# US 8,542,439 B2 · Method and device for scanning-microscopy imaging of a specimen

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- Number: US 8,542,439 B2
- Document ID: US8542439B2
- Status: Granted
- Kind: B2
- Application number: 13/347,756
- Filed: 2012-01-11
- Published: 2012-07-12
- Granted: 2013-09-24
- Earliest claimed: 2011-01-11
- Assignees: Leica Microsystems CMS GmbH
- Applicants: Holger Birk; Bernd Widzgowski; Holger Nissle
- Inventors: Holger Birk; Bernd Widzgowski; Holger Nissle
- Web page: https://patentgrep.com/patent/US8542439B2

## CPC

- G02B 21/008: {Details of detection or image processing, including general computer control}
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G02B 21/0076: {arrangements using fluorescence or luminescence}
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

A method and a device for scanning-microscopy imaging of a specimen (28) are described. Provision is made that a plurality of specimen points are scanned by means of a scanning beam (14) in successive scanning time intervals, the intensity of the radiation emitted from the respectively scanned specimen point is repeatedly sensed within the associated scanning time interval, an intensity mean value is determined, as a mean value image point signal, from the intensities sensed in the respectively scanned specimen point, and the mean value image point signals are assembled into a mean value raster image. Provision is further made for additionally determining an intensity variance value, as a variance image point signal, from the intensities sensed in the respectively scanning specimen points, and for assembling the variance image point signals into a variance raster image signal.

## Claims

### Claim 1

A method for scanning-microscopy imaging of a specimen (28), comprising the following steps:
scanning a plurality of specimen points by means of a scanning beam (14) in successive scanning time intervals,
repeatedly sensing the intensity of the radiation emitted from the respectively scanned specimen point within the associated scanning time interval,
determining an intensity mean value, as a mean value image point signal, from the intensities sensed in the respectively scanned specimen point, and
assembling the mean value image point signals into a mean value raster image signal,
wherein an intensity variance value is additionally determined, as a variance image point signal, from the intensities sensed in the respectively scanned specimen point, and
the variance image point signals are assembled into a variance raster image signal.

### Claim 2 (depends on 1)

The method according to claim 1, wherein
an evaluation image point signal is generated on the basis of the respective mean value image point signal and the associated variance image point signal, and
the generated evaluation image point signals are assembled into an evaluation raster image signal.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the intensity of the radiation emitted within each scanning time interval is digitized n times in successive sub-intervals in order to sense n intensity values, n being a whole number greater than 1 and the sub-intervals each being equal to 1/n times the scanning time interval.

### Claim 4 (depends on 3)

The method according to claim 3, wherein the n intensity values are sensed by the fact that an analog signal corresponding to the emitted radiation is converted by means of an analog-digital converter n times within the respective scanning time interval, in each case with a predetermined conversion time that is equal to the sub-interval, into a digital signal representing the respective intensity value.

### Claim 5 (depends on 3)

The method according to claim 3, wherein the n intensity values are sensed by the fact that the number of photons that correspond to the emitted radiation is determined by means of a photon counter n times within the respective scanning time interval, in each case with a predetermined counting time that is equal to the sub-interval.

### Claim 6 (depends on 1)

The method according to claim 1, wherein
the respective intensity mean value x is determined according to formula (1) below:
x
=
1
n
⁢
∑
i
=
1
n
⁢
x
i
(
1
)
and the respective intensity variance value V is determined according to formula (2) below:
V
=
1
n
-
1
⁢
∑
i
=
1
n
⁢
(
x
i
-
x
_
)
2
,
(
2
)
where the variable
∑
i
=
1
n
⁢
(
x
i
-
x
_
)
2
is preferably determined according to formula (3)
∑
i
=
1
n
⁢
(
x
i
-
x
_
)
2
=
∑
i
=
1
n
⁢
x
i
2
-
n
⁢
⁢
x
_
2
(
3
)
or according to formula (3′) below:
∑
i
=
1
n
⁢
(
x
i
-
x
_
)
2
=
∑
i
=
1
n
⁢
x
i
2
-
1
n
⁢
(
∑
i
=
1
n
⁢
x
i
)
2
(
3
′
)
where
xi denotes the intensities sensed within the respective scanning time interval, and
n denotes a whole number, greater than 1, that indicates the number of intensities xi sensed within the respective scanning time interval.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the quotient of the intensity variance value V and the intensity mean value x is determined as an evaluation image point signal.

### Claim 8 (depends on 6)

The method according to claim 6, wherein the number of photons Nph from the respective specimen point that are detected within the associated scanning time interval is determined, as an evaluation image point signal, as a function of the intensity mean value x and the intensity variance value V according to formula (4) below:
N
ph
=
x
_
2
V
.
(
4
)

### Claim 9

A device for scanning-microscopy imaging of a specimen, comprising:
a scanning unit (12, 18) that is embodied to scan a plurality of specimen points by means of a scanning beam (14) in successive predetermined scanning time intervals,
a sensing unit (36, 38) that is embodied to sense the intensity of the radiation emitted from the respectively scanned specimen point repeatedly within the associated scanning time interval,
a calculation unit (40) that is embodied to determine an intensity mean value, as a mean value image point signal, from the intensities sensed in the respectively scanned specimen point, and
an evaluation unit (42) that is embodied to assemble the mean value image point signals into a mean value raster image signal,
wherein
the calculation unit (40) is further embodied to additionally determine an intensity variance value, as a variance image point signal, from the intensities sensed in the respective sensed specimen point, and
the evaluation unit (42) is further embodied to assemble the variance image point signals into a variance raster image signal.

### Claim 10 (depends on 9)

The device according to claim 9, wherein the evaluation unit (42) is further embodied to generate an evaluation raster image signal on the basis of the mean value raster image signal and the variance raster image signal, and to assemble the generated evaluation image point signals into an evaluation raster image signal.

### Claim 11 (depends on 8)

The device according to claim 8, wherein the sensing unit (36, 38) encompasses a digitizing means (38) that is embodied to digitize the intensity of the radiation detected within each scanning time interval n times in successive sub-intervals in order to sense n intensity values, n being a whole number greater than 1 and the sub-intervals each being equal to 1/n times the scanning time interval.

### Claim 12 (depends on 11)

The device according to claim 11, wherein the digitizing means encompasses an analog-digital converter (38) that is embodied to sense the n intensity values by the fact that it converts an analog signal corresponding to the emitted radiation n times within the respective scanning time interval, in each case with a predetermined conversion time that is equal to the sub-interval, into a digital signal representing the respective intensity value.

### Claim 13 (depends on 11)

The device according to claim 11, wherein the digitizing means encompasses a photon counter that is embodied to sense the n intensity values by the fact that it determines the number of photons that correspond to the emitted radiation n times within the respective scanning time interval, in each case with a predetermined counting time that is equal to the sub-interval.

### Claim 14 (depends on 9)

The device according to claim 9, wherein the calculation unit (40) encompasses a freely programmable logic module, in particular an FPGA, a DSP, or an ASIC.

### Claim 15 (depends on 9)

A confocal scanning microscope (10) having a device according to claim 9.