filters, filter wheel · CPC title

• G02B21/16

  adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)} · CPC title

• G02B21/361

  Optical details, e.g. image relay to the camera or image sensor (G02B21/364 takes precedence; illumination details G02B21/06 and subgroups) · CPC title

• G02B21/368

  details of associated display arrangements, e.g. mounting of LCD monitor · CPC title